東北医科薬科大学
分子生体膜研究所年報
2018年度 研究成果報告書
2018 Annual Report ofInstitute of Molecular Biomembrane and Glycobiologyࡈᣵᣜ  ᖺᗘࡣࠊᮏᏛศᏊ⏕య⭷◊✲ᡤࡀୡヰேࢆົࡵࠊ➨㸱㸵ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ
఍ࢆ௝ྎᅜ㝿ࢭࣥࢱ࣮࡛  ᭶㸰㸶Ѹ㸱㸮᪥࡟㛤ദ࠸ࡓࡋࡲࡋࡓࠋ௝ྎ࡛ࡢ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍ࡣࠊ᫛࿴㸳㸶ᖺ㸦➨㸴ᅇ ᯇ⏣࿴㞝ඛ⏕㸧ࠊᖹᡂ㸳ᖺ㸦➨㸯㸳ᅇ 㕥ᮌⱱ⏕ඛ⏕㸧ࠊᖹᡂ㸯㸶ᖺ㸦➨㸰㸴ᅇ ᐑᇛጁᏊඛ⏕㸧ࡑࡋ࡚௒ᖺࡢ➨㸱㸵ᅇ࡛㸲ᅇ┠ࡢ㛤ദ࡜࡞ࡾࡲࡍࠋ㸯㸰ᖺ๓ࡢ➨㸰㸴ᅇ࡛ࡣࠊᐑᇛ┴❧ࡀࢇࢭࣥࢱ࣮ࠊᮾ໭኱Ꮫࠊ
ᘯ๓኱Ꮫࠊᮾ໭⸆⛉኱Ꮫ㸦⌧ᮾ໭་⛉⸆⛉኱Ꮫ㸧ࡢ⢾㉁⛉Ꮫ◊✲⪅ࡀୡヰே࡜࡞ࡾࠊ௒ᅇ࡜ྠࡌ௝ྎᅜ㝿ࢭࣥࢱ࣮࡛㛤ദࡉࢀࡲࡋࡓࠋࡇࡢ➨㸰㸴ᅇᖺ఍ࡀዎᶵ࡜
࡞ࡾࠊᮾ໭⢾㙐◊✲఍ࡀⓎ㊊ࡋࠊẖᖺ୍ᅇࡢ◊✲఍ࢆ㛤ദࡋάືࡋ࡚࠸ࡲࡍ㸦85/
KWWSWRKRNXWWRXVDVWULNLQJO\FRP㸧ࠋ⌧ᅾࠊᮾ໭⢾㙐◊✲఍࡟ཧຍࡋ࡚࠸ࡿ◊✲⪅ࡢ◊✲ᶵ㛵ࡣࠊୖグࡢ㸲◊✲ᶵ㛵࡟ຍ࠼࡚ࠊ໭࠿ࡽ㮚ᥭ㒓⭈◊✲ᡤࠊᒾᡭ་⛉኱Ꮫࠊ⚟ᓥ┴❧་⛉኱Ꮫࠊ᪂₲኱Ꮫࠊ᪂₲⸆⛉኱Ꮫࠊ㛗ᒸᢏ⾡⛉Ꮫ኱Ꮫࠊ⩌㤿኱Ꮫࡢィ㸯1 ◊✲ᶵ㛵࡟Ⓨᒎࡋࠊ➨㸱㸵ᅇᖺ఍ࡢࣉࣟࢢ࣒ࣛጤဨࠊ⤌⧊ጤဨࢆᢸᙜࡋ
࡚࠸ࡓࡔࡁࡲࡋࡓࠋࡇࡇ࡟ཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ
௒ᅇࡢᖺሗ࡛ࡣࠊྛ◊✲㒊㛛ࡢ◊✲άືሗ࿌࡟ຍ࠼࡚ࠊ➨㸱㸵ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ
఍ࡢෆᐜሗ࿌ࠊ➨㸰ᅇ⟽Ᏺ௝୍㑻㈹ࡢዡບ㈹࠾ࡼࡧඃ⚽ㄽᩥ㈹ࡢࡈ⤂௓ࢆ࠸ࡓࡋ
ࡲࡍࠋ ⓙᵝ࠿ࡽࡢ࡞࠾୍ᒙࡢࡈᣦᑟࠊࡈ㠴᧡ࡢ࡯࡝⾺ᚰࡼࡾ࠾㢪࠸⏦ࡋ࠶ࡆࡲࡍࠋ

ᖹᡂ㸱㸯ᖺ㸱᭶      ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤ࣭ᡤ㛗ᮾ໭⢾㙐◊✲఍࣭఍㛗஭ࣀཱྀ ோ୍
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 ᡤ㛗ᣵᣜ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸯ศᏊ⏕య⭷◊✲ᡤ࣭ᡤ㛗 ஭ࣀཱྀ ோ୍ ➨㸱㸵ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍ࡢ㛤ദሗ࿌࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸲 ᒣᕝẸኵඛ⏕ࢆംࢇ࡛࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸵ᮾ໭་⛉⸆⛉኱Ꮫᐈဨᩍᤵ 㕥ᮌ᫂㌟ ⟽Ᏺ௝୍㑻㈹࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸯㸰ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤࠕ⟽Ᏺ௝୍㑻㈹ࠖつ⣙
ᖹᡂ 30 ᖺᗘ ⟽Ᏺ௝୍㑻㈹ዡບ㈹✄᳃ ၨ୍㑻㸦ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤ㸧ඃ⚽ㄽᩥ㈹┠㯮 ᗣὒ㸦ᮾ໭኱Ꮫ኱Ꮫ㝔㎰Ꮫ◊✲⛉㸧ඃ⚽ㄽᩥ㈹ᮧୖ ཭ኴ㸦⚟ᓥ┴❧་⛉኱Ꮫ་Ꮫ㒊㸧 ◊✲ሗ࿌࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸰㸯ᶵ⬟⑓ែศᏊ㒊㛛
ࠕGM3 ࢞ࣥࢢࣜ࢜ࢩࢻࡢ⏕⌮ⓗព⩏ࡢゎ᫂࡟ྥࡅ࡚ࠖ஭ࣀཱྀ ோ୍㸪✄᳃ ၨ୍㑻㸪Ọ⚟ ṇ࿴㸪⊁㔝 ⿱⪃⏕య⭷᝟ሗ㒊㛛
ࠕ⏕ά⩦័⑓࡟㛵㐃ࡋࡓ G ࢱࣥࣃࢡ㉁ඹᙺཷᐜయࡢᶵ⬟࡜⢾㙐࠾ࡼࡧཷᐜయ┦஫స⏝࡟ࡼࡿㄪ⠇ࠖᮾ ⚽ዲ㸪୰ᕝ ဴே㸪㯮⏣ ႐ᖾ⣽⬊ไᚚ㒊㛛
ࠕ⢾㙐࡟ࡼࡿࡀࢇ⣽⬊ࡢᶵ⬟࡜୰ᯡ⚄⤒⣔࡟࠾ࡅࡿ⅖⑕཯ᛂࡢไᚚࠖ㢳 ᘓᅜ㸪⚟⏣ ཭ᙪ㸪ఀᕥ἞ ▱ᘺศᏊㄆ㆑㒊㛛
ࠕࣞࢡࢳࣥࡢᢠ⭘⒆࣓࢝ࢽࢬ࣒ࡢゎ᫂࡜ࡀࢇ⸆≀἞⒪࡬ࡢᛂ⏝ࠖ⣽㔝 㞞♸㸪Ⳣཎ ᰤ⣖㸪❧⏣ ᓅ⏕ Ꮫ఍Ⓨ⾲グ㘓࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸱㸵 Ꮫ⾡ㄽᩥ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸲㸰3➨㸱㸵ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍ࡢ㛤ദሗ࿌஭ࣀཱྀ ோ୍      ᖹᡂ㸱㸮ᖺ㸶᭶㸰㸶ࠥ㸱㸮᪥࡟㛤ദࡉࢀࡓ௒ᅇࡢ➨ 37 ᅇᖺ఍࡛ࡣࠕḟୡ௦་⒪࡬ࡢ⢾㙐ࡢ㈉⊩ࠖࢆࢸ࣮࣐࡟ᥖࡆࠊ⢾㙐ᕤᏛ࡜⢾㙐⏕≀Ꮫ࡟࠾ࡅࡿ᪥ᮏࡢᙉࡳ
ࢆ⏕࠿ࡋࠊ࠸࠿࡟ḟୡ௦་⒪࡟ྥࡅࡓ་⒪㠉࿨࡟㈉⊩ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࡢ࠿ࠋࡑ
ࡢ⌧≧࡜ᮍ᮶ࢆᵝࠎ࡞どⅬ࠿ࡽᒎᮃࡍࡿᶵ఍࡟ࡋࡓ࠸࡜ᛮ࠸ࡲࡋࡓࠋཧຍ⪅ࡣࠊ኱ኚ⦕㉳ࡢⰋ࠸ 555 ྡࡨࡗࡓࡾ࡛ࠊ୍⯡࠿ࡽࡢཧຍ⪅ࡶ 2 ๭ࢆ༨ࡵࠊ┒఍࡜࡞ࡾࡲࡋࡓࠋ≉ูㅮ₇࡜ࡋ࡚ 2 㢟ࠊࣞࢪ࢙ࣥࢻࣞࢡࢳ࣮ࣕ2 㢟ࠊࢩ࣏ࣥࢪ࣒࢘㸯௳ࠊ⏨ዪඹྠཧ⏬௻⏬ 1 㢟ࠊዡບ㈹ཷ㈹ㅮ₇ 1 㢟ࠊඃ⚽ㅮ₇㈹➨ 2 ḟᑂᰝ 7 㢟ࠊ୍⯡₇㢟 77 㢟ࠊ࣏ࢫࢱ࣮₇㢟 168 㢟ࡢⓎ⾲ࡀ⾜ࢃࢀࡲࡋࡓࠋ 㸯᪥┠ࡢᮅࡣࠊ୍⯡₇㢟࡜୪⾜ࡋ࡚ࠊ௒ᅇึࡵ࡚௻⏬ࡉࢀࡓⱝᡭ◊✲⪅ࢆᑐ㇟࡜ࡋࡓඃ⚽ㅮ₇㈹ೃ⿵⪅㸵ྡࡢㅮ₇ࡀ࠾ࡇ࡞ࢃࢀࡲࡋࡓࠋ࠸ࡎࢀࡢⓎ⾲ෆᐜࡶࡍࡤࡽࡋࡃࠊⓎ⾲⪅ࡢᑗ᮶ࡀᴦࡋࡳ࡛ࡍࠋࡑࡢᚋࡢ⥲఍࡛ࡣࠊ῝℩఍㛗ཬࡧᲚཎ๪
఍㛗࠿ࡽࠊ᪥ᮏ⢾㉁Ꮫ఍ࡢάືࡀሗ࿌ࡉࢀࡓ࠶࡜ࠊ᮶ᖺࡢྡྂᒇ኱఍ࡢୡヰே௦⾲ࡢ໭ᓥ೺ඛ⏕࠿ࡽࠊグᛕࡍ࡭ࡁ⢾㉁Ꮫ఍㸲㸮࿘ᖺ࡟ᙜࡓࡿ➨㸱㸶ᅇᖺ఍ࢆࠕ୙
ᝨࠖ࡜࠸࠺࣮࣮࢟࣡ࢻ࡛⾜࠺࡜ࡢᢪ㈇ࢆ㏙࡭ࡽࢀࡲࡋࡓࠋ
⥆࠸࡚ࠊ➨㸰㸯ᅇ⢾㉁Ꮫ఍ዡບ㈹࠾ࡼࡧ➨㸰㸮ᅇ࣏ࢫࢱ࣮㈹ࡢᤵ㈹ᘧࡀ⾜ࢃࢀࡲࡋࡓࠋᖹᡂ㸱㸮ᖺᗘࡢཷ㈹ࡣࠊᮾ໭་⛉⸆⛉኱Ꮫ ་Ꮫ㒊ࡢୖᮧ⪽ᚿ༤ኈ㸦ཷ㈹ࢱ࢖ࢺ
ࣝ㸸ࢫࣇ࢕ࣥࢦ⢾⬡㉁ྜᡂ㓝⣲ࡢ⣽⬊ෆືែゎᯒ㸧࡛ࡍࠋୖᮧ༤ኈࡣࠊ௒࠿ࡽ㸰㸮ᖺ๓ࠊ⚾ࡀ໭ᾏ㐨኱Ꮫ⸆Ꮫ㒊ຓᩍᤵ᫬௦ࡢ኱Ꮫ㝔⏕࡛࠶ࡾࠊࡇࡢཷ㈹ࢆᚰࡼࡾ
Ꮀࡋࡃᛮ࠸ࡲࡍࠋ
⏨ዪඹྠཧ⏬௻⏬࡛ࡣࠊᮾ໭኱Ꮫࡢᰩཎ࿴ᯞඛ⏕ࡀࠕዪᛶ◊✲⪅ά㌍࡬ࡢᮇᚅ࡜Ṍࡳࠖ࡟ࡘ࠸࡚ㅮ₇ࡉࢀࡲࡋࡓࠋᮏ௻⏬ࡣࠊ⩌㤿኱Ꮫࡢᯇᑿ୍㑻ඛ⏕ࡀ࠾ୡヰࡉࢀࠊᰩཎඛ⏕ࡢዪᛶ◊✲⪅࡜ࡋ࡚ࡢ⏕ࡁ᪉ࢆඛ⏕ࡈ⮬㌟ࡢయ㦂࡜ே⏕ほࢆࡶ࡜࡟⋡┤࡟࠾ヰࡋࡉࢀ኱ኚዲホ࡛ࡋࡓࠋ  ≉ูㅮ₇࡛ࡣࠊᮾ໭኱Ꮫ ᮾ໭࣓ࢹ࢕࣭࣓࢝ࣝ࢞ࣂࣥࢡᶵᵓࡢᶵᵓ㛗࡛࠶ࡿᒣᮏ㞞அඛ⏕࠿ࡽࠊࠕᮾ໭࣓ࢹ࢕࣭࣓࢝ࣝ࢞ࣂࣥࢡィ⏬ࡢ┠ᶆ࡜㐍ᤖ≧ἣ㸫ୡ⏺᭱ඛ➃ࡢࣂ࢖࢜ࣂࣥࢡࡢᵓ⠏ࢆ┠ᣦࡋ࡚㸫ࠖ࡜㢟ࡋ࡚ࠊᅽᕳࡢࡈㅮ₇ࢆ࠸ࡓࡔࡁࡲ
ࡋࡓࠋ㸵ᖺ๓ࠊᮾ᪥ᮏ኱㟈⅏࠿ࡽࡢ་⒪᚟⯆ࢆ┠ᣦࡋ࡚㛤ጞࡉࢀࡓᮾ໭࣓ࢹ࢕࢝
࣭࣓ࣝ࢞ࣂࣥࢡᵓ᝿ࡀࠊ௒ࡸࠊୡ⏺ࢆ࣮ࣜࢻࡍࡿ㑇ఏᏊゎᯒ࣭ࢤࣀ࣒◊✲ࢆ⤌ࡳྜࢃࡏࡓࠕࢤࣀ࣒ࢥ࣮࣍ࢺ◊✲ࠖ࡟ᡂ㛗ࡋࠊḟୡ௦ࡢ་⒪ࢆ≌ᘬࡋ࡚ࡃᡂᯝࡀ⥆ࠎ࡜㐩ᡂࡉࢀ࠾ࡾࠊྠࡌ௝ྎ࡟ఫࡴ୍ே࡜ࡋ࡚኱ኚᎰࡋࡃᛮ࠸ࡲࡍࠋ᫝㠀ࠊ⢾㙐◊
4✲ࢆࡇࡢኊ኱࡞ࣉࣟࢪ࢙ࢡࢺຍ࠼࡚࠸ࡓࡔࡁࡓ࠸ࠊཧຍࡋࡓ࠸࡜ᛮࢃࡎࠊࣇࣟ࢔
࠿ࡽⓎゝࡉࡏ࡚㡬ࡁࡲࡋࡓࠋ
ḟࡢ≉ูㅮ₇ࡣࠊ㐲⸨⋢ኵඛ⏕࡛ࡍࠋࠕ⢾㙐⏕ྜᡂ␗ᖖ࡟ࡼࡿඛኳᛶ➽ࢪࢫࢺࣟࣇ࢕࣮ࠖ࡜㢟ࡋ࡚ࡢㅮ₇࠾㢪࠸ࡋࠊᏛኈ㝔㈹ཷ㈹࡟⮳ࡗࡓ◊✲ࡢ⤒⦋ࢆࡌࡗࡃ
ࡾ㸯᫬㛫࠾ヰࡋ࠸ࡓࡔ࠸ࡓࡢࡣࠊ⢾㉁Ꮫ఍࡛ࡣึࡵ࡚࡛ࠊඛ⏕ࡢ኱Ꮫ㝔⏕᫬௦࠿ࡽࡢ◊✲ே⏕ࢆᴦࡋࡃࡑࡋ࡚⯆࿡῝ࡃᣏ⫈࡛ࡁࡲࡋࡓࠋ௒ᚋࡶࠊࡲࡍࡲࡍࡢࡈά
㌍ࢆ㢪ࡗ࡚࠸ࡲࡍࠋ
㸰᪥┠ࡢᮅ㸶᫬༙࠿ࡽࡣࠊࣞࢪ࢙ࣥࢻࣞࢡࢳ࣮ࣕࢆ㕥ᮌ㑥ᙪඛ⏕࡟࠾㢪࠸ࡋࠊࠕᏊ౪ࡢ㑇ఏᛶ⚄⤒⑌ᝈ㸸 ᪥ᮏ࠿ࡽࡢⱝ࠸◊✲⪅࡜୍⥴࡟㎺ࡗࡓ㊰ࠖ࡜ࡋ࡚ࡈㅮ
₇㈷ࡾࡲࡋࡓࠋඛ⏕ࡣࠊࠕከࡃࡢᑠඣࡢ⚄⤒⣔ࡢ௦ㅰ⑌ᝈ࡟㛵ࡾࡲࡋࡓࡀࠊᩓₔ࡟࡞ࡿࡢࢆ㑊ࡅࡿࡓࡵ࡟ࠊࡇࡢ࠾ヰ࡛ࡣࠊࢸ࣮࣐ࢆ⤠ࡗ࡚ࠊ1970 ᖺ௨᮶ࠊࠕ᰾㓟᫬
௦ࠖࡶ㏻ࡋ࡚⚾ࡢ◊✲ᐊࡢ୺࡞ࢸ࣮࣐ࡢ୍ࡘ࡛࠶ࡗࡓྂ඾ⓗ࡞㑇ఏᛶ⬻ⓑ㉁ኚᛶ
⑕࡛࠶ࡿࢡࣛ࣋⑓ (globoid cell leukodystrophy, GLD) ࡢ⑓⌮ᶵᗎࡢゎ᫂ࢆ㏣ࡗ࡚᮶ࡓ㐨ࢆ㎺ࡗ࡚ࡳࡓ࠸࡜ᛮ࠸ࡲࡍࠋ
ࠖ࡜ࡉࢀࠊඛ⏕ࡢ⡿ᅜ࡛ࡢ㸲㸮ᖺ࡟Ώࡿ◊✲ே⏕ࡢ୍➃ࢆ࠾ヰࡋࡃࡔࡉ࠸ࡲࡋࡓࠋࡈㅮ₇ࡢ୰࡛ࠊ࣑ࢩ࢞ࣥ኱Ꮫࡢ Norman
Radin ᩍᤵ࡜ࡢ◊✲஺ὶࡢ࠾ヰࡋࡀ࠶ࡾࡲࡋࡓࠋࡑࢀࡣࠊ1970 ᖺ௦ึ㢌ࡢࡇ࡜ࠊ
ࠕRadin ᩍᤵࡣࠊࡲࡔᮍⓎ⾲ࡢ࢞ࣛࢡࢺࢩࣝࢭ࣑ࣛࢲ࣮ࢮࡢ࢔ࢵࢭ࢖ἲࢆᛌࡃᥦ
౪ࡋ࡚ࡃࢀࡓ࠾࠿ࡆ࡛ࠊࢡࣛ࣋⑓ࡀ࢞ࣛࢡࢺࢩࣝࢭ࣑ࣛࢲ࣮ࢮࡢḞᦆ࡛࠶ࡿࡇ࡜
ࢆ࠸ࡕ᪩ࡃド᫂ࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋᙼࡢຓࡅࡀ࡞࠿ࡗࡓࡽࠊࡶࡗ࡜ࡶࡗ࡜᫬㛫ࡀ࠿࠿ࡗ࡚࠸ࡓࠋࠖ࡜㏙࡭ࡽࢀࡲࡋࡓࠋ⚾ࡣ Radin ◊࡟ 1985-87 ᖺᅾ⡠ࡋࠊࢢࣝࢥ
ࢩࣝࢭ࣑ࣛࢻྜᡂ㓝⣲㜼ᐖ๣ࡢ㛤Ⓨ◊✲ࡀࡁࡗ࠿ࡅ࡛⢾㙐◊✲⪅࡜ࡋ࡚ࡢ㐨ࢆṌࡳጞࡵࡓࡇ࡜࠿ࡽࠊࡇࡢ㐓ヰ⪺ࡃࡇ࡜ࡀ࡛ࡁࠊᎰࡋࡃ㄂ࡾ࡟ᛮ࠸ࡲࡋࡓࠋ
㸰᪥┠༗๓୰ࡢḟࡢࣉࣟࢢ࣒ࣛࡣࠊྡྂᒇ኱ࡢ㛛ᯇ೺἞ඛ⏕࡜ᒸᓮ◊✲ᶵᵓࡢຍ⸨᫭୍ඛ⏕࡟࣮࢜࢞ࢼ࢖ࢨ࣮࡟࠾㢪࠸ࡋ࡚ࡢࣃࢿࣝࢹ࢕ࢫ࢝ࢵࢩࣙࣥᙧᘧࡢࢩ
࣏ࣥࢪ࣒࢘ࠕ⢾㙐㛵㐃኱ᆺ◊✲ࡢ⌧≧࡜ᑗ᮶ᒎᮃ࡛ࠖࡍࠋ
ࠕ࡝࠺ࡍࡿ㸟ࢽࢵ࣏ࣥ-20ᖺᚋࡢ⢾㙐⛉Ꮫࢆ⪃࠼ࡿ- ࢩ࣏ࣥࢪ࣒࡛࢘㆟ㄽࡋࡼ࠺㸟୺ᙺࡣᏛ఍ཧຍ⪅࡛ࡍࠋࠖ࡜㢟ࡋࡓ㛛ᯇඛ⏕ࡀసᡂࡉࢀࡓࣃࣥࣇࣞࢵࢺࢆᏛ఍ึ᪥࠿ࡽᏛ఍ཧຍ⪅඲ဨ࡟ཷ௜࡛㓄ᕸࡋࠊ5 ྡࡢࢩ࣏ࣥࢪࢫࢺࠊ3 ྡࡢᣦᐃウㄽ⪅࡜ࣇࣟ࢔࡜ࡢᵝࠎ࡞ゅᗘ࠿ࡽάⓎ࡞ウㄽࡀணᐃㄪ࿴ࡣ௻ᅗࡏࡎ࠾ࡇ࡞ࢃࢀࡲࡋࡓࠋ㆟ㄽࢆ㏻ࡋ࡚ࠊ⢾㙐Ꮫ⏺
࠾ࡼࡧྛேࡢ୰࡟ࠊ࠾ࡰࢁࡆ࡛ࡶክ࠶ࡿ࠸ࡣ┠ᣦࡍ࡭ࡁࡶࡢࢆᥥࡅ࡚࠸ࡓࡔࡅࡓ
࡞ࡽࠊࡇࡢࢩ࣏ࣥࢪ࣒࢘ࡢព⩏ࡣ࠶ࡗࡓ࡜ᛮ࠸ࡲࡍࠋ
༗ᚋࡣࠊ࢚ࢡࢫ࣮࢝ࢩࣙࣥ㸦ᯇᓥ᥈ゼࠊ⿕⅏ᆅゼၥࡸᐑᇛࢽࢵ࢝࢘࢖ࢫ࣮࢟ᕤሙぢᏛࠊ௝ྎ᪂

࡜Ỉ᪘㤋࡞࡝㸧ࠊࡑࡋ࡚㸯㸷᫬࠿ࡽࡣ᠓ぶ఍ࡀ⾜ࢃࢀࡲࡋࡓࠋỤ㝧ࢢࣛࣥࢻ࣍ࢸ࡛ࣝ⾜ࢃࢀࡓ᠓ぶ఍࡛ࡣࠊୡヰேࡢ⚾࠿ࡽࠊከᩘࡢཧຍ⪅ࡢⓙᵝ࡬ࡢឤㅰࡢព࡜ᒣᕝẸኵඛ⏕࠿ࡽࠕ⊛ᬬ࡜㊊ࡢලྜࡀࡼࢁࡋࡃ࡞࠸ࡢ࡛ࠊṧᛕ࡞
5ࡀࡽ௝ྎ࡟ࡣ⾜࠿࡞࠸ࠋ࡛ࡁࢀࡤࠊ᫖ᖺᇳ➹ࡋࡓᮾ኱་⛉◊๰❧㸯㸰㸳࿘ᖺ࣭ᨵ⤌㸳㸮࿘ᖺグᛕㄅ࡬ࡢᐤ✏ᩥ—൅ࡢ㟷᫓᫬௦-ఏᰁ⑓◊✲ᡤࡢ㡭—ࢆⓙᵝ࡟࠾㓄ࡾࡋ࡚࠸ࡓࡔࡁࡓ࠸ࠋࠖ࡜ඛ㐌㟁ヰࢆ࠸ࡓࡔ࠸ࡓࡇ࡜ࢆሗ࿌࠸ࡓࡋࡲࡋࡓࠋḟ࡟ࠊࣞࢪ࢙ࣥࢻࣞࢡࢳ࣮ࣕࢆ࠸ࡓࡔ࠸ࡓ㕥ᮌ㑥ᙪඛ⏕࡟࠾ゝⴥࢆ࠸ࡓࡔࡁࠊ⥆࠸࡚࣮࣐࢝ࣝ࢖࣮ࣖ࢔࣮࣡ࢻࡢཷ㈹ࡀỴᐃࡉࢀࡓ㇂ཱྀ┤அඛ⏕ࡢ࠿ࡽ࠾ゝⴥࢆ࠸ࡓࡔࡁ
ࡲࡋࡓࠋ஝ᮼࡢࡈⓎኌࡣࠊྡ㄃఍ဨࡢ㛗㇂⣧ᏹඛ⏕࡟࠸ࡓࡔࡁࡲࡋࡓࠋᐑᇛࡢᆅ
㓇ࠊ∵ࢱࣥࠊ᪪ࡢ㨶௓㢮࡞࡝ࡢ㒓ᅵࡢ࿡࡟ࡈ‶㊊࠸ࡓࡔࡅࡓ࡛ࡋࡻ࠺࠿㸽ࡇࡢグ஦ࢆ᭩࠸࡚࠸ࡿࡢࡣ㸯㸮᭶࡛ࡍࡀࠊ㸯㸯᭶࠿ࡽࡢ௝ྎࡣ∻⾃ࡢࢩ࣮ࢬࣥࡀጞࡲࡾ
ࡲࡍࠋࡲࡓࠊ
ࠕࡍࡎࡵ㋀ࡾࠖࢆ-௝ྎࡍࡎࡵ㋀ࡾఀ㐩ࡢ⯙-ࡉࢇࡢࣉࣟࡢᣦᑟࡶࡼࢁ
ࡋࡃࠊⓙᵝ࡜ᴦࡋࡃࡈ୍⥴࡟㋀ࡿࡇ࡜ࡀ࡛ࡁࡓࡢࡣ኱ኚⰋ࠸ᛮ࠸ฟ࡟࡞ࡾࡲࡋࡓࠋ
㸱᪥┠㸶᫬༙࠿ࡽࡣࠊ㛗㇂⣧ᏹඛ⏕ࡢࣞࢪ࢙ࣥࢻࣞࢡࢳ࣮ࣕࡣࠕ㑏ඖᮎ➃⢾ṧᇶࡢ⺯ගᶆ㆑࡟ࡼࡿゎᯒ࡛ࠖࠊ࡞ࡐ 2-aminoptridine㸦PA㸧ࢆ⺯ග๣࡜ࡋ࡚㑅ࡤࢀࡓ⤒⦋࡜Ⓨᒎࡢ㐣⛬ࢆࠊඛ⏕ࡀᙳ㡪ࢆཷࡅࡽࢀࡓㅖඛ㍮ࡢྡゝ࡜ඹ࡟ㄒࡗ࡚࠸
ࡓࡔࡁࡲࡋࡓࠋࣇࣟ࢔࠿ࡽࡣࠊ➟஭⊩୍ඛ⏕ࡀ⢾ࡢᶆ㆑ヨ⸆࡜ࡋ࡚ PA ࢆ㑅ࡤࢀ࡞࠿ࡗࡓࡽࣇࣟࣥࢱࣝ࢔ࣇ࢕ࢽࢸ࢕࣮ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ࡢᡂຌࡣ࡞࠿ࡗࡓ࡜Ⓨゝࡉࢀࡲࡋࡓࠋ
ᮏᖺ఍ࡣࠊከࡃࡢᏛ఍࠿ࡽࡢඹദࠊ༠㈶ࠊᚋ᥼ࢆ࠸ࡓࡔࡁࠊࡲࡓࠊࡈ᥼ຓࠊᒎ
♧ࠊᗈ࿌࡟࡚ࡈᨭ᥼࠸ࡓࡔࡁࡲࡋࡓ㈈ᅋࠊᅋయࠊ௻ᴗ࡟῝ࡃឤㅰ⏦ࡋୖࡆࡲࡍࠋ
ࡲࡓࠊࣛࣥࢳࣙࣥࢭ࣑ࢼ࣮ࢆඹദ࠸ࡓࡔࡁࡲࡋࡓࢧ࣮ࣔࣇ࢕ࢵࢩ࣮ࣕࢧ࢖࢚ࣥࢸ
࢕ࣇ࢕ࢵࢡ࡜ᓥὠ〇సᡤ࡟ᚰ࠿ࡽ࠾♩⏦ࡋୖࡆࡲࡍࠋࡑࡋ࡚ࠊࣉࣟࢢ࣒ࣛጤဨࡢ
ඛ⏕᪉ࠊ࢔ࢻࢫ࣮ࣜࠊJTBࠊᮾ໭་⛉⸆⛉኱ศᏊ⏕య⭷◊✲ᡤࡢࢫࢱࢵࣇࠊᏛ⏕࢔ࣝࣂ࢖ࢺࡢⓙࡉࢇࡢࡈ༠ຊ࡟࠾♩⏦ࡋୖࡆࡲࡍࠋ
ࡇࡢ࣏࣮ࣞࢺࢆᇳ➹୰࡟ࠊᒣᕝẸኵඛ⏕ࡀࡈ㏽ཤࡉࢀࡲࡋࡓࠋ㸶᭶࡟⢾㉁Ꮫ఍
࡬ࡢࡈฟᖍࡢ࠾㢪࠸࡛ࠊᒣᕝඛ⏕࡜࠾㟁ヰ࡛࠾ヰࡋ࠸ࡓࡋࡲࡋࡓࡇ࡜᫖᪥ࡢࡼ࠺
࡟ᛮ࠸ฟࡋ࡚࠾ࡾࡲࡍࠋඛ⏕ࡢ࠾Ꮉᵝ࠿ࡽࡣࠊࠕ௒࡜࡞ࡗ࡚ࡣ௝ྎ࡟⾜ࡅ࡞࠿ࡗࡓࡇ࡜ࡀṧᛕ࡛࡞ࡾࡲࡏࢇࠋᮏேࡢỴ᩿࡛ࡣ࠶ࡾࡲࡋࡓࡀࠊⓙࡉࡲ࡟࠾఍࠸࡛ࡁ
࡚࠸ࢀࡤࠊⰋ࠸ᛮ࠸ฟ࡟࡞ࡗࡓ࠿࡜ᛮ࠸ࡲࡍࠋࠖ࡜ࡢ࠾ゝⴥࢆ࠸ࡓࡔࡁࡲࡋࡓࠋ ࡇࡇ࡟ㅽࢇ࡛ࠊࡈෞ⚟ࢆ࠾♳ࡾ࠸ࡓࡋࡲࡍࠋ
ḟ㡯࡟ᒣᕝඛ⏕ࡢ୍␒ᘵᏊ࡛࠶ࡽࢀࡿᮏᏛᐈဨᩍᤵࡢ㕥ᮌ᫂㌟ඛ⏕࡟ࠕᒣᕝẸኵඛ⏕ࢆംࢇ࡛ࠖࢆ⥛ࡗ࡚㡬ࡁࡲࡋࡓࠋ
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ᮾ໭་⛉⸆⛉኱Ꮫᐈဨᩍᤵ 㕥ᮌ᫂㌟ඛ⏕ࡣ 2018 ᖺ 10 ᭶ 7 ᪥ࠊ96 ṓ࡛௚⏺ࡉࢀࡲࡋࡓࠋ┤㏆ࡲ࡛ࠊṌ⾜ࡀᑡࡋ㞴ࡋࡃ࡞ࡗ࡚࠸ࡓࡇ࡜ࢆ㝖ࡁࠊඛ⏕ࡢ≉ᚩࡢ୍ࡘ࡛࠶ࡗࡓᗘࢆ㉺ࡋ࡚ࡢ≀஦࡬ࡢዲወᚰࡶ⾶࠼ࡎࠊࡍࡇࡪࡿ࠾ඖẼ࡛ࡋࡓࠋ㣗ḧࡶ᪲┒࡛࠶ࡗࡓ࡜࠾⪺ࡁࡋ࡚࠾ࡾࡲ
ࡍࠋ⚾ࡀ࠾ぢ⯙࠸ࡋࡓ 9 ᭶ 25 ᪥ࠊ10 ᭶㸳᪥ࡣࠊព㆑ࡀ࡞࠸࡜ࡣ࠸࠼ࠊᐷጼࡣᬑ
ẁࡢඛ⏕ࡢ࠾ጼ࡟ኚࢃࡾࡣ࡞ࡃࠊࡲࡔࡲࡔࠊ኱୔ኵࡔ࡜ࠊᛮࡗ࡚࠸ࡓ▮ඛࡢࡇ࡜࡛ࡋࡓࠋඛ⏕ࡢ⏕ᾭࡢ୍᫬ᮇࢆඹ᭷ࡉࡏ࡚࠸ࡓࡔ࠸ࡓ⪅ࡢ⣲ᮔ࡞ឤ᝿࡜ࡋ࡚ࠊඛ
⏕ࡢ⏕ᾭࡣࡲࢀ࡟ࡳࡿᴟᙬⰍ࡟࠶ࡩࢀࡓࡶࡢ࡛࠶ࡗࡓࡇ࡜࡟␲࠸ࢆᣢࡕࡲࡏࢇࠋ
ඛ⏕ࡣ 1921 ᖺ 10 ᭶ 19 ᪥㸦኱ṇ 10 ᖺ㸧ࠊᮾ໭ᖇᅜ኱Ꮫ་Ꮫ㒊ෆ⛉Ꮫᒣᕝ❶ኴ㑻ᩍᤵࡢ஧⏨࡜ࡋ࡚ࠊ௝ྎᕷ࡛⏕ࡲࢀࠊ௝ྎ➨஧୰㸦⌧௝ྎ➨஧㧗ᰯ㸧ࠊᪧไ➨஧㧗➼Ꮫᰯࢆ༞ᴗࡉࢀࡿࡲ࡛ࠊ௝ྎ࡛㐣ࡈࡉࢀࡲࡋࡓࠋ௝ྎࡣඛ⏕ࡢᨾ㒓࡛ࡋࡓࠋ஭ࣀཱྀோ୍ඛ⏕ࡣୡヰே௦⾲ࢆࡉࢀࡓ➨ 37 ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸦2018 ᖺ 8 ᭶
28-30 ᪥㸧࡟ᒣᕝඛ⏕ࢆᣍᚅࡉࢀࡲࡋࡓࠋඛ⏕ࡣ኱ኚ஌ࡾẼ࡛ࠊᴦࡋࡳ࡟ࡉࢀ࡚
࠸ࡲࡋࡓࡀࠊ⊛ᬬ࡜యㄪࢆᠱᛕࡉࢀࠊ㛫㝿࡟࡞ࡗ࡚᩿ᛕࡉࢀࡲࡋࡓࠋ኱ኚᚰṧࡾ
ࡢࡈᵝᏊ࡛࠶ࡗࡓ࡜࠾⪺ࡁࡋ࡚࠾ࡾࡲࡍࠋ
ඛ⏕ࡣ 1944 ᖺ 9 ᭶(᫛࿴ 19 ᖺ)࡟ᮾிᖇᅜ኱Ꮫ་Ꮫ㒊ࢆ༞ᴗࡉࢀࡲࡍࡀࠊᏛ⏕ࡢ᫬࠿ࡽఏᰁ⑓◊✲ᡤ㸦⌧་⛉Ꮫ◊✲ᡤ㸧ࡢ᭷ᶵ໬Ꮫࡢ◊✲ᐊ࡛ࠊ໬Ꮫྜᡂࡢᐇ
㦂ࢆయ㦂ࡋࡲࡍࠋఏᰁ⑓◊✲ᡤࡀ㑅ᢥࡉࢀࡓࡢࡣࠊ㛗඗ࡢᒣᕝ㑥ኵᩍᤵ㸦ᮾிᖇᅜ኱Ꮫ་Ꮫ㒊༞ᴗᚋࠊ㡰ኳᇽ኱Ꮫෆ⛉Ꮫᩍᤵ㸧ࡢᚋᢲࡋࡀ࠶ࡗࡓ࡜ࡢࡇ࡜࡛ࡍࠋ
ࡑࢀ࡜࡜ࡶ࡟ࠊ᭷ᶵ໬Ꮫࢆ㑅ᢥࡋࡓࡢࡣࠊ་Ꮫࡢ◊✲ࢆ᭷ᶵ໬Ꮫࡢ▱㆑ࢆᇶ♏࡜
ࡋ࡚ᐇ㊶ࡍࡿỴពࡀ࠶ࡗࡓ࡜ࠊ⪺࠸࡚࠸ࡲࡍࠋࡇࡢᐇ⩦୰࡟ࠊ⇿Ⓨࡢ஦ᨾࢆ㉳ࡇࡋࡲࡍࠋࢺ࢚ࣝࣥࢆࢽࢺࣟ໬ࡋࠊ⏕ᡂࡋࡓ࢜ࣝࢺ࡜ࣃࣛࢽࢺࣟࢺ࢚ࣝࣥࢆῶᅽ⵨
␃࡛ศ㞳ࡋࠊఱᗘࡶ⧞ࡾ㏉ࡋ࡚ 100g ࡯࡝ࡢࣃࣛࢽࢺࣟࢺ࢚ࣝࣥࢆ㈓ࡵࡲࡋࡓࠋ
ࣃࣛࢽࢺࣟࢺ࢚ࣝࣥࡢ࣓ࢳࣝᇶࢆ㓟໬ࡋࠊࣃࣛࢽࢺࣟᏳᜥ㤶㓟࡟ࡍࡿࡓࡵ࡟ࠊ100㹥࡯࡝ࡢ↓Ỉࢡ࣒ࣟ㓟ࢆຍ࠼ࡿ࡜ࠊࣂ࣮ࣥ࡜኱⇿Ⓨ……ࠋ༶ධ㝔ࡉࢀࠊࡑࡢᚋࠊ1㐌㛫ࡄࡽ࠸ࡣព㆑ࡀ࡞ࡃࠊࡦ࡝࠸③ࡳ࡟࡞ࡸࡲࡉࢀࠊ⏕Ṛࡢࡣࡊࡲࢆࡉࡲࡼ࠸ࠊ
⏕㑏ࡉࢀࡲࡍࠋ⚾ࡢᏛ⏕᫬௦ࡣࠊᏛ఍࡛ᐟἩࡍࡿ᪑㤋࡛ࠊඛ⏕࡜୍⥴࡟࠾㢼࿅࡟ࡣ࠸ࡿᶵ఍ࡀ࠶ࡗࡓࡾࡋࡲࡋࡓࡀࠊඛ⏕ࡢ⬚㒊࡜⭡㒊ࡢ༙ศࡣࡦ࡝࠸ࢣࣟ࢖ࢻ࡛
࠾࠾ࢃࢀ࡚࠸ࡲࡋࡓࠋࡇࡢ᫬ࠊぶ㌟࡟ᚰ㓄ࡉࢀࡓࠊ⸆⛉ᑓ㛛Ꮫᰯฟ㌟࡛ᐇ㦂ຓᡭࢆࡉࢀ࡚࠸ࡓዪᛶࡀ࠾ࡽࢀࠊࡇࡢ᪉ࠊᜨὠᏊኵேࠊ࡜ඛ⏕ࡣ⏕ᾭࢆඹ࡟ࡉࢀࡲࡍࠋ
ࡇࡢ஦ᨾ࡟ࡶ࠿࠿ࢃࡽࡎࠊඛ⏕ࡣ༞ᴗᚋ࡟ఏᰁ⑓◊✲ᡤࡢ໬Ꮫ㒊࡟ධࡽࢀࠊᯞ
௜ࡁ⬡⫫㓟ࡢྜᡂࠊࡑࡢᐇ㦂ື≀యෆ࡛ࡢ௦ㅰࡢ◊✲ࢆ⾜࠸ࠊ་Ꮫ༤ኈ࡜࡞ࡽࢀ
7ࡲࡍࠋఏᰁ⑓◊✲ᡤࡣ໭㔛ᰘ୕㑻ࡀ⚾❧ఏᰁ⑓◊✲ᡤ࡜ࡋ࡚๰タࡋࠊࡑࡢᚋෆົ
┬ࡢᅜ❧ఏᰁ⑓◊✲ᡤ࡜࡞ࡾࠊ⣛వ᭤ᢡࢆ⤒࡚ࠊᮾிᖇᅜ኱Ꮫࡢᡤ⟶࡜࡞ࡗࡓࡇ
࡜ࡀ▱ࡽࢀ࡚࠸ࡲࡍࠋఏᰁ⑓◊✲ᡤ࡛ࡣࠊᡓᚋࡲࡶ࡞ࡃࡲ࡛ࠊ◚യ㢼ࠊࢪࣇࢸࣜ
࢔࡞࡝ឤᰁ⑕࡟ᑐࡍࡿᢠ⾑Ύࡀ〇㐀ࡉࢀ࡚࠸ࡲࡋࡓࠋ኱㔞ࡢ⾑Ύࢆᚓࡿࡓࡵ࡟ࠊ
࣐࢘࡟ᢠཎࢆᢞ୚ࡋࠊ᥇⾑ࡋ࡚ᚓࡽࢀࡓ⾑ᾮࢆจᅛࡉࡏࠊᅛࡲࡗࡓ⾑⌫ᡂศ࡜ࣇ࢕ࣈࣜࣥࢆ㝖ࡁࠊᢠ⾑Ύࢆ࠼ࡲࡍࠋจᅛ≀ࡣࠊ↓⏝ࡢ㛗≀࡜ࡋ࡚ࠊᤞ࡚ሙ࡟ᅔࡗ
࡚࠸ࡓࡔࢁ࠺ࡇ࡜ࡀ᝿ീ࡛ࡁࡲࡍࠋඛ⏕ࡣࡇࡢᗫᲠ≀࡟╔┠ࡋࡲࡍࠋᏛ⏕᫬௦࡟໬Ꮫࡢ◊✲ᐊ࡟ཧຍࡋࡓ⤒⦋ࡣୖグࡢ࡜࠾ࡾ࡛ࡍࡀࠊࡶ࠺୍ࡘẼ࡟࡞ࡗ࡚࠸ࡓ◊✲ศ㔝ࡣᕝ႐⏣ឡ㑻ᩍᤵࡢ࢘࢖ࣝࢫᏛࡔࡗࡓࡼ࠺࡛ࡍࠋ࢖ࣥࣇ࢚ࣝࣥࢨ࢘࢖ࣝࢫ
ࡣ㉥⾑⌫ࢆจ㞟ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡲࡍࠋࡉࡽ࡟ື≀㛫࡛㉥⾑⌫ࡢච␿཯ᛂ࡟
✀ᕪࡀ࠶ࡿࡇ࡜ࡶ▱ࡽࢀ࡚࠸ࡲࡋࡓࠋఏᰁ⑓◊✲ᡤࡢඛ⏕ࡢ࿘㎶࡛ࡣࠊឤᰁ⑕ࡸ
ච␿ࡀ㛵ᚰ஦࡛ࡋࡓࠋୖグࡢ஧ࡘࡢࡇ࡜ࡀඛ⏕ࡢ୰࡛ࠊ⼥ྜࡋࡓࡼ࠺࡛ࡍࠋᇵࡗ
ࡓ⬡⫫㓟ࡢྜᡂࠊศ㞳ࠊᵓ㐀ゎᯒࡢᡭἲࢆ౑ࡗ࡚ࠊᅔࡾࡶࡢࡢ࣐࢘ࡢจᅛሢ࠿ࡽ
࣐࢘㉥⾑⌫࡟≉␗࡞≀㉁ࢆᡭ࡟ධࢀࡿࡇ࡜ࡣฟ᮶࡞࠸࠿࡜⪃࠼ࡓࡼ࠺࡛ࡍࠋ⏕⌮㣗ሷỈ୰࡛จᅛሢࢆᡭ࡛ࡶࡳ࡯ࡄࡋࠊ㐟㞳ࡋ࡚ࡁࡓ㉥⾑⌫ࢆ㐲ᚰࡋ࡚㞟ࡵࠊࡑࡇ࡬ࠊ㓑㓟࡛㓟ᛶ࡟ࡋࡓỈࢆ኱㔞࡟ຍ࠼࡚ࠊపᙇฎ⌮ࡋࡲࡍࠋຍ࠼ࡓỈࡢ㔞ࡀᑡ࡞࠸࡜ࠊᨺฟࡉࢀࡓ࣊ࣔࢢࣟࣅࣥ࡟ࡼࡿẚ㔜ୖ᪼ࡢࡓࡵ࠿㉥⾑⌫⭷ሢࡢỿẊࡀ┠ど࡛ࡁࡲࡏࢇࠋ኱㔞ࡢỈ࡛࠶ࢀࡤࠊ┠ど࡛ୖΎࢆྲྀࡾ㝖ࡃࡇ࡜ࡀ࡛ࡁࡲࡍࠋỿẊࢆ㐲ᚰࡋࠊỿᰝࡢ stroma ࢆ㞟ࡵࠊ෾⤖஝⇱ࡋࡲࡍࠋࡇࡢᮦᩱ࠿ࡽ᭷ᶵ⁐፹࡛⬡㉁ࢆᢳฟࡋ࡚ࠊ࢔ࢭࢺࣥ୙⁐ࠊ࢚ࢸ࣮ࣝ୙⁐ࠊࣆࣜࢪࣥྍ⁐⏬ศࢆ㞟ࡵࠊᵝࠎ࡞ศᯒ࡟౪ࡋࡲࡋࡓࠋࡇࢀࡽࡢᐇ㦂⤖ᯝ࠿ࡽᚓࡽࢀࡓ⢾⬡㉁ࡣ hematoside ࡜࿨ྡࡉࢀ ࠊ ᵓ 㐀 ࡣ sphingosine(d18:1)[-lignoceric acid (24:0)]-galactose-galactoseprehemataminic acidࠊprehemataminic acid ࡣⅣ⣲ 9 ಶࡢ᪂ࡋ࠸⢾࡛࠶ࡿ࡜ J.
Biochem. 38, 199-212, 1951 ࡟ሗ࿌ࡉࢀࡲࡋࡓࠋprehemataminic acid ࡣᙜ᫬ᵓ㐀ࡀ୙࡛᫂࠶ࡗࡓ neuraminic acid ࠶ࡿ࠸ࡣ sialic acid ࡜ྠࡌᵓ㐀ࡢ༢⢾࡛࠶ࡿࡇ࡜ࡀࡑࡢᚋ᫂ࡽ࠿࡟ࡉࢀࡲࡍࠋ
Sialic acid ࡢṇࡋ࠸ᵓ㐀ࡣ 1955 ᖺ࡟ Gottschalk࡟ࡼࡗ࡚Ⅳ⣲ 9 ಶࡢ༢⢾࡜ࡋ࡚ሗ࿌ࡉࢀࡲࡍࠋ࣐࢘㉥⾑⌫ࡢ hematoside ࡢṇࡋ
࠸

ᵓ

㐀

ࡣ

ceramide(d18:1-24:0)-glucose-galactose-(4-O-acetyl)

N-glycolylneuraminic acid ࡛࠶ࡿࡇ࡜ࡀࡑࡢᚋሗ࿌ࡉࢀࡲࡍࡀࠊᵝࠎ࡞ᶵჾศᯒ
ࡢᡭἲࡀ౑࠼࡞࠿ࡗࡓ᫬௦࡟⏕య⭷ࡢࣔࢹࣝ࡜ࡶ࡞ࡿ㉥⾑⌫⭷ࡢᡂศ࡜ࡋ࡚⢾⬡㉁ hematoside ࡀᏑᅾࡍࡿࡇ࡜ࢆⓎぢࡋࡓࡇ࡜ࡣࠊṔྐ࡟ṧࡿ◊✲࡛࠶ࡿࡇ࡜ࢆ
♧ࡋ࡚࠸ࡲࡍࠋ
◊✲ࡣࣄࢺ㉥⾑⌫⭷⢾⬡㉁ࡢゎᯒ࡟ᒎ㛤ࡉࢀࠊgloboside ࡜࿨ྡࡉࢀࡓ⢾⬡㉁ࡀ
༢㞳ࡉࢀࡲࡍࠋGloboside ࡢ࿨ྡࡣඛ⏕࡟ࡼࡿࡶࡢ࡛ࡍࡀࠊࣄࢺ㉥⾑⌫ࡢ୺せ⢾⬡㉁ࡢᵓ㐀ࡣ Klenk ࡟ࡼࡿⓎ⾲ࡀඛ࡛࠶ࡾࡲࡋࡓࠋGlobo-series ࡢྡ๓ࡣ8

GalD1-4GalE1-4GlcE1-Cer ࡢ㦵᱁ᵓ㐀ࢆᣢࡘ⢾⬡㉁࡟௒࡛ࡶ౑ࢃࢀ࡚࠸ࡲࡍࠋ᪥
ᮏ⏕໬Ꮫ఍኱఍࡛ࡇࢀࡽࡢ⤖ᯝࢆⓎ⾲ࡋࡓ㝿࡟ࠊᮾ໭኱Ꮫ་Ꮫ㒊་໬Ꮫᩍᐊṇ᐀୍ᩍᤵࡀࠊgloboside ࡟ ABO ᘧ⾑ᾮᆺࡢᢠཎάᛶࡣ࡞࠸ࡢ࠿࡜㉁ၥࡉࢀࡓ࡜ࠊ⪺࠸࡚࠸ࡲࡍࠋṇ᐀୍ඛ⏕ࡣ⟽Ᏺ௝୍㑻ඛ⏕ࡢᜠᖌ࡛ࠊ⢾㙐⏕≀Ꮫࡢඛ㥑⪅ࡢ࠾୍ே࡛ࡍࠋࡇࡢ㉁ၥࡀዎᶵ࡟࡞ࡾࠊࣄࢺ㉥⾑⌫⢾⬡㉁ࡢ A ᆺࠊB ᆺᢠཎάᛶࡀㄪ࡭
ࡽࢀࡲࡋࡓࠋ⢾⬡㉁ࡣỈ⁐ᾮ୰࡛⃭ࡋࡃ᧠ᢾࡍࡿࡇ࡜࡟ࡼࡾ࣑ࢭࣝࢆᙧᡂࡋࠊᢠ
ཎ⢾⬡㉁ࡣ multivalency ࢆ⋓ᚓࡋ࡚ࠊᢠయ࡜ࡢ཯ᛂᛶࡣ㣕㌍ⓗ࡟ୖ᪼ࡍࡿࡇ࡜ࡀ
௒࡛ࡣ▱ࡽࢀ࡚࠸ࡲࡍࠋᢠయ࡜⢾⬡㉁Ỉ⁐ᾮࢆ཯ᛂࡉࡏࠊࡑࡢᚋ࡟㉥⾑⌫ࡢᠱ⃮ᾮࢆຍ࠼࡚ࠊ㉥⾑⌫จ㞟㜼Ṇάᛶࢆᐃࡋࡲࡍࠋࣄࢺ㉥⾑⌫⢾⬡㉁⏬ศࡣᙜ᫬ᑟ

ධࡉࢀጞࡵࡓࢩࣜ࢝ࢤࣝࡢ࣒࢝ࣛࢡ࣐ࣟࢺ࡛ࢡ࣒ࣟࣟ࣍ࣝ-࣓ࢱࣀ࣮ࣝΰᾮ࡛ศ
㞳⁐ฟࡉࢀࠊศ⏬ࡋࡓ⏬ศ࡛จ㞟㜼Ṇάᛶࡀㄪ࡭ࡽࢀࡲࡋࡓࠋࡑࡢ⤖ᯝࠊgloboside(GalNAcE1-3GalD1-4GalE1-4GlcE1-Cer)ࡼࡾࡶ⢾㙐ࡢ㛗࠸⢾⬡㉁ࡢ⏬ศ࡟㜼Ṇάᛶࡢ࠶ࡿࡇ࡜ࡀぢฟࡉࢀࠊ1953 ᖺࠊ1960 ᖺ࡟Ⓨ⾲ࡉࢀࡲࡍࠋࡑࡢᚋࠊෆ㈗ࠊ
Marcus ࡢ◊✲࡛ globoside ࡣ P ᘧ⾑ᾮᆺᢠཎ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀࡲࡍࠋ
ࡉࡽ࡟ࡀࢇ㛵㐃ᢠཎ࡜ࡋ࡚ࡢ⢾⬡㉁ࡢ◊✲ࡀ⟽Ᏺඛ⏕࡟ࡼࡾ⢭ຊⓗ࡟⾜ࢃࢀࠊከࡃࡢ⣲ᬕࡽࡋ࠸◊✲ᡂᯝࡀⓎ⾲ࡉࢀࡲࡋࡓࠋABO ᘧ⾑ᾮᆺ࡟㛵ࡋ࡚ࡣࠊศᏊ⏕≀Ꮫⓗ࡞ศᏊ࣓࢝ࢽࢬ࣒ࡀ 1990 ᖺ࡟⟽Ᏺ௝୍㑻ඛ⏕ࡢ◊✲࡛᏶ᡂࡉࢀࡲࡍࠋ
⢾⬡㉁ࡢᶵ⬟ࡣከࡃࡢ◊✲⪅࡟ࡼࡗ࡚◊✲ࡉࢀ࡚ࡁࡲࡋࡓࠋࡑࡢ୰࡛ࡶ⚄⤒⣽⬊ࡢ⚄⤒✺㉳ࢆቑຍࡉࡏࡿࠊఙ㛗ࡉࡏࡿ࡜࠸࠺ᡂᯝࡀὀ┠ࡉࢀࡲࡍࠋ1980 ᖺ௦࡟ࡣࠊ࢖ࢱࣜ࢔ࡢ〇⸆௻ᴗࡀㄪ〇ࡋࡓ࢘ࢩ⬻ࡢ࢞ࣥࢢࣜ࢜ࢩࢻࢆ➽⫗ὀᑕࡋ࡚ࠊከ
Ⓨᛶ◳໬⑕ࠊ⢾ᒀ⑓࡞࡝࡟ຠᯝࡀ࠶ࡿ࡜࠸࠺◊✲⤖ᯝࡀⓎ⾲ࡉࢀࡲࡋࡓࠋㄆ▱⑕ࡢᝈ⪅ࡢ⬻ᐊෆ࡟࢞ࣥࢢࣜ࢜ࢩࢻࢆὀධࡋࠊຠᯝࡀ࠶ࡗࡓ࡜࠸࠺ሗ࿌ࡶ࡞ࡉࢀࡲ
ࡋࡓࠋࡋ࠿ࡋࠊ➽⫗ὀᑕ࡟ࡼࡿᢞ୚࡟ࡼࡾࠊ࢘ࢩ⬻ࡢ࢞ࣥࢢࣜ࢜ࢩࢻ〇๣࡟ྵࡲࢀ࡚࠸ࡓᚤ㔞ࡢ N-glycolylneuraminic acid ࢆྵࡴ࢞ࣥࢢࣜ࢜ࢩࢻࡀᢠཎ࡜࡞ࡾࠊ
ᢠ࢞ࣥࢢࣜ࢜ࢩࢻᢠయࡀ⏘⏕ࡉࢀࠊࡑࢀ࡟ࡼࡿࢠࣛࣥࣂࣞ⑕ೃ⩌ࡢⓎ⑕ࡀㄆࡵࡽ
ࢀࠊ୍㐃ࡢ◊✲ࡣ୰Ṇࡉࢀࡲࡋࡓࠋ஭ࣀཱྀඛ⏕ࡢ◊✲ᐊ࡛ࡣᒣᕝඛ⏕ࡀⓎぢࡉࢀࡓ hematoside (GM3, N-acetylneuraminic acid-Gal-GlcCer)ࡢ⏕⌮Ꮫࠊ⑓⌮Ꮫⓗస⏝ࡀ◊✲ࡉࢀ࡚࠾ࡾࠊ኱ኚὀ┠ࡉࢀࡿᡂᯝࡀሗ࿌ࡉࢀ࡚ࡁ࡚࠸ࡲࡍࠋࡇࡇ௝ྎ
ࡢᆅ࡛ࠊhematoside ࡢᶵ⬟ࡀ᫂ࡽ࠿࡟ࡉࢀࠊ⑌ᝈࡢデ᩿ࡸ἞⒪࡟᭷ຠ࡛࠶ࡿࡇ࡜
ࡀ♧ࡉࢀࡿ᪥ࡀ᮶ࡿࡇ࡜ࢆ㢪ࡗ࡚ࡸࡳࡲࡏࢇࠋᒣᕝඛ⏕ࡶぢᏲࡗ࡚ࡃࢀ࡚࠸ࡿ࡜
ಙࡌࡲࡍࠋ


9

ᒣᕝẸኵඛ⏕ ࡈ␎Ṕ኱ṇ 10 ᖺ 10 ᭶ 19 ᪥ࠊ௝ྎ࡛⏕ㄌ (すᬺ 1921 ᖺ)᫛࿴ 19 ᖺ ᮾிᖇᅜ኱Ꮫ་Ꮫ㒊་Ꮫ⛉༞ᴗ
᫛࿴ 19 ᖺ 10 ᭶ ᮾிᖇᅜ኱Ꮫఏᰁ⑓◊✲ᡤ ධᡤ
᫛࿴ 34 ᖺ 10 ᭶ ᮾி኱Ꮫఏᰁ⑓◊✲ᡤ໬Ꮫ◊✲㒊ᩍᤵ
᫛࿴ 41 ᖺ 4 ᭶ ᮾி኱Ꮫ་Ꮫ㒊ᩍᤵ㸦⏕໬Ꮫㅮᗙᢸᙜ㸧  
᫛࿴ 48 ᖺ 9 ᭶ ᪥ᮏ⏕໬Ꮫ఍఍㛗㸦㹼᫛࿴ 49 ᖺ 10 ᭶㸧᫛࿴ 57 ᖺ 4 ᭶ ㈈ᅋἲே ᮾி㒔⮫ᗋ་Ꮫ⥲ྜ◊✲ᡤᡤ㛗㸦㹼ᖹᡂ 3 ᖺ 3 ᭶㸧᫛࿴ 62 ᖺ 12 ᭶ ᪥ᮏᏛኈ㝔఍ဨ
ᖹᡂ 3 ᖺ 4 ᭶ ᮾி⸆⛉኱ᏛᏛ㛗 (~ᖹᡂ 7 ᖺ 3 ᭶)ᖹᡂ 14 ᖺ 7 ᭶ ㈈ᅋἲே ᚤ⏕≀໬Ꮫ◊✲఍఍㛗 㸦~ᖹᡂ 22 ᖺ 3 ᭶㸧᫛࿴ 30 ᖺ 10 ᭶ ᪥ᮏ⏕໬Ꮫ఍ዡບ㈹ཷ㈹᫛࿴ 50 ᖺ 1 ᭶ ᮅ᪥㈹ཷ㈹᫛࿴ 57 ᖺ 4 ᭶ ᪥ᮏᏛኈ㝔㈹ཷ㈹ᖹᡂ 3 ᖺ 1 ᭶ ᮾி㒔ᩥ໬㈹ཷ㈹ᖹᡂ 3 ᖺ 11 ᭶ ໏஧➼⍞ᐆ❶ཷ❶2014 ᖺ 11 ᭶ ᩥ໬ຌປ⪅ཧ⪃ᩥ⊩㐲⸨⋢ኵࠊ▼⏣⚽἞ࠊ໭ᓥ ೺ (2015) JSCR Newsletter 19-2, 8-12ᒣᕝẸኵ (1981) ⢾⬡㉁≀ㄒ Ꮫ⾡ᩥᗜ 562 ㅮㄯ♫
T. Yamakawa and S. Suzuki (1951) The chemistry of the lipids of
posthemolytic residue or stroma of erythrocytes. I. Concerning theether-insoluble lipids of lyophilized horse blood stroma. J. Biochem. 38,
199-212
A. Suzuki (2009) Tamio Yamakawa: Down of glycobiology. J. Biochem. 146,
149-156


10

写真2015 年 8 月 2 日

シアル酸研究会・JCGG による文化功労者の顕彰をお祝いする会で談笑される先生。上野精養軒梅の間。鈴木邦彦先生撮影。

11ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤࠕ⟽Ᏺ௝୍㑻㈹ࠖ㸦Sen-itiroh Hakomori Glycoscience Award㸧つ⣙                           ᖹᡂ 30 ᖺ 9 ᭶ 22 ᪥ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤ஭ࣀཱྀ ோ୍
ྡ⛠⟽Ᏺ௝୍㑻㈹ᤵ୚ᶵ㛵ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤ┠ⓗ⏕≀⣔໬Ꮫ⣔ࢆၥࢃࡎᗈࡃ⢾㙐⛉Ꮫࢆᑓᨷࡋࠊ᪥ኪດຊࢆ⥆ࡅ࡚࠸ࡿᮾ໭࢚ࣜ࢔ࡢ◊✲⪅ࢆ㢧ᙲࡍࡿࡇ࡜࡟ࡼࡾࠊ᪥ᮏࡢ⢾㙐⛉Ꮫ◊✲ࡢቑ㐍ࢆᅗࡿࠋ㈹ࡢ✀ู

ዡບ㈹   1 ྡ㢧ᙲᴙ࠾ࡼࡧ๪㈹ඃ⚽ㄽᩥ㈹ 1ࠥ2 ྡ 㢧ᙲᴙ࠾ࡼࡧ๪㈹ᑐ㇟㸯㸧ዡບ㈹㸸ᮾ໭࢚ࣜ࢔㸦᪂₲┴࠾ࡼࡧ⩌㤿┴ࢆྵࡴ㸧࡛◊✲࡟ᚑ஦ࡍࡿᛂເ᫬ 45 ṓࡲ࡛ࡢ◊✲⪅㸰㸧ඃ⚽ㄽᩥ㈹㸸ྠᆅᇦ࡛◊✲࡟ᚑ஦ࡋ㸪⏦ㄳ᫬Ⅼ࡛኱Ꮫ㝔⏕ࡶࡋࡃࡣ༤ኈ◊✲ဨ࡛࠶ࡿ⪅㸨࠸ࡎࢀࡶᣦᐃࡉࢀࡓᮇ㛫ෆ࡟Ⓨ⾲㸦accepted ࡛ࡶྍ㸧ࡉࢀࡓᏛ⾡ㄽᩥ࡟ᑐࡋ࡚ᑂᰝ࣭ᤵ୚ࡍࡿࠋ

㑅⪃᪉ἲ⮬⸀࠾ࡼࡧ௚⸀࡟ࡼࡿࡶࡢ࡜ࡋࠊᮾ໭⢾㙐◊✲఍ୡヰேࡀᑂᰝࡍࡿࠋホ౯ࢆⅬᩘ໬㸦ḟ㡯ཧ↷㸧ࡋ࡚Ỵࡍࡿࠋ

ᤵ㈹᪉ἲ

ᮾ໭⢾㙐◊✲఍㛤ദ᫬࡟ᤵ㈹࠾ࡼࡧཷ㈹ㅮ₇ࢆ⾜࠺ࠋ㐠⏝᪉ἲ㢧ᙲᴙ࠾ࡼࡧ๪㈹ࡢ㉎ධ㈝ࡣࠊ⟽Ᏺ㈹ᇶ㔠ࢆཎ㈨࡜ࡋࠊศᏊ⏕య⭷◊✲ᡤࡀࡇࢀࢆᨭᘚࡍࡿࠋ

ࡑࡢ௚
 

஦ົᒁࡣศᏊ⏕య⭷◊✲ᡤෆ࡟⨨ࡃࠋ12ᑂᰝ㸦㸯㸧ᛂເ㈨᱁࠾ࡼࡧᛂເ᪉ἲ  ᮾ໭ 6 ┴ࠊ᪂₲┴࠾ࡼࡧ⩌㤿┴㸦ᮾ໭⢾㙐◊✲఍࢚ࣜ࢔㸧࡛◊✲ࢆ⾜ࡗ࡚࠸ࡿ 45 ṓ௨ୗࡢ⢾㙐⛉Ꮫ◊✲⪅㸦኱Ꮫ㝔⏕ࠊ༤ኈ◊✲ဨࡣ⮬ືⓗ࡟ඃ⚽ㄽᩥ㈹࡬ࡢࣀ࣑ࢿ࣮ࢺ࡜࡞ࡿ㸧ࠋᑐ㇟ㄽᩥࡣࠊࡑࡢᖺᗘෆ㸦ࡓࡔࡋᛂເ⥾ษ௨๓㸧࡟ Impact factor ࡀ௜୚ࡉࢀ࡚࠸ࡿⱥᩥᏛ⾡㞧ㄅ㸦ᰝㄞ᭷㸧࡟ฟ∧ࠊࡲ
ࡓࡣᥖ㍕ࡀỴᐃࡉࢀ࡚࠸ࡿཎⴭㄽᩥ㸦accepted ࡛ࡶྍ㸧࡜ࡋࠊᛂເ⪅ࡀ➹㢌ⴭ⪅࡜࡞ࡗ࡚࠸ࡿࡶࡢ࡜ࡍࡿࠋせ᪨࿴ヂ࡜ㄽᩥ PDF ࢆῧ࠼࡚᪤ᐃࡢ࢚ࣥ
ࢺ࣮ࣜࣇ࢛࣮࣒࡟グධࡋ࡚࣓࣮ࣝ࡟࡚ᛂເࡍࡿࠋ໬Ꮫ⣔࣭⏕≀⣔ࡣၥࢃ࡞࠸ࠋ
⮬⸀࠾ࡼࡧ௚⸀࡝ࡕࡽ࡛ࡶཷࡅ௜ࡅࡿࠋᛂເᮇ㛫➼࡟ࡘ࠸࡚ࡣࡑࡢ㒔ᗘỴᐃࡋࠊ࿘▱ࡍࡿࠋ
᭩㢮㏦௜ඛ㸸ᮾ໭་⛉⸆⛉኱Ꮫ ศᏊ⏕య⭷◊✲ᡤ ᡤ㛗 ஭ࣀཱྀ ோ୍࣓࣮ࣝ࢔ࢻࣞࢫ㸸jin@tohoku-mpu.ac.jp㸦㸰㸧࢚ࣥࢺ࣮ࣜࣇ࢛࣮࣒㸦ูῧ㸧㸦㸱㸧ᑂᰝጤဨ࠾ࡼࡧᑂᰝ᪉ἲ
㸯㸧ศᏊ⏕య⭷◊✲ᡤᡤ㛗ࡀᑂᰝဨ㛗ࢆົࡵࡿࠋ
㸰㸧ᮾ໭⢾㙐◊✲఍ୡヰேࡀᑂᰝဨ࡜࡞ࡿࠋࡓࡔࡋࠊୡヰேࡀ᥎⸀⪅ࡲࡓࡣ⮬⸀⪅ࡢᡤᒓ㈐௵⪅࠶ࡿ࠸ࡣㄽᩥࡢඹⴭ⪅࡛࠶ࡿሙྜࡣᑂᰝ࡟ཧຍࡋ
࡞࠸ࠋ㸱㸧ᮇ㝈ෆ࡟ᥦฟࡉࢀࡓㄽᩥ࡟ࡘ࠸࡚஦ົᒁࡀ୍ḟᑂᰝ㸦ୗグᇶ♏Ⅼ࡟ࡼࡿ㸧ࢆ⾜࠸ࠊዡບ㈹࠾ࡼࡧඃ⚽ㄽᩥ㈹ࡑࢀࡒࢀୖ఩㸱ሗࢆ㑅ᐃࡍࡿࠋ㸲㸧ୖ఩㸱ሗ࡟ࡘ࠸࡚ᑂᰝဨࡀୗグホ౯㡯┠ࢆ᥇Ⅼࡋࠊ඲ဨ㸦ࡑࡢ㒔ᗘேᩘࡣ␗࡞ࡿ㸧ࡢホ౯ᖹᆒⅬ㸩ᇶ♏Ⅼ㸦100 Ⅼ‶Ⅼࠊ1 Ⅼᮍ‶ࡣᅄᤞ஬ධ㸧࠿ࡽ᭱ୖ఩⪅ࢆཷ㈹⪅࡜ࡋ࡚Ỵᐃࡍࡿࠋ㸳㸧᭱㧗Ⅼࡀ」ᩘฟࡓሙྜࡣᑂᰝဨ㛗ࡢุ᩿࡟ጤࡡࡿࠋ ᇶ♏Ⅼ
 Impact factor




ホ౯᭱㧗Ⅼ
⊂๰ᛶ   
10 Ⅼ࢖ࣥࣃࢡࢺ㸦㔜せᛶ㸧  10 Ⅼㄽᩥᵓᡂ  
10 Ⅼᑗ᮶ᛶ   
10 Ⅼ

2.0 ᮍ‶ 10 Ⅼ
4.0 ᮍ‶ 20 Ⅼ
6.0 ᮍ‶ 30 Ⅼ
8.0 ᮍ‶ 40 Ⅼ
10 ᮍ‶ 50 Ⅼ
10 ௨ୖ 60 Ⅼ

13

平成 30 年度 箱守仙一郎賞（Sen-itiroh Hakomori Glycoscience Award 2018）
東北糖鎖研究会（http://tohokut-tousa.strikingly.com）の世話人による厳正な審査の結果、第二回箱守仙一郎賞の研究奨励賞および優秀論文賞の受賞者が決定し、弘前で行われた第１２回東北糖鎖研究会で授賞式および奨励賞受賞講演が行われました。
奨励賞
稲森 啓一郎東北医科薬科大学分子生体膜研究所 機能病態分子学 准教授
ガングリオシド生合成不全は肥満モデル KKAy マウスのレプチンおよびメ
ラノコルチンシグナルを改善する
Deficient ganglioside synthesis restores responsiveness to leptin and melanocortin
signaling in obese KKAy mice
優秀論文賞
目黒 康洋
東北大学大学院農学研究科 生物有機化学分野 博士後期課程二年
Glycosyl Bunte Salts: A Class of Intermediates for Sugar Chemistry
Organic Letters, 20, 76-79 (2018)村上 友太
福島県立医科大学医学部脳神経外科学講座 博士研究員
Spontaneous intracranial hypotension is diagnosed by a combination of
lipocalin-type prostaglandin D synthase and brain-type transferrin in cerebrospinal
fluid
Biochim Biophys Acta Gen Subj, 1862, 1835-1842 (2018)

左より、井ノ口所長，村上友太博士、稲森啓一郎博士、目黒康洋さん(D2)

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ዡບ㈹
࢞ࣥࢢࣜ࢜ࢩࢻ⏕ྜᡂ୙඲ࡣ⫧‶ࣔࢹࣝ KKAy ࣐࢘ࢫࡢࣞࣉࢳࣥ࠾ࡼࡧ࣓ࣛࣀࢥࣝࢳࣥࢩࢢࢼࣝࢆᨵၿࡍࡿ
Deficient ganglioside synthesis restores responsiveness to leptin andmelanocortin signaling in obese KKAy mice✄᳃ ၨ୍㑻㸦ᮾ໭་⛉⸆⛉኱Ꮫ ศᏊ⏕య⭷◊✲ᡤ ᶵ⬟⑓ែศᏊᏛ࣭෸ᩍᤵ㸧
GM3 ࡣ a ࠾ࡼࡧ b ⣔ิ࢞ࣥࢢࣜ࢜ࢩࢻࡢ๓㥑య࡛ࠊ⫧‶ࣔࢹࣝື≀ࡢ⬡⫫⣽⬊ࡸࠊ2 ᆺ⢾ᒀ⑓ࡲࡓࡣ⬡㉁␗ᖖ⑕࡛࠶ࡿ⫧‶ᝈ⪅ࡢ⾑Ύ୰࡛ቑຍࡀࡳࡽࢀࡿࠋ
GM3 ྜᡂ㓝⣲(GM3S)ࢆࣀࢵࢡ࢔࢘ࢺ(KO)ࡋࡓ C57BL/6 ࣐࢘ࢫ࡛ࡣࠊ࢖ࣥࢫࣜ
ࣥឤཷᛶࡢஹ㐍࡜ࠊ㧗⬡⫫㣗ㄏᑟᛶࡢ࢖ࣥࢫࣜࣥ᢬ᢠᛶࡢⓎ⑕ࡀ㍍ῶࡉࢀࡿࠋࡋ
࠿ࡋࠊᦤ㣗࡜௦ㅰࡢ୰ᯡᛶไᚚ࡟࠾ࡅࡿ GM3 ࡜㛵㐃࢞ࣥࢢࣜ࢜ࢩࢻࡢ⑓ែ⏕⌮ⓗᙺ๭࡟ࡘ࠸࡚ࡣ୙࡛᫂࠶ࡿࠋᡃࠎࡣࠊ㯤Ⰽ⫧‶࣐࢘ࢫ KKAy ࡢ GM3S 㑇ఏᏊࢆ KO ࡋࡓࣔࢹ࣐ࣝ࢘ࢫ(KKAy GM3S KO)࡟࠾࠸࡚ࠊ⫧‶⑓ែࡀⴭࡋࡃᨵၿࡉࢀࡿࡇ࡜ࢆぢฟࡋࡓࠋKKAy ࣐࢘ࢫࡀ㐣㣗࡛㔜ᗘࡢ⫧‶ࢆⓎ⑕ࡍࡿࡢ࡟ᑐࡋࠊKKAy
GM3S KO ࣐࢘ࢫࡣⴭ᫂࡞య㔜࡜ᦤ㣵㔞ࡢῶᑡࠊ࠾ࡼࡧࠊ⪏⢾⬟࡜࢖ࣥࢫࣜࣥឤ
ཷᛶࡢⴭࡋ࠸ᨵၿࢆ♧ࡋࡓࠋKKAy ࣐࢘ࢫ࡛ࡣࠊࣞࣉࢳࣥࡢ⭡⭍ෆᢞ୚࡟ᑐࡍࡿどᗋୗ㒊ࢽ࣮ࣗࣟࣥࡢᛂ⟅ᛶࡀ኱ᖜ࡟ῶᙅࡋ࡚࠸ࡓࡀࠊKKAy GM3S KO ࣐࢘ࢫ࡛ࡣࣞࣉࢳࣥ࡟ᑐࡍࡿᛂ⟅ᛶࡀ༑ศಖࡓࢀ࡚࠸ࡓࠋ࣐࢘ࢫどᗋୗ㒊⏤᮶⚄⤒⣽⬊ᰴࢆ⏝࠸ࡓᐇ㦂࡛ࡣࠊGM3S Ḟᦆ⣽⬊࡟࠾࠸࡚ࣞࣉࢳࣥ౫Ꮡࡢ ERK ࣜࣥ㓟໬ࡀஹ㐍ࡋ࡚࠸ࡓࠋࡉࡽ࡟ࠊKKAy GM3S KO ࣐࢘ࢫࡣẟⰍࡢኚ໬ࢆ࿊ࡋ࡚࠾ࡾࠊ
GM3S ࡣ࣓ࣛࣀࢥࣝࢳࣥࢩࢢࢼࣝ࡟ࡶ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋᡃࠎࡢⓎぢ࡟ࡼࡾࠊGM3 㛵㐃࢞ࣥࢢࣜ࢜ࢩࢻࡀࣞࣉࢳࣥ࠾ࡼࡧ࣓ࣛࣀࢥࣝࢳࣥࢩࢢࢼࣝ࡟࠾࠸࡚㔜せ࡞ᙺ๭ࢆࡶࡘࡇ࡜ࡀ♧ࡉࢀࡓ(1)ࠋ
  ,QDPRUL.,WR+7DPXUD<1LWWD7<DQJ;1LKHL:6KLVKLGR),PD]X67VXNLWD6<DPDGD7.DWDJLUL + ,QRNXFKL - 'HILFLHQW JDQJOLRVLGH V\QWKHVLV UHVWRUHV UHVSRQVLYHQHVV WR OHSWLQ DQGPHODQRFRUWLQVLJQDOLQJLQREHVH..$\PLFH J Lipid Res  ࠙⤒Ṕࠚᖹᡂ 7 ᖺ㸸஑ᕞ኱Ꮫ⌮Ꮫ㒊༞ᴗ㸪ᖹᡂ 9 ᖺ㸸஑ᕞ኱Ꮫ኱Ꮫ㝔⌮Ꮫ◊✲⛉ಟኈㄢ⛬ಟ஢㸪ྠᖺ ᪥ᮏᏛ⾡᣺⯆఍ ≉ู◊✲ဨ㸪ᖹᡂ 13 ᖺ㸸஑ᕞ኱Ꮫ኱Ꮫ㝔་Ꮫ⣔◊✲⛉༤ኈㄢ⛬ಟ஢㸦༤ኈ࣭⌮Ꮫ㸧㸪ྠᖺ ኱㜰኱Ꮫ኱Ꮫ㝔་Ꮫ⣔◊✲⛉ ༤ኈ◊✲ဨ㸪ᖹᡂ 16 ᖺ㸸ྠ ≉௵ຓᡭ㸦O-࣐ࣥࣀ࣮ࢫᆺ⢾㙐ࡢ⏕ྜᡂ࡟㛵ࢃࡿ⢾㌿⛣㓝⣲ࡢ◊✲㸧㸪ᖹᡂ 18 ᖺ㸸⡿ᅜ࢔࢖࢜࣡኱Ꮫ་Ꮫ㒊࣭ࣁ࣮࣡ࢻࣄ࣮ࣗ
ࢬ་Ꮫ◊✲ᡤ Postdoctoral fellow㸪ᖹᡂ 24 ᖺ㸸ྠ Associate㸦ࢪࢫࢺࣟࢢࣜ࢝ࣥࡢ࣑ࣛࢽࣥ⤖ྜᛶ⢾㙐ࡢ⏕ྜᡂ࡟㛵ࡍࡿ◊✲㸧㸪ᖹᡂ 25 ᖺ㸸ᮾ໭⸆⛉኱Ꮫ ศᏊ⏕య⭷◊✲ᡤ ᶵ⬟⑓ែศᏊᏛᩍᐊ ෸ᩍᤵ㸦ᖹᡂ 28 ᖺ ኱Ꮫྡࡀᮾ໭་⛉⸆⛉኱Ꮫ࡟ᨵ⛠㸧ࠋ⌧ᅾࠊ⢾⬡㉁࢞ࣥࢢࣜ࢜ࢩࢻࡢ⏕⌮ᶵ⬟࡟㛵ࡍࡿ◊✲࡟ᚑ஦ࠋ

15

࠙ཷ㈹ࡢឤ᝿࡜ᢪ㈇ࠚ
 ࡇࡢᗘࠊᖹᡂ㸱㸮ᖺᗘ⟽Ᏺ௝୍㑻㈹ዡບ㈹ࢆ㈷ࡾࡲࡋࡓࡇ࡜ࠊ኱ኚගᰤ࡟Ꮡࡌ
ࡲࡍࠋ⟽Ᏺඛ⏕ࡣࡌࡵᮾ໭⢾㙐◊✲఍ࡢ⟽Ᏺ㈹ᑂᰝጤဨࡢඛ⏕᪉࡟῝ࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ⢾㙐◊✲࡟ᦠࢃࡗ࡚௨᮶ࠊ⟽Ᏺඛ⏕ࡢ೧኱࡞ࡿࡈᴗ⦼࡜⢾㙐⏕≀Ꮫ◊✲࡟࠾ࡅࡿከ኱࡞ࡈຌ⦼ࢆᣏぢࡋ࡚࠾ࡾࡲࡋࡓࡀࠊࡑࡢ⟽Ᏺඛ⏕ࡢ࠾ྡ๓ࢆෙࡋ
ࡓ㈹ࢆ࠸ࡓࡔࡅࡲࡋࡓࡇ࡜ࠊ῝ࡃឤㅰ⏦ࡋୖࡆࡲࡍ࡜࡜ࡶ࡟ࠊ኱ኚ㌟ࡢᘬࡁ⥾ࡲࡿᛮ࠸ࡀ࠸ࡓࡋࡲࡍࠋ ⢾㙐◊✲࡟ධࡗ࡚࠸ࡃࡁࡗ࠿ࡅࡣࠊ኱Ꮫ㝔᫬௦࡟஑ᕞ኱Ꮫ࣭ᒾỌ㈆᫛ඛ⏕㸪ᕝ⏿ಇ୍㑻ඛ⏕ࡢࡈᣦᑟࡢࡶ࡜⾜ࡗࡓ࢝ࣈࢺ࢞ࢽࡢ⏕య㜵ᚚࣞࢡࢳࣥࡢᶵ⬟ゎᯒ࡛ࡋࡓࠋࡑࡢᚋࠊ኱㜰኱Ꮫ࣭㇂ཱྀ┤அඛ⏕ࡢࡶ࡜࡛⢾㌿⛣㓝⣲࡟㛵ࡍࡿ◊✲࡟ᦠࢃࡾࡲࡋࡓࠋࡑࡇ࡛ぢࡘࡅࡓ᪂つࡢ㓝⣲ࡀ O-࣐ࣥࣀ࣮ࢫᆺ⢾㙐ࡢ⏕ྜᡂ࡟㛵୚ࡋ࡚࠸ࡓࡇ࡜࠿ࡽࠊᙜ᫬ࠊࢪࢫࢺࣟࢢࣜ࢝ࣥࡢ⢾㙐␗ᖖ࡜➽ࢪࢫࢺࣟࣇ࢕࣮ࡢ㛵ಀ࡟ࡘ࠸࡚◊✲ࢆ᥎ࡋ㐍ࡵ࡚࠸ࡓ࢔࢖࢜࣡኱Ꮫ࣭Prof. Kevin Campbell ࡢ◊✲ᐊ࡟㣕ࡧ㎸ࡳࡲࡋࡓࠋࡑࡇ࡛ࡣࢪࢫࢺࣟࢢࣜ࢝ࣥࡢ O-࣐ࣥࣀ࣮ࢫᆺ⢾㙐ୖ࡟┤᥋ࣜ࢞ࣥࢻ⤖ྜᛶከ⢾ࢆ௜ຍࡍࡿ⢾㌿⛣㓝⣲ࡢάᛶྠᐃࢆ⾜࠸ࡲࡋࡓࠋ⌧ᅾࡣࠊᮾ໭་⛉⸆⛉኱Ꮫ࣭஭ࣀཱྀோ୍ᩍᤵࡢࡶ࡜ࠊ⢾⬡㉁࢞ࣥࢢࣜ࢜ࢩࢻࡢ⏕⌮ᶵ⬟࡟ࡘ࠸࡚ࠊ
≉࡟ࢩ࢔ࣝ㓟㌿⛣㓝⣲࡛࠶ࡿ GM3 ྜᡂ㓝⣲ࡢḞᦆ࣐࢘ࢫࢆ⏝࠸࡚⫧‶⑓ែ࡞࡝ࡢ⑌ᝈ࡟࠾ࡅࡿ⑓ែ⏕⌮ⓗᙺ๭ࡢゎ᫂ࢆ┠ᣦࡋ࡚࠸ࡲࡍࠋ⢾ࢱࣥࣃࢡ㉁⢾㙐࠿ࡽ⢾⬡㉁࡬࡜◊✲ෆᐜࡀ኱ࡁࡃࢩࣇࢺࡋࠊᅄ༑ࢆ㐣ࡂ࡚࠿ࡽࡢ᪂ࡓ࡞ᣮᡓ࡟ᮍࡔ࡟
ⱞ㜚ࡋ࡚࠾ࡾࡲࡍࡀࠊ⢾㙐ࡢ⏕⌮ᶵ⬟ࡢゎ᫂ࢆ┠ᣦࡍ࡜࠸ࡗࡓⅬ࡛ࡣ୍㈏ᛶࢆࡶ
ࡗ࡚◊✲࡟ྲྀࡾ⤌ࢇ࡛࠾ࡾࡲࡍࠋ௒ᅇࡢཷ㈹ᑐ㇟࡜࡞ࡗࡓㄽᩥ࠿ࡽࡣࠊ࢞ࣥࢢࣜ
࢜ࢩࢻࡢ୰ᯡᛶࡢᦤ㣗࣭௦ㅰㄪ⠇࡟࠾ࡅࡿᙺ๭࡜࡜ࡶ࡟ࠊண᝿እ࡟ࡶẟࡸ⓶⭵ࡢⰍ⣲⏘⏕ㄪ⠇࡟࠾ࡅࡿ㛵୚ࡀ♧၀ࡉࢀࡲࡋࡓࠋࡋ࠿ࡋ࡞ࡀࡽࠊ࣓࢝ࢽࢬ࣒ࡢ㠃࡛ࡣ୙᫂࡞㒊ศࡀከࡃࡲࡔࡲࡔࢃ࠿ࡽ࡞࠸ࡇ࡜ࡤ࠿ࡾ࡛ࠊ௒ᚋࡦ࡜ࡘࡦ࡜ࡘࢆ᫂ࡽ
࠿࡟ࡋ࡚࠸ࡁࡓ࠸࡜⪃࠼࡚࠾ࡾࡲࡍࠋ
᭱ᚋ࡟ࠊࡇࡢ◊✲ࢆ⾜࠺࡟࠶ࡓࡾࠊ஭ࣀཱྀᩍᤵࢆࡣࡌࡵࠊࡈᣦᑟ࣭ࡈ༠ຊ࠸ࡓ
ࡔࡁࡲࡋࡓඹⴭ⪅ࡢඛ⏕᪉࣭኱Ꮫ㝔⏕ࠊࡈᨭ᥼ࡃࡔࡉࡗࡓ◊✲ᐊࡢ࣓ࣥࣂ࣮࡟ᚰ
ࡼࡾឤㅰ⏦ࡋୖࡆࡲࡍࠋ௒ᅇࡢཷ㈹ࢆບࡳ࡟ࠊ⢾㙐⛉Ꮫࡢࡉࡽ࡞ࡿⓎᒎ࡟ᑡࡋ࡛
ࡶ㈉⊩ࡋ࡚࠸ࡅࡲࡍࡼ࠺ᘬࡁ⥆ࡁ⢭㐍ࡋ࡚ཧࡾࡓ࠸ᡤᏑ࡛ࡍࠋ௒ᚋ࡜ࡶࡈᣦᑟࡈ
㠴᧡ࡢ࡯࡝ࡼࢁࡋࡃ࠾㢪࠸⏦ࡋୖࡆࡲࡍࠋ✄᳃ ၨ୍㑻16ඃ⚽ㄽᩥ㈹
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Organic Letters, 20, 76-79 (2018)┠㯮 ᗣὒ㸦ᮾ໭኱Ꮫ኱Ꮫ㝔㎰Ꮫ◊✲⛉࣭༤ኈᚋᮇㄢ⛬஧ᖺ㸧ᡃࠎࡣ⢾㉁ྜᡂ࡟࠾ࡅࡿ୰㛫య࡜ࡋ࡚ࠊࡇࢀࡲ࡛࡟๓౛ࡢ↓࠸ࢳ࢜◲㓟⢾ࢆ๰〇ࡋࡓࠋࡇࡢ᪂つ⢾ㄏᑟయ࡛࠶ࡿࢳ࢜◲㓟⢾ࢆࠊ࢔ࣝ࢟ࣝࣈࣥࢸሷࡢⓎぢ⪅࡛࠶ࡿ
19 ୡ⣖ࡢࢻ࢖ࢶே໬Ꮫ⪅ Hans Bunte ࡟ᅉࢇ࡛ࢢࣜࢥࢩࣝࣈࣥࢸሷ࡜ྡ௜ࡅࡓࠋ
ࡍ࡞ࢃࡕࠊ࣒࣍ࣝ࢔࣑ࢪࣥᆺ⬺Ỉ⦰ྜ๣ࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡾࠊỈ⁐ᾮ୰࡟࡚↓ಖㆤ⢾࡜Ᏻ౯࡞ࢳ࢜◲㓟ࢼࢺ࣒ࣜ࢘࠿ࡽࠊ୍ẁ㝵࡛᪂つ⢾໬ྜ≀࡛࠶ࡿࢢࣜࢥࢩࣝ
ࣈࣥࢸሷࡢྜᡂࢆ㐩ᡂࡋࡓࠋᮏ཯ᛂࡣỈ⁐ᾮ୰࠿ࡘ Ԩࡢ ࿴࡞᮲௳ୗ࡛㐍⾜ࡋࠊỈ⁐ᾮ୰࡛ࡢ཯ᛂ࡛࠶ࡿࡓࡵ↓ಖㆤ࢜ࣜࢦ⢾࡟ࡶၥ㢟࡞ࡃ㐺⏝࡛ࡁࠊಙ㢗ᛶࡢ㧗࠸ỗ⏝ⓗྜᡂἲ࡛࠶ࡿࠋࡉࡽ࡟ࠊࢢࣜࢥࢩࣝࣈࣥࢸሷࡣࡇࢀࡲ࡛ᮍሗ࿌ࡢ໬ྜ≀࡛࠶ࡿࡇ࡜࠿ࡽࠊᐁ⬟ᇶኚ᥮࡟ࡼࡿᵝࠎ࡞᭷⏝⢾㉁ࡢྜᡂ୰㛫య࡜ࡋ࡚ࡢ฼⏝ࢆヨࡳࡓࠋᮏἲࡣࠊᚑ᮶ἲ࡛ࡣከẁ㝵཯ᛂࡀᚲせ࡛࠶ࡗࡓࢢࣜࢥࢩࣝࢳ࣮࢜ࣝࠊࢪ
ࢫࣝࣇ࢕ࢻ⢾ࠊOࢢࣜࢥࢩࢻࠊ࢔ࣥࣄࢻࣟ⢾࡞࡝ᵝࠎ࡞⢾㉁࡬ࡢኚ᥮ࡀᐜ᫆
࡛࠶ࡿࡇ࡜࠿ࡽࠊᴟࡵ࡚฼⏝౯್ࡢ㧗࠸⏬ᮇⓗ࡞ྜᡂ཯ᛂ࡛࠶ࡿࠋ
࠙ཷ㈹ࡢឤ᝿࡜ᢪ㈇ࠚ
ࡇࡢᗘࡣࠊ➨஧ᅇ⟽Ᏺ௝୍㑻㈹࣭ඃ⚽ㄽᩥ㈹ࢆ㈷ࡾ୍◊✲ဨ࡜ࡋ࡚኱ኚගᰤ࡟
Ꮡࡌࡲࡍࠋ⟽Ᏺඛ⏕ࡣ࣓ࢳࣝ໬࡟ࡼࡿከ⢾ศᯒἲࡢ㛤Ⓨࡶ᭷ྡ࡛ࡍࡀ⚾ࡢᑓ㛛࡜㛵㐃ࡍࡿኳ↛࠾ࡼࡧ⏕య㛵㐃⢾㙐ࡢ᥈⣴࣭ホ౯࡟࠾࠸࡚⣲ᬕࡽࡋ࠸◊✲ᡂᯝࢆᣏぢࡋ࡚࠸ࡲࡍࠋୡ⏺࡟ඛ㥑ࡅ࡚⢾㙐໬Ꮫࡢᒎ㛤࡟ከ኱࡞ࡿຌ⦼ࢆ୚࠼ࡓࡇ࡜࡛ྡ
㧗࠸⟽Ᏺඛ⏕ࡢྡ๓ࢆෙࡋࡓ㈹ࢆ㡬ࡃࡇ࡜ࡣ኱ኚᜍ⦰࡛ࡣ࠶ࡾࡲࡍࡀࠊࡇࡢ㈹ࢆ
ບࡳ࡟⚾⮬㌟ࡶ⢾㙐໬ᏛࡢⓎᒎ࡟㈉⊩࡛ࡁࡿࡼ࠺ດࡵ࡚࠸ࡁࡓ࠸࡜ᛮࡗ࡚࠾ࡾࡲࡍࠋ ⚾ࡣṇ⏣᫴୍㑻ᩍᤵࡢୗࠊ1,2-࢔ࣥࣄࢻࣟ⢾ࡢᙧᡂࢆ⤒⏤ࡍࡿ᪂つࢢࣜࢥࢩࣝ
໬཯ᛂࡢ㛤Ⓨ◊✲࡟ᚑ஦ࡋ࡚࠸ࡲࡋࡓࠋ◊✲ࢸ࣮࣐ࢆ⮬ࡽタᐃࡋࠊᐇ㦂ࠊศᯒࠊࡑࡋ࡚ㄽᩥᇳ➹࡜࠸࠺୍㐃ࡢ⤒㦂ࢆ⤒࡚ࠊ◊✲ࡢዟ῝ࡉࢆᐇឤࡍࡿ࡜࡜ࡶ࡟ࠊ◊✲ᡂᯝࢆᚓࡿࡇ࡜ࡀ࠸࠿࡟ᅔ㞴࠿ࢆ③ឤࡋ࡚࠸ࡲࡍࠋࡋ࠿ࡋ࡞ࡀࡽࠊᅔ㞴࡟┤㠃ࡍࡿࡓࡧ࡟ヨ⸆ࢆᡭ࡟ྲྀࡗ࡚ᐇ㦂ࢆ⧞ࡾ㏉ࡋ⾜ࡗ࡚ࡁࡲࡋࡓࠋࡑࡢࡼ࠺࡞◊✲⏕άࡢ୰࡛௒ᅇࡢཷ㈹ෆᐜ࡛࠶ࡿ᭷ᶵ/↓ᶵࣁ࢖ࣈࣜࢵࢻᵓ㐀ࢆᣢࡘ᪂つ⢾ㄏᑟయࢢࣜࢥࢩࣝࣈࣥࢸሷࡢⓎぢ࡟⮳ࡾࡲࡋࡓࠋᮏ཯ᛂࡣᗈ⠊࡞↓ಖㆤ࢜ࣜࢦ⢾࡟ࡶၥ㢟࡞ࡃ㐺⏝࡛ࡁࠊࡉࡽ࡟ࢢࣜࢥࢩࣝࢳ࣮࢜ࣝࠊࢪࢫࣝࣇ࢕ࢻ⢾ࠊO-ࢢࣜࢥࢩࢻ࡞
࡝࡬ࡢኚ᥮ࡶᐜ࡛᫆࠶ࡿࡇ࡜࠿ࡽ฼⏝౯್ࡢᴟࡵ࡚኱ࡁ࡞⏬ᮇⓗ཯ᛂ࡛࠶ࡿ࡜⪃࠼࡚࠸ࡲࡍࠋࡇࡢⓎぢࡢឤືࡣ◊✲⪅࡜ࡋ୍࡚⏕ᛀࢀࡽࢀ࡞࠸㈈⏘࡟࡞ࡿ࡛ࡋࡻ
17࠺ࡀࡇࡢࡼ࠺࡞⤒㦂ࡀ࡛ࡁࡓࡢࡶࡦ࡜࠼࡟ṇ⏣ඛ⏕ࡢ⮬⏤࡞Ẽ㢼ࡢୗ࡛◊✲ࢆ⾜࠼ࡓ࠾࠿ࡆ࡛࠶ࡿ࡜ᛮࡗ࡚࠾ࡾࡲࡍࠋ
ಟኈㄢ⛬᫬࡟࠾࠸࡚ࠊ᭷ᶵྜᡂ㛵㐃ࡢᏛ఍࡟࡚≉࡟ኳ↛≀ࡢ඲ྜᡂ◊✲࡟㛵ࡋ࡚ᣏ⫈ࡍࡿᶵ఍ࡀከࡃ࠶ࡾࡲࡋࡓࠋ⨾ࡋ࠸!ࡑࡢᙜ᫬ࠊ⚾࡟ࡣ඲ྜᡂࡢ⤒㦂ࡣ࠶ࡾࡲࡏࢇ࡛ࡋࡓࡀࠊࡉ࡞ࡀࡽⱁ⾡ⓗ࡞⤮⏬ࢆぢ࡚࠸ࡿ࠿ࡢࡼ࠺࡟┤ほⓗ࡟⨾ࡋ࠸࡜ឤࡌࡲࡋࡓࠋࡶࡋࠊ⮬ศࡀࡇࡢࡼ࠺࡞໬Ꮫኚ᥮ࢆ⮬⏤⮬ᅾ࡟᧯ࡿࡇ࡜ࡀ࡛ࡁࠊ᭱
⤊┠ⓗ≀ࢆᡭ࡟ࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜ࡋࡓࡽࠊࡇࢀࡲ࡛࡟࡞࠸㐩ᡂឤࢆᚓࡽࢀࡿ࡜
☜ಙࡋࡲࡋࡓࠋ⚾ࡣࡇࡢࡼ࠺࡞⤒⦋࠿ࡽ᭷ᶵྜᡂ࡞ࡽࡧ࡟඲ྜᡂ◊✲࡟㨩஢ࡉࢀࠊ⌧ᅾ࡛ࡣ᱓ཎ㔜ᩥᩍᤵ(ᮾ໭኱Ꮫ㎰Ꮫ)ࡢୗ࡛⢾ྵ᭷ኳ↛≀ࡢ඲ྜᡂ࡟ᚑ஦ࡋ࡚࠸
ࡲࡍࠋ඲ྜᡂ࡟㛵㐃ࡋࡓ་⸆࣭㎰⸆ࡢ㛤Ⓨࡣձኳ↛≀࡞࡝ࠊ᭷⏝࡞⏕⌮స⏝ࢆᣢࡘ⸆๣ࡢೃ⿵≀㉁ࡢ᥈⣴࣭㑅ᢤղྜᡂ࡟ࡼࡿᵓ㐀᭱㐺໬ճ⮫ᗋヨ㦂ࢆ⤒࡚⾜ࢃࢀࡿࡇ࡜ࡀከࡃࠊղࢆᢸ࠺඲ྜᡂ◊✲⪅ࡣ୰᰾ⓗ࡞Ꮡᅾ࡛ࡍࠋࡑࡢࡓࡵࠊ඲ྜᡂࢆ
ᚿࡍ⚾ࡣᵝࠎ࡞㡿ᇦࡢ◊✲⪅ࢆ⤖ࡧࡘࡅࡿ࡟㊊ࡿᗈ࠸▱㆑࡜ᢏ㔞ࢆ⵳࠼ࠊ◊✲ᡂᯝࢆ♫఍࡟㑏ඖ࡛ࡁࡿࡼ࠺ດࡵ࡚࠸ࡁࡓ࠸࡜⪃࠼࡚࠾ࡾࡲࡍࠋྜᡂ໬Ꮫ⪅࡜ࡋ࡚ࡢᢏ㔞࡜▱㆑࡟☻ࡁࢆ࠿ࡅࠊᑗ᮶ⓗ࡟ࡣ⢾㉁ྜᡂࡢᇶ♏◊✲࠿ࡽ♫఍࡬ࡢ㑏ඖࡲ࡛ࢆໟᣓࡍࡿ㡿ᇦᶓ᩿ⓗ࡞◊✲࡟ᚑ஦ࡋࡓ࠸࡜⪃࠼࡚࠾ࡾࡲࡍࠋ
᭱ᚋ࡟◊✲ࡢࡈᣦᑟࢆ㡬࠸ࡓṇ⏣᫴୍㑻ᩍᤵ࡜◊✲ᐊࡢⓙᵝ࡟῝ࡃឤㅰ࠸ࡓࡋ
ࡲࡍࠋࡇࢀࡲ࡛ࡢ཯ᛂ㛤Ⓨ࡜ࡣ␗࡞ࡿⓎ᝿ࡸᐇ㦂ᡭἲ࡟‶ࡕࡓ᪂ࡓ࡞⎔ቃ࡟࡚⌧ᅾࡣ⏕≀άᛶኳ↛≀ࡢ඲ྜᡂ࡟ᣮᡓࡋ࡚࠸ࡲࡍࠋࡇࡢ᪂ኳᆅ࡛࠶ࡿᮾ໭኱Ꮫ㎰Ꮫ◊✲⛉࡟࠾࠸࡚ࡶ௒ᅇࡢཷ㈹ࢆບࡳ࡟᪂ࡓ࡞◊✲ᡂᯝࢆሗ࿌࡛ࡁࡿࡼ࠺⢭୍ᮼດຊ࠸ࡓࡋࡲࡍࠋ┠㯮 ᗣὒ
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Biochim Biophys Acta Gen Subj  ᮧୖ ཭ኴ㸦⚟ᓥ┴❧་⛉኱Ꮫ་Ꮫ㒊⬻⚄⤒እ⛉Ꮫㅮᗙ࣭༤ኈ◊✲ဨ㸧࠙ㄽᩥせ᪨ࠚ
≉Ⓨᛶప㧊ᾮᅽ⑕ೃ⩌㸦⬻⬨㧊ᾮ₃ฟ⑕㸧ࡣ⬻⬨㧊ᾮ㸦㧊ᾮ㸧₃ฟ࡞࡝࡟ࡼࡾ㉳❧ᛶ㢌③ࠊࡵࡲ࠸➼ࡢ୙ᐃឋッࢆ♧ࡍ⑌ᝈ࡛࠶ࡿࠋ05,ࠊ&7 ࡞࡝࡛デ᩿ࡉࢀࡿࡀࠊ
⏬ീୖࡢኚ໬ࡀஈࡋ࠸⑕౛ࡀ࠶ࡾࠊࡇࡢሙྜデ᩿ࡀᴟࡵ࡚ᅔ㞴࡜࡞ࡿࠋᮏ◊✲ࢢ࣮ࣝࣉࡣ㧊ᾮ୰ࡢࣂ࢖࣐࣮࣮࢜࢝ࢆ᳨⣴ࡍࡿ◊✲ࢆ⾜ࡗࡓࠋᮏ⑌ᝈࡢ㧊ᾮࢱࣥࣃ
ࢡ㉁ࢆ⣔⤫ⓗ࡟ㄪ࡭࡚ࠊ⬻ᆺࢺࣛࣥࢫࣇ࢙ࣜࣥཬࡧ࣏ࣜ࢝ࣜࣥᆺࣉࣟࢫࢱࢢࣛࣥ
ࢹ࢕ࣥ ' ྜᡂ㓝⣲ࡢቑຍࢆ♧ࡋࠊ୧⪅ࢆ⑌ᝈ࣐࣮࣮࢝࡜ࡋࡓሙྜ࡟≉Ⓨᛶప㧊ᾮᅽ⑕ೃ⩌ࡀឤᗘ ࠊ≉␗ᗘ ࡛デ᩿ࡉࢀࡿࡇ࡜ࢆ♧ࡋࡓࠋ ࡇࡢㄽᩥࡣ≉Ⓨᛶప㧊ᾮᅽ⑕ೃ⩌࡟࠾ࡅࡿࣂ࢖࣐࣮࣮࢜࢝ࡢୡ⏺ึࡢሗ࿌࡛࠶ࡿࠋ ௜グ㸸ᅜ❧◊✲㛤Ⓨἲே᪥ᮏ་⒪◊✲㛤Ⓨᶵᵓ㸦$0('㸧ࡢࣉࣞࢫ࣮ࣜࣜࢫ
  KWWSVZZZDPHGJRMSQHZVUHOHDVHBKWPO ࠙⤒Ṕࠚᖹᡂ  ᖺ  ᭶ ⚟ᓥ┴❧་⛉኱Ꮫ་Ꮫ㒊 ༞ᴗᖹᡂ  ᖺ  ᭶ ⚟ᓥ┴❧་⛉኱Ꮫ་Ꮫ㒊⬻⚄⤒እ⛉Ꮫㅮᗙ ධᒁᖹᡂ  ᖺ  ᭶ ⚟ᓥ┴❧་⛉኱Ꮫ኱Ꮫ㝔་Ꮫ◊✲⛉༤ኈㄢ⛬ ಟ஢㸦ྠ኱Ꮫ་Ꮫ㒊⏕໬Ꮫㅮᗙ ᶫᮏᗣᘯᩍᤵࡢࡶ࡜ࠊ⬻⬨㧊ᾮࣂ࢖࣐࣮࣮࢜࢝ࡢ◊✲㸧ᖹᡂ  ᖺ  ᭶ ⚟ᓥ┴❧་⛉኱Ꮫ་Ꮫ㒊⬻⚄⤒እ⛉Ꮫㅮᗙ ຓᩍ
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19

࠙ཷ㈹ࡢឤ᝿࡜ᢪ㈇ࠚ
ࡇࡢࡓࡧࠊ⟽Ᏺ௝୍㑻㈹ඃ⚽ㄽᩥ㈹ࢆ㈷ࡾࡲࡋ࡚኱ኚගᰤ࡟Ꮡࡌࡲࡍࠋ⟽Ᏺඛ⏕ࡣࡌࡵᮾ໭⢾㙐◊✲఍ㄽᩥ㈹ᑂᰝጤဨࡢඛ⏕᪉࡟ᚚ♩⏦ࡋୖࡆࡲࡍࠋ
ཷ㈹ㄽᩥࡣࠊ⚾ࡀ኱Ꮫ㝔◊✲࡛ᙜㅮᗙ㸦㰻⸨Ύᩍᤵ㸧ࡀྠ኱Ꮫ⏕໬Ꮫㅮᗙ㸦ᶫᮏᗣᘯᩍᤵ㸧࡜ඹྠ◊✲ࢆ⾜ࡗ࡚࠸ࡿ⬻⬨㧊ᾮ㸦㧊ᾮ㸧ࣂ࢖࣐࣮࣮࢜࢝࡟ࡘ࠸࡚
ࡢ◊✲ෆᐜࡢ୍㒊࡛ࡍࠋ⚾ࡀ◊✲࡟㛵ࢃࡽࡏ࡚࠸ࡓࡔࡃ๓ẁ㝵࡛ࠊ㧊ᾮ࡜⾑Ύ࡜ࡢᛶ≧ࡢ㐪࠸࡜ࡋ࡚ࠊ㧊ᾮ୰ࡢࢺࣛࣥࢫࣇ࢙ࣜࣥ㸦7I㸧࡜࠸࠺ࢱࣥࣃࢡ㉁࡟ࡣࠊ
⾑Ύᆺ⢾㙐ࢆࡶࡘ⾑Ύᆺ 7I ࡜ࡣู࡟ࠊ㧊ᾮ୰࡟≉᭷ࡢ⬻ᆺ⢾㙐ࢆࡶࡘ⬻ᆺ 7I ࡀᏑᅾࡍࡿࠊ࡜࠸࠺ࡇ࡜ࡀぢ࠸ࡔࡉࢀ࡚࠾ࡾࡲࡋࡓࠋ⬻⬨㧊ᾮࡢ⏕⌮Ꮫ࡜ࡋ࡚ࡣࠊ
ࡇࡢᩘ༑ᖺ᮶ᩍ⛉᭩࡟グ㍕ࡉࢀ࡚࠸ࡓ EXONIORZ ㄝ㸦ഃ⬻ᐊෆࡢ⬦⤡ྀ࡛㧊ᾮࡀ⏘⏕ࡉࢀ࡚ࠊࡑࡢᚋ㧊ᾮࡣ࣮ࣔࣥࣟᏍࢆ⤒⏤ࡋࠊ➨Ϫ⬻ᐊࠊ➨ϫ⬻ᐊࢆ㏻ࡾࠊࡸࡀ
࡚ࡃࡶ⭷ୗ⭍ෆ࡟ᗈࡀࡾࠊࡃࡶ⭷㢛⢏࠿ࡽ྾཰ࡉࢀୖ▮≧㟼⬦Ὕ࡬ὶࢀࡿࠊ࡜࠸
࠺㧊ᾮࡢ⏘⏕ࡸ྾཰࡟㛵ࡍࡿ௬ㄝ㸧ࡀ㏆ᖺ␲ၥどࡉࢀࠊ᪂ࡓ࡞▱ぢࡀቑ࠼࡚ࡁࡓ
᫬ᮇ࡛࠶ࡗࡓࡇ࡜࠿ࡽࠊ㧊ᾮ࡟⯆࿡ࢆᙉࡃᣢࡕࡲࡋࡓࠋ
ཷ㈹ㄽᩥෆᐜࡣࠊ≉Ⓨᛶప㧊ᾮᅽ⑕ೃ⩌ࡢ㧊ᾮデ᩿ࡣࠊ࡜ࡶ࡟㧊ᾮ≉᭷ࡢ≀㉁࡛࠶ࡿࠊ⬻ᆺ 7Iࠊ࠾ࡼࡧࠊ࣏ࣜ࢝ࣜࣥᆺࣉࣟࢫࢱࢢࣛࣥࢹ࢕ࣥ ' ྜᡂ㓝⣲ࡢ⤌ࡳྜࢃࡏ࡟ࡼࡗ࡚㧗࠸デ᩿⢭ᗘุ࡛᩿ྍ⬟࡜࡞ࡿࡇ࡜࡜ࠊ㧊ᾮ⏕⌮Ꮫࡢ⪃ᐹ࡛ࡍࠋ≉Ⓨᛶప㧊ᾮᅽ⑕ೃ⩌ࡣࠊデ᩿ᅔ㞴࡞⑌ᝈ࡛࠶ࡾ᭷⑓⋡ࡶ㧗ࡃ࠶ࡾࡲࡏࢇࡀࠊࡑ
ࡢデ⒪ࢆᑓ㛛ⓗ࡟⾜ࡗ࡚࠸ࡿᒣ⋤⑓㝔ࡢ㧗ᶫᾈ୍ඛ⏕࡜⨾㤿㐩ኵඛ⏕࡜ࡢඹྠ◊✲࡟㛵ࢃࡿᶵ఍ࢆࠊᶫᮏᩍᤵࡢࡈ㧗㓄࡟ࡼࡾࠊᖾ㐠࡟ࡶ࠸ࡓࡔࡁࡲࡋࡓࠋᮏ⑌ᝈ
࡟㝈ࡽࡎࠊ୰ᯡ⚄⤒⣔⑌ᝈࡢ᪥ᖖデ⒪࡛ࡣࠊ⮫ᗋ⑕≧ࡀ⏬ീᡤぢࢆࡣࡌࡵ࡜ࡋࡓ᳨ᰝᡤぢ࡛ㄝ࡛᫂ࡁ࡞࠸࡜࠸࠺≧ἣ࡟㐼㐝ࡍࡿࡇ࡜ࡀ࠶ࡾࡲࡍࠋᮏ◊✲ࡀࠊ㧊ᾮ㛵㐃⑌ᝈࡢ⑓ែゎ᫂࠶ࡿ࠸ࡣࠊ⮫ᗋデ᩿ࡢ୍ຓ࡟࡞ࢀࡤࠊࡇࢀ௨ୖࡢ႐ࡧࡣ࠶ࡾ
ࡲࡏࢇࠋ᭱ᚋ࡟ࠊᶫᮏᩍᤵࠊ㰻⸨ᩍᤵࢆࡣࡌࡵࠊ୧ㅮᗙࡢඛ⏕᪉࡟ࡣࡇࡢሙࢆ೉ࡾࡲࡋ࡚῝ㅰ⏦ࡋୖࡆࡲࡍࠋ௒ᚋࡣࠊ㧊ᾮࠊ࡜ࡃ࡟⢾㙐࡟╔┠ࡋࡓᇶ♏◊✲ࡣⓎᒎ㏵ୖ࡟࠶ࡿ≧ែ࡛࠶ࡿࡓࡵࠊࡉࡽ࡟ྠᵝࡢ◊✲࡟㛵ࢃࡗ࡚࠸ࡁࡓ࠸࡜⪃࠼࡚࠾ࡾࡲࡍࠋ
௒ᅇࡢཷ㈹ࢆບࡳ࡟◊㛑ࢆ㔜ࡡ࡚࠸ࡁࡓ࠸࡜ᛮࡗ࡚࠾ࡾࡲࡍࠋ௒ᚋ࡜ࡶࡈᣦᑟࡈ
㠴᧡ࡢ࡯࡝ࡼࢁࡋࡃ࠾㢪࠸࠸ࡓࡋࡲࡍࠋ
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21

GM3 ࢞ࣥࢢࣜ࢜ࢩࢻࡢ⏕⌮ⓗព⩏ࡢゎ᫂࡟ྥࡅ࡚
ᶵ⬟⑓ែศᏊ㒊㛛  ஭ࣀཱྀ ோ୍࢞ࣥࢢࣜ࢜ࢩࢻࣇ࢓࣑࣮ࣜ⏕ྜᡂࡢ᭱ึࡢศᏊࡣࠊࣛࢡࢺࢩࣝࢭ࣑ࣛࢻ࡟ࢩ࢔ࣝ㓟ࡀ㌿⛣ࡋ࡚⏕ᡂࡍࡿ GM3 ࢞ࣥࢢࣜ࢜ࢩࢻ࡛࠶ࡿࠋࡑࡶࡑࡶ GM3 ࡣ㸪⢾⬡㉁◊✲ࡢⲡศࡅ࡛࠶ࡿᒣᕝẸኵ༤ኈࡀ࣐࢘㉥⾑⌫࠿ࡽ࣐࣊ࢺࢩࢻ࡜ࡋ࡚ྠᐃࡉࢀࡓࡶ
ࡢ࡛࠶ࡿࠋᡃࠎ࡟࢞ࣥࢢࣜ࢜ࢩࢻ◊✲ࡢࡁࡗ࠿ࡅࢆ୚࠼࡚ࡃࢀࡓࡢࡣࠊ1998 ᖺ࡟
GM3 ྜᡂ㓝⣲㸦GM3S㸪ST3GAL5㸧ࡢ㑇ఏᏊࢡ࣮ࣟࢽࣥࢢ࡟ཧ⏬ࡋࡓࡇ࡜࡛࠶
ࡿࠋ2018 ᖺᗘࡣࠊࢪࣙࣥࢫ࣍ࣉ࢟ࣥࢫ኱ࡢ Ronald L. Schnaar ᩍᤵࡢ౫㢗࡛ GM3࢞ࣥࢢࣜ࢜ࢩࢻࡢ⏕≀Ꮫⓗព⩏࡟ࡘ࠸࡚ࡢ➹⪅ࡽࡢ㸰㸮ᖺ㛫ࡢ◊✲ࢆ “Biology
of GM3 ganglioside”࡜ࡋ࡚௨ୗࡢࡼ࠺࡟㞟኱ᡂࡋࡓ⥲ㄝࢆฟ∧ࡋࡓ(1)ࠋ
1. Molecular biology of GM3S/ST3GAL5
2. Insulin resistance in adipocytes and macrophages
3. Leptin signaling and resistance in hypothalamus
4. T-Cell immunity and immune disorders
5. GM3S deficiency and hearing loss௒ᖺᗘࡢ◊✲ᡂᯝ࡜ࡋ࡚ࠊ㸲ࡘࡢࢺࣆࢵࢡ࡟ࡘ࠸࡚⤂௓ࡍࡿࠋϨ㸬࣓ࣛࣀࢥࣝࢳࣥࢩࢢࢼࣝ࡟࠾ࡅࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢ㛵୚㯤Ⰽ⫧‶࣐࢘ࢫ KKAy ࡟࠾ࡅࡿ GM3 ྜᡂ㓝⣲(GM3S)ࡢࣀࢵࢡ࢔࢘ࢺ(GM3S
KO)࡛ࡣࠊKKAy ࣐࢘ࢫࡢ㐣㣗࡜⫧‶㸪⪏⢾⬟␗ᖖ࠾ࡼࡧ࢖ࣥࢫࣜࣥ᢬ᢠᛶࡢⴭ
᫂࡞ᨵၿࡀࡳࡽࢀࡓࠋKKAy ࣐࢘ࢫ࡛ࡣ
ࣞࣉࢳࣥ⭡⭍ෆᢞ୚࡟ᑐࡍࡿどᗋୗ㒊ᘪ≧᰾ࢽ࣮ࣗࣟࣥࡢᛂ⟅ᛶࡀ኱ᖜ࡟ῶ
ᙅࡋ࡚࠸ࡓࡀࠊKKAy GM3S KO ࣐࢘ࢫ࡛ࡣࣞࣉࢳࣥ࡟ᑐࡍࡿᛂ⟅ᛶࡀ༑ศಖ
ࡓࢀ࡚࠸ࡓࠋࡲࡓ㸪࣐࢘ࢫどᗋୗ㒊⏤᮶⚄⤒⣽⬊ᰴ࡟࠾ࡅࡿ GM3S Ḟᦆ⣽⬊࡛ࡣࠊࣞࣉࢳࣥ౫Ꮡࡢ ERK ࣜࣥ㓟໬ࡀஹ㐍ࡋ࡚࠸ࡓࠋࡉࡽ࡟ࠊKKAy/GM3S KO࣐࢘ࢫࡣẟⰍࡢኚ໬㸦㯤Ⰽń⅊Ⰽ㸧ࢆ࿊ࡋ࡚࠾ࡾࠊGM3 㛵㐃࢞ࣥࢢࣜ࢜ࢩࢻࡣ
ࣞࣉࢳࣥ࡜࣓ࣛࣀࢥࣝࢳࣥࢩࢢࢼࣝ࡟࠾࠸࡚㔜せ࡞ᙺ๭ࢆࡶࡘࡇ࡜ࡀ♧၀ࡉࢀࡓ(2)ࠋ

22ϩ㸬⭠⟶࠿ࡽࡢࢥࣞࢫࢸ࣮ࣟࣝ྾཰࡟࠾ࡅࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢᙺ๭
௒ᅇᡃࠎࡣࠊGM3 ྜᡂ㓝⣲(GM3S)ࢆࣀࢵࢡ࢔࢘ࢺ㸦KO㸧ࡋࡓ࢞ࣥࢢࣜ࢜ࢩ
ࢻࡀ⏘⏕ࡉࢀ࡞࠸⣽⬊(GM3S KO ⣽⬊)ࢆసᡂࡋࠊNPC1L1 ࢆ௓ࡋࡓࢥࣞࢫࢸࣟ
࣮ࣝ྾཰࡟࠾ࡅࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢ⏕⌮Ꮫⓗព⩏࡟ࡘ࠸࡚ゎᯒࡋࡓࠋእ࠿ࡽ୚࠼
ࡓࢥࣞࢫࢸ࣮ࣟࣝࡣࠊ⣽⬊⭷ୖ࡟࠶ࡿ NPC1L1 ࡟⤖ྜࡍࡿ࡜ࠊࢥࣞࢫࢸ࣮ࣟࣝ౫
Ꮡⓗ࡟ NPC1L1࣭ࢥࣞࢫࢸ࣮ࣟࣝ」ྜయࡢ⣽⬊ෆ⛣⾜(࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ)ࡀ㉳ࡇࡾࠊయෆ࡟ࢥࣞࢫࢸ࣮ࣟࣝࡀྲྀࡾ㎸ࡲࢀࡿࠋ࡜ࡇࢁࡀࠊGM3S KO ⣽⬊࡛ࡣࢥࣞࢫࢸ࣮ࣟࣝ౫Ꮡⓗ࡞ NPC1L1 ࡢ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫࡀᢚไࡉࢀࠊ⣽⬊ෆࢥࣞࢫࢸ࣮ࣟࣝྵ㔞ࡢቑຍࡶ᭷Ⅽ࡟ᢚไࡉࢀ࡚࠸ࡓࠋ⥆࠸࡚ࠊ࣐࢘ࢫ࡟࠾࠸࡚ࡶࠊ⣽⬊࡛ࣞ࣋ࣝࡢᐇ㦂⤖ᯝ࡜୍⮴ࡍࡿࡼ࠺࡟ࠊGM3S KO ࣐࢘ࢫࡣࢥࣞࢫࢸ࣮ࣟࣝ㈇Ⲵ
࡟ࡼࡾᘬࡁ㉳ࡇࡉࢀࡿ⾑୰ࡢࢥࣞࢫࢸ್࣮ࣟࣝࡢୖ᪼ࡀⴭࡋࡃᢚไࡉࢀ࡚࠸ࡓࠋ
ࡉࡽ࡟ࠊ㧗ࢥࣞࢫࢸ࣮ࣟࣝ⾑⑕ࣔࢹࣝ࡜ࡋ࡚⏝࠸ࡽࢀࡿ ApoE ኚ␗࣐࢘ࢫ࡟࠾࠸
࡚ࠊApoE ኚ␗ GM3SKO ࣐࢘ࢫࢆస〇ࡋࡓ࡜ࡇࢁࠊApoE ኚ␗࣐࢘ࢫࡀ♧ࡍ㧗ࢥࣞࢫࢸ࣮ࣟࣝ⾑⑕ࡣ ApoE ኚ␗ GM3SKO ࣐࢘ࢫ࡛ࡣ㢧ⴭ࡟ᨵၿࡉࢀࠊ⭠⟶࠿ࡽࡢࢥࣞࢫࢸ࣮ࣟࣝ྾཰⋡࠾ࡼࡧᑠ⭠⤧ẟ࡟࠾ࡅࡿ NPC1L1 ࡢ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩ
ࢫࡶᢚไࡉࢀࡓࠋ௒ᅇࡢ◊✲࠿ࡽ GM3 ࢆࡣࡌࡵ࡜ࡍࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢⓎ⌧ࢆᢚไࡍࡿࡇ࡜࡛ࠊNPC1L1 ࡢᶵ⬟పୗࢆᘬࡁ㉳ࡇࡋࠊ㧗ࢥࣞࢫࢸ࣮ࣟࣝ⾑⑕ࡢⓎ⑕ࡀᢚไࡉࢀࡿྍ⬟ᛶࡀぢฟࡉࢀࡓࠋ࢞ࣥࢢࣜ࢜ࢩࢻࡢⓎ⌧ไᚚࡀ㧗ࢥࣞࢫࢸࣟ
࣮ࣝ⾑⑕࡟ᑐࡍࡿ᪂つ἞⒪ᶆⓗ࡜ࡋ࡚๰⸆࡟ࡘ࡞ࡀࡿࡇ࡜ࡀᮇᚅࡉࢀࡿ(3)ࠋ
Ϫ㸬T ⣽⬊ศ໬࡟࠾ࡅࡿࢫࣇ࢕ࣥࢦ࣑࢚ࣜࣥࡢᶵ⬟T ⣽⬊ࡣ⣽⬊ᛶච␿ࡢ୰ᚰⓗᙺ๭ࢆᢸ࠺ࣜࣥࣃ⌫ࡢ୍⩌࡛࠶ࡾࠊ⬚⭢࡟࠾࠸࡚ከẁ㝵ࡢ⏕ᏑቑṪ࣭ศ໬ࢆ⤒࡚ᡂ⇍ࡋࠊᮎᲈ࡬⛣ฟࡋ࡚ච␿཯ᛂࢆᢸࡗ࡚࠸ࡿࠋ
ᮏ◊✲࡛ࡣࠊᙧ㉁⭷ୖ࡛ࢫࣇ࢕ࣥࢦ࣑࢚ࣜࣥ㸦SM㸧࡜ࢥࣞࢫࢸ࣮ࣟࣝ࡟ࡼࡾᙧᡂ
ࡉࢀࡿᚤᑠ㡿ᇦ㸦⬡㉁࣐࢖ࢡࣟࢻ࣓࢖ࣥ㸧ࡢ T ⣽⬊ࡢศ໬㐣⛬࡟࠾ࡅࡿ⏕⌮ⓗព⩏ࢆゎ᫂ࢆヨࡳࡓࠋ⬚⭢⣽⬊ࡢ SM Ⓨ⌧㔞ࡣ୍㐃ࡢศ໬㐣⛬࡛኱ࡁࡃኚືࡋࠊࡑ
ࢀࡣࢭ࣑ࣛࢻ࠿ࡽ SM ࢆྜᡂࡍࡿ㓝⣲࡛࠶ࡿ SM ྜᡂ㓝⣲ 1㸦SMS1㸧ࡢⓎ⌧࡛ࣞ࣋ࣝཝᐦ࡟ไᚚࡉࢀ࡚࠸ࡿࡇ࡜ࢆぢ࠸ࡔࡋࡓࠋศ໬ࡢึᮇẁ㝵࠿ࡽ SM Ⓨ⌧ࡣᚎࠎ࡟పୗࡋࠊṇ㈇ࡢ㑅ᢥࡢ๓ᚋ࡛ SM Ⓨ⌧ࡀ෌ᗘ኱ࡁࡃቑຍࡋࠊSM ࣐࢖ࢡࣟࢻ࣓࢖ࣥⓎ⌧㔞ࡣ 10 ಸ௨ୖቑຍࡋࠊṇ㈇ࡢ㑅ᢥ࡟࠾ࡅࡿ SM ࣐࢖ࢡࣟࢻ࣓࢖ࣥ
ࡢ㛵୚ࡀ⪃࠼ࡽࢀࡓࠋSMS1 Ḟᦆ࣐࢘ࢫ⏤᮶⣽⬊࡛ࡣ⣽⬊Ṛࡀஹ㐍ࡋࠊTCR ่⃭
ᚋࡢ TCR ㏆఩ࡢࢩࢢࢼࣝศᏊ࡛࠶ࡿ ZAP-70 ࡢࣜࣥ㓟໬ࡀஹ㐍ࡋࠊ㈇ࡢ㑅ᢥ࡟ࡘ࡞ࡀࡿ ERK5 ࡢࣜࣥ㓟໬ࡸ࢔࣏ࢺ࣮ࢩࢫಁ㐍ศᏊ࡛࠶ࡿ Bim㸪ERK5 ࡢୗὶࡢఏ
㐩ศᏊࡢ Nur77 ࡢⓎ⌧ࡶ㢧ⴭ࡟ቑຍࡋ࡚࠾ࡾࠊSMS1 Ḟᦆ⬚⭢⣽⬊࡛ࡣ TCR ࢩࢢࢼࣝఏ㐩ࡀஹ㐍ࡍࡿࡇ࡜ࡀุ᫂ࡋࡓࠋSMS1 Ḟᦆ⬚⭢⣽⬊࡛ࡣ TCR ่⃭ࡢ㜈್ࡀୗࡀࡾ TCR ࢩࢢࢼࣝᙉᗘࡀቑᙉࡉࢀ࡚㈇ࡢ㑅ᢥࡀஹ㐍ࡋࡓ࡜⪃࠼ࡽࢀࡿࠋ
ࡇࡢࡇ࡜ࡣ TCR ࢩࢢࢼࣝᙉᗘࡢㄪ⠇࡟ SM ࡟ᐩࢇࡔ⬡㉁࣐࢖ࢡࣟࢻ࣓࢖ࣥࡀᐤ
23୚ࡍࡿࡇ࡜ࢆ♧၀ࡋ࡚࠾ࡾࠊ⬚⭢⣽⬊ศ໬࡟࠾࠸࡚ SM Ⓨ⌧㔞ࡢቑῶࡣཝᐦ࡟ไ
ᚚࡉࢀࠊṇ㈇ࡢ㑅ᢥࢆỴᐃࡍࡿ TCR ่⃭ࡢ㜈್ࢆ㐺ṇ໬ࡋ࡚࠸ࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ௒ᚋࠊ⮬ᕫච␿⑌ᝈᝈ⪅ࡢ T ⣽⬊࡟࠾ࡅࡿ SM Ⓨ⌧ࡢኚ໬ࢆゎᯒࡍࡿࡇ࡜
࡛ࠊ᪂ࡓ࡞デ᩿ࡸ἞⒪ἲࡢ㛤Ⓨࡀᮇᚅࡉࢀࡿ(4)ࠋ
ϫ㸬⢾ᒀ⑓ᛶ⭈⑕࡟࠾ࡅࡿࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡢᙺ๭
ᮏ◊✲࡛ࡣࠊ㧗⬡⫫㣗ࢆ KK ࣐࢘ࢫ࡟ 8 㐌㛫㈇Ⲵࡋ⢾ᒀ⑓ᛶ⭈⑕ࣔࢹࣝࢆసᡂ
ࡋࡓࠋࡇࡢ㧗⬡⫫㣗㈇Ⲵ KK ࣐࢘ࢫࡢ⭈⮚࡛ࡣࠊࢢࣟ࣎⣔⢾⬡㉁ Gb3 ࡢⓎ⌧ࡀⴭࡋࡃቑຍࡋ࡚࠸ࡓࠋࡑࡢศᏊ✀ゎᯒࢆ, ᾮయࢡ࣐ࣟࢺࢢࣛࣇ-ࢱࣥࢹ࣒㉁㔞ศᯒィ
(LC-MS/MS)ࢆ⏝࠸࡚⾜ࡗࡓ⤖ᯝࠊGb3 ࡢࢭ࣑ࣛࢻ㒊ศࡢ࢔ࢩࣝ㙐࡟㣬࿴ࡢᴟ㛗
㙐(C22, C23, C24)᭷ࡍࡿศᏊ✀ࡀ≉࡟ቑຍࡋ࡚࠸ࡿࡇ࡜ࢆぢฟࡋࡓࠋࡇࡢ⤖ᯝࡼࡾ, ࢢࣟ࣎⣔⢾⬡㉁ࡢ⑓ែᙧᡂ࡬ࡢ㛵୚ࡀ♧၀ࡉࢀࡿࡇ࡜࠿ࡽ,⢾ᒀ⑓ᛶ⭈⑕ࡢ

⑓ែᙧᡂ࡟㔜せ࡞⅖⑕཯ᛂ࡟࠾ࡅࡿ, ࢢࣟ࣎⣔⢾⬡㉁ࡢ⅖⑕ច㉳άᛶࡢ᭷↓ࢆࣄࢺ༢⌫ࡸ࣐࢘ࢫ㦵㧊⏤᮶࣐ࢡࣟࣇ࢓࣮ࢪࢆ⏝࠸࡚ホ౯ࡋࡓ. ࡑࡢ⤖ᯝ, ᴟ㛗㙐ࢢࣟ࣎⣔⢾⬡㉁ࡣ, Toll-like receptor 4 (TLR4)ࣜ࢞ࣥࢻ(LPS, HMGB1)ࡢᏑᅾୗ,TLR4 㑅ᢥⓗ࡞࣏ࢪࢸ࢕ࣈࣔࢪ࣮ࣗࣞࢱ࣮࡜ࡋ࡚⅖⑕ಁ㐍άᛶࢆᣢࡘࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊᙼࡣࣞࣉࢳࣥཷᐜయࡀኚ␗ࡋࡓ db/db ࣐࢘ࢫ࡛ࡣ Gb3 ࡢⓎ⌧ࡀపࡃࠊࣞࣉࢳࣥࡢ⭡⭍ෆᢞ୚࡛ࠊGb3 ࡢⓎ⌧ࡀᅇ᚟ࡍࡿࡇ࡜ࢆぢ࠸ࡔࡋ࡚࠸ࡿࠋ㧗⬡⫫㣗㈇Ⲵ࡟ࡼࡾ⫧኱ࡋࡓ⬡⫫⤌⧊࠿ࡽศἪಁ㐍ࡉࢀࡓࣞࣉࢳࣥࡀࠊ⭈⮚ࡢࢢࣟ࣎⣔⢾⬡㉁ࡢⓎ⌧ࢆቑຍࡉࡏ, ቑຍࡋࡓࢢࣟ࣎⣔⢾⬡㉁ࡀࠊTLR4 ࡟స⏝ࡍࡿࡇ
࡜࡛⅖⑕཯ᛂࢆಁ㐍ࡋ, ⢾ᒀ⑓ᛶ⭈⑕ࡢ⑓ែᙧᡂ࡟㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀᮏ◊✲ࡼࡾ♧၀ࡉࢀࡓࠋࡇࢀࡽࡢ◊✲ᡂᯝࡣࠊ⢾ᒀ⑓ᛶ⭈⑕ࡢ᪂ࡓ࡞デ᩿ἲࡸ἞⒪⸆㛤Ⓨ࡟ࡘ࡞ࡀࡿࡇ࡜ࡀᮇᚅࡉࢀࡿ.
2018 ᖺᗘ࡟Ⓨ⾲ࡋࡓㄽᩥ
1. Inokuchi J, Inamori K, Kabayama K, Nagafuku M, Uemura S, Go S,
Suzuki A, Ohno I, Kanoh H, Shishido F. Biology of GM3 ganglioside (Book
Chapter in Gangliosides in Health and Diseases). Progress in Molecular

Biology and Translational Science(Schnaar
RL.,
Lopez
PHH.,
eds,
Elsevior)
page152-195
ISBN978-0-12-81234-6(https://doi.org/10.1016/bs.pmbts.2017.10.004),
2018.
2. Inamori K, Ito H, Tamura Y, Nitta T,Yang X, Nihei W, Shishido F, Imazu S,
Tsukita S, Yamada T. Katagiri H, Inokuchi J. (2018) Deficient gangliosidesynthesis restores responsiveness to leptin and melanocortin signaling inobese KKAy mice. J Lipid Res. 59, 1472-1481, 2018.
3. Nihei W, Nagafuku M, Hayamizu H, Odagiri Y, Tamura Y, Kikuchi Y,
Veillon L, Kanoh H, Inamori K, Arai K, Kabayama K, Fukase K, Inokuchi J.
24

NPC1L1-dependent intestinal cholesterol absorption requires gangliosideGM3 in membrane microdomains J Lipid Res. 59, 2181-2187, 2018.
4. Toshima K, Nagafuku M, Okazaki T, Kobayashi T, Inokuchi J. Plasma
membrane sphingomyelin modulates thymocyte development by inhibiting
TCR-induced apoptosis. International Immunology
doi:10.1093/intimm/dxy082, 2018.
5. Nakayama H, Nagafuku M, Akemi Suzuki A, Iwabuchi K. and Inokuchi J.
The regulatory roles of glycosphingolipid-enriched lipid rafts in immune
systems. FEBS letters doi:10.1002/1873-3468.13275, 2018.
6. Ọ⚟ ṇ࿴㸪㇏ᓥ ࠿࠾ࡿ㸪ᇼෆ 㞙㸪஭ࣀཱྀ ோ୍㸬ࢫࣇ࢕ࣥࢦ࣑࢚࣐ࣜࣥ࢖ࢡ
ࣟࢻ࣓࢖ࣥࡣ Jurkat ⣽⬊ࡢ T ⣽⬊ᢠཎཷᐜయ౫Ꮡᛶࡢάᛶ໬ࢆ㈇࡟ไᚚࡍࡿ㸬ᮾ໭་⛉⸆⛉኱Ꮫ◊✲ㄅ 65, 29-42, 2018.
7. Nitta T, Kanoh H, Inamori K, Suzuki A, Takahashi T, and Inokuchi J.
Globo-series
glycosphingolipids
enhancelipopolysaccharide-induced
inflammation and play a pathophysiological role in diabetic nephropathy
Glycobiology 29, 260-268, 2019.
8. Hiraoka M, Ohkawa E, Abe A, Murata M, Go S, Inokuchi J, Ohguro H.
Visual function in mice lacking GM3 synthase. Curr Eye Res
https://doi.org/10.1080/02713683.2019.1576206, 2019.
25⏕ά⩦័⑓࡟㛵㐃ࡋࡓ G ࢱࣥࣃࢡ㉁ඹᙺཷᐜయࡢᶵ⬟࡜⢾㙐࠾ࡼࡧཷᐜయ┦஫స⏝࡟ࡼࡿㄪ⠇⏕య⭷᝟ሗ㒊㛛  ᮾ ⚽ዲ
1. GPCRǂ ┦⿵࢔ࢵࢭ࢖ࡢᨵⰋ
 ㏆ᖺࠊGPCR ࡢάᛶᐃἲࡢ 1 ࡘ࡜ࡋ࡚ ǂ ⿵᏶ࡀ౑ࢃࢀࡿࡼ࠺࡟࡞ࡗ࡚ࡁࡓ

(1-3)ࠋࡇࢀࡣࠊ኱⭠⳦ࡢ ǃ ࢞ࣛࢡࢺࢩࢲ࣮ࢮ (LacZ)ࢆࢥ࣮ࢻࡍࡿ lacZ ࡢᑐ❧㑇
ఏᏊࡢ 1 ࡘ࡛࢔࣑ࣀᮎ➃௜㏆ࡢ 11-41 ࢔࣑ࣀ㓟㓄ิࢆḞኻࡋ ǃ ࢞ࣛࢡࢺࢩࢲ࣮ࢮ
άᛶࢆᣢࡓ࡞࠸ lacZƦM15㸦ǚ ࣇࣛࢢ࣓ࣥࢺࢆࢥ࣮ࢻࡋ࡚࠸ࡿ㸧ࡢ⏘≀࡜ࠊࡑࢀࢆ┦⿵ࡋ࡚ ǃ ࢞ࣛࢡࢺࢩࢲ࣮ࢮάᛶࢆ᚟ඖࡉࡏࡿ LacZ ࢱࣥࣃࢡ㉁ࡢ࢔࣑ࣀᮎ➃50 ࢔࣑ࣀ㓟ṧᇶ⛬ᗘࢆࢥ࣮ࢻࡍࡿ lacZǂ ࡢ⏘≀㸦ǂ ࣇࣛࢢ࣓ࣥࢺ㸧ࡢ཯ᛂࢆ฼⏝ࡋࡓ⤖ྜᣦᶆ࢔ࢵࢭ࢖࡛࠶ࡿࠋᮏ᮶ࠊ኱⭠⳦࡛฼⏝ࡉࢀ࡚ࡁࡓࡶࡢ࡛࠶ࡿࡀࠊ࣍
ங㢮ࡢ⣽⬊࡛฼⏝࡛ࡁࡿࠋලయⓗ࡟ࡣࠊᐃᑐ㇟ࡢ GPCR ࡢ C ᮎ➃࡟ ǂ ࣇࣛࢢ࣓ࣥࢺࢆࠊάᛶ໬࡟ࡼࡾ GPCR ࡜⤖ྜࡍࡿ࢔ࣞࢫࢳࣥ➼ࡢࢱࣥࣃࢡ㉁ࡢ C ᮎ➃࡟ ǚ ࣇࣛࢢ࣓ࣥࢺࢆࡑࢀࡒࢀ⼥ྜࡋ࡚ᇵ㣴⣽⬊࡟Ⓨ⌧ࡉࡏࠊࣜ࢞ࣥࢻ่⃭࡟ࡼࡾ
GPCR ࡜࢔ࣞࢫࢳࣥࡀ⤖ྜࡍࡿ࡜ࡑࢀࡽ࡟⼥ྜࡋࡓ ǂ ࣇࣛࢢ࣓ࣥࢺ࡜ ǚ ࣇࣛࢢ࣓ࣥࢺࡀ⤖ྜࡋ࡚ ǃ ࢞ࣛࢡࢺࢩࢲ࣮ࢮάᛶࢆ⾲ࡍࡢ࡛ࠊࡇࢀ࡟
ࢺࢩࢲ࣮ࢮࡢᇶ㉁ࢆᑟධࡍࢀࡤࠊ⤖ྜࡀᐃྍ⬟࡞ ǃ ࢞ࣛࢡᐃ࡛ࡁࡿ㸦ୗᅗ㸧ࠋ

Arrestin ⤖ྜࢆᣦᶆ࡜ࡋࡓ GPCR άᛶ໬ࡢᐃᚑ᮶ἲ࡛ࡣ G ࢱࣥࣃࢡ㉁ࡢୗὶࡢ཯ᛂࢆ ࡿࡓࡵ࡟≉␗ᛶࡀ⦆࠿ࡗࡓࡀࠊᮏἲ
࡛ࡣ┠ⓗࡢ GPCR ࡟≉ᐃࡋ࡚ࢩࢢࢼࣝࢆࣔࢽࢱ࣮࡛ࡁࡿ฼Ⅼࡀ࠶ࡿࠋ୍㒊ࡣ࢔ࢵࢭ࢖࢟ࢵࢺ࡜ࡋ࡚ᕷ㈍ࡉࢀ࡚࠸ࡿࡀࠊ┠ⓗ㑇ఏᏊࢆⓎ⌧ࡉࡏࡓᇵ㣴⣽⬊࡜ࡋ࡚ࡢࡳධᡭྍ⬟࡛࠶ࡿࠋᚑࡗ࡚ࠊ௵ពࡢ GPCR ࡜࢔ࣞࢫࢳࣥ➼ࡢ⤌ࡳྜࢃࡏ࡛◊✲ࡍࡿࡓࡵࠊᡃࠎࡣࠊ⊂⮬࡟ࣉࣛࢫ࣑ࢻ࣋ࢡࢱ࣮ࢆస〇ࡋࡓࠋpAcGFP1-N1 (Clontech)ࡢ AcGFP1 ࢆ ǂ ࣇࣛࢢ࣓ࣥࢺࠊࡲࡓࡣࠊǚ ࣇࣛࢢ࣓ࣥࢺ࡟⨨᥮ࡋࡓ࣋ࢡࢱ࣮ࢆ⏝26

࠸ࡿ࡜࠸࠺⡆౽࡞ࡶࡢ࡛࠶ࡿ(4)ࠋᙜึࡣࠊ┠ⓗ㑇ఏᏊⓎ⌧⣽⬊ࢆ 96 well plate࡟෌᧛✀ᚋ㸯᫬㛫࡛ࣜ࢞ࣥࢻ่⃭ࡍࡿ࡜࠸࠺᪉ἲࢆ࡜ࡗࡓࡓࡵࠊ᧛✀᧯సࡢ≀⌮ⓗ࡞่⃭࡟ࡼࡾ⣽⬊࠿ࡽ┦ᙜ㔞ࡢ ATP ࡀศἪࡉࢀࠊATP ࢆࣜ࢞ࣥࢻ࡜ࡍࡿ P2Y2ཷᐜయ➼ࡢ࢔ࢵࢭ࢖࡟ࡣ୙౽࡛ᕤኵࢆせࡋࡓࡀࠊࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ࡜ྠ᫬࡟
᧛✀ࡋ࡚ 24 ᫬㛫ᚋ࡟ࣜ࢞ࣥࢻ่⃭ࡍࡿ᪉ἲ࡟ࡼࡾࡇࡢၥ㢟ࡣᨵၿࡉࢀࡓ(5)ࠋᮏ
ἲࢆ⏝࠸࡚ࠊP2Y2R ࡢ࣍ࣔࢲ࢖࣐࣮ᙧᡂ࡟࠾ࡅࡿࢪࢫࣝࣇ࢕ࢻᯫᶫࡢᐤ୚ࢆ᫂ࡽ
࠿࡟ࡋࡓ(5)ࠋ
 ⣽⬊ෆ࡟Ⓨ⌧ࡉࢀࡓ ǃ ࢞ࣛࢡࢺࢩࢲ࣮ࢮࢆ

ᐃࡍࡿࡓࡵ࡟ࡣࠊ⣽⬊⭷ࢆ⁐࠿ࡋ࡚ ᇶ ㉁ ࢆ ຍ ࠼ ࡿ ᚲ せ ࡀ ࠶ ࡿ ࠋ ᳨ ฟ ឤ ᗘ ࡶ ᚲ せ ࡞ ࡢ ࡛ Gal-Screen (Applied
Biosystems) ࡢࡼ࠺࡞Ⓨගᇶ㉁ࡀᚲせ࡛࠶ࡗࡓࠋࡇࡢᇶ㉁ࡣ኱ኚ㧗౯࡛࠶ࡿࡇ࡜
࡜࡜ࡶ࡟ࠊ96 well plate ࡜ࡢᖸ΅ࡀ࠶ࡗ࡚Ⓨගࡀ୙ᆒ୍࡟࡞ࡿ࡜࠸࠺ၥ㢟ࡀ࠶ࡗ
ࡓࠋ96 well plate ࡢ୙ᆒ୍ᛶࡣ ELISA ➼࡛ࡶ⤒㦂ࡍࡿࡇ࡜࡛ࠊ⌧ᅾࡢ〇㐀ᢏ⾡࡛ࡣ᏶඲࡟ࡣゎỴ࡛ࡁ࡞࠸ࡑ࠺ࡔࠋࡇࡢၥ㢟ࢆᨵၿࡍࡿࡓࡵࠊⓎගᇶ㉁࡟᥮࠼࡚
⺯ගᇶ㉁࡛࠶ࡿ 4-methylumbelliferone (4MU) ࡢ౑⏝ࢆヨࡳࡓࠋ⣽⬊⭷ࢆ⁐࠿ࡍ
ࡓࡵ࡟ SDS ࢆῧຍࡋࡓᇶ㉁⁐ᾮ࡜ࡍࡿࡔࡅ࡛ 4MU ࡢศゎࡀ
GalScreen ࡜ࡶ㐯Ⰽ࡞࠸ᐃ࡛ࡁࡓࠋᐃࡀྍ⬟࡛࠶ࡾࠊ96 well plate ࡜ࡢᖸ΅ࡶᑡ࡞࠿ࡗࡓࠋ

ࡇ࠺ࡋ࡚㧗౯࡛୙Ᏻᐃ࡞ᇶ㉁ࢆ⏝࠸ࡎ࡜ࡶ࢔ࢵࢭ࢖ࡀྍ⬟࡜࡞ࡗࡓࠋ
2. GPCR 㛫ぶ࿴ᛶ࡟ࡼࡿࣈࣛࢪ࢟ࢽࣥ B2 ཷᐜయࡢ⬡㉁ࣛࣇࢺ࡬ࡢࣜࢡ࣮ࣝࢺ
(6)
 GPCR ࡜⬡㉁ࣛࣇࢺࡢ㛵ಀࡣ࠸ࡃࡘ࠿▱ࡽࢀ࡚࠸ࡿࡀࠊATP/UTP ཷᐜయ P2Y2
(P2Y2R)ࡣ࡯ࡰࡍ࡭࡚ࡀ⬡㉁ࣛࣇࢺ࡟ᒁᅾࡍࡿ(7)ࠋࣈࣛࢪ࢟ࢽࣥ B2 ཷᐜయ(B2R)
ࡣࠊ⬡㉁ࣛࣇࢺ࡟ࡣ 25-30%⛬ᗘศ⏬ࡉࢀࡓࠋ୧⪅ࢆඹⓎ⌧ࡋࡓ࡜ࡇࢁࠊP2Y2R
ࡢศᕸࡣ୙ኚ࡛࠶ࡗࡓࡀࠊB2R ࡣࡼࡾከࡃࡀࣛࣇࢺ⏬ศ࡟⛣⾜ࡋࡓࠋࡇࡢࡇ࡜ࡣ
୧ཷᐜయࡢ┦஫స⏝ࡀ B2R ࢆ⬡㉁ࣛࣇࢺ࡟ࣜࢡ࣮ࣝࢺࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸
ࡿࠋ15 ศ㛫ࡢ UTP ่⃭ᚋࠊ୧⪅ࡣ⣽⬊⭷⾲㠃࠿ࡽ࡯࡜ࢇ࡝ᾘኻࡋ⣽⬊ෆ࡬⛣⾜ࡋࡓࠋ15 ศ㛫ࡢࣈࣛࢪ࢟ࢽ่⃭࡛ࣥࡣ୧⪅ࡢ㒊ศⓗ࡞࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫീࡀඹ
↔Ⅼ⺯ග㢧ᚤ㙾࡛ほᐹࡉࢀࡓࠋ௨๓ሗ࿌ࡋࡓࡼ࠺࡟(8)ࠊGT1b ࢞ࣥࢢࣜ࢜ࢩࢻ࡜
ࢥࣥࢻࣟ࢖ࢳࣥ◲㓟 C ࡛㐃⥆่⃭ࡍࡿ࡜ B2R ࡣ⣽⬊⭷⾲㠃࠿ࡽᾘኻࡍࡿ࡜࡜ࡶ
࡟ P2Y2R ࡶᾘኻࡋࡓࠋࡇࡢࡼ࠺࡟ࠊP2Y2R ࡜ B2R 㛫࡟ࡣࢩࢢࢼࣝࢡࣟࢫࢺ࣮ࢡ
ࡀᏑᅾࡋࠊ࢔ࣞࢫࢳࣥࡢࣜࢡ࣮ࣝࢺ࣭࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡛ᣲືࢆඹ࡟ࡍࡿࡀࠊ
ࡇࡢ୧⪅ࡢ┦஫స⏝ࡣ⬡㉁ࣛࣇࢺୖ࡛㉳ࡇࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋࡲࡓࠊ⣽⬊⾲㠃࡬⛣⾜࡛ࡁࡎከࡃࡣᑠ⬊య࡟␃ࡲࡿ P2Y2R ࡢ N ⤖ྜ⢾㙐Ḟᦆኚ␗యࡶࣛࣇ
ࢺ⏬ศ࡟ศ⏬ࡉࢀࡿ(7)ࡀࠊB2R ࡣࡇࡢኚ␗యࡢඹⓎ⌧࡟ࡼࡗ࡚ࡶࣛࣇࢺ⏬ศ࡟㞟✚ࡋࠊከࡃࡣኚ␗య࡜ྠᵝ࡟ᑠ⬊య࡟␃ࡲࡗࡓࠋࡋࡓࡀࡗ࡚ࠊᑠ⬊యࡢ⬡㉁ࣛࣇ
ࢺ࡟࠾࠸࡚୧⪅ࡣࡍ࡛࡟┦஫స⏝ࡋࠊࣛࣇࢺ࡟ぶ࿴ᛶࡢ㧗࠸ P2Y2R ࡟≌ᘬࡉࢀࡿࡇ࡜࡟ࡼࡗ࡚ B2R ࡣࣛࣇࢺ࡟⛣⾜ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ
27

3. ǃ2 ࢔ࢻࣞࢼࣜࣥཷᐜయ (ǃ2AR) ࢩࢢࢼࣝ࡟ࡼࡿࣄ࢔ࣝࣟࣥ㓟ྜᡂಁ㐍 (9, 10) ࣄ࢔ࣝࣟࣥ㓟㸦HA㸧ࡣࠊ⣽⬊እ࣐ࢺࣜࢵࢡࢫࡢᵓᡂศᏊ࡜ࡋ࡚ࠊ⤌⧊ࡢᙎᛶࡸ
≀ᛶ⥔ᣢ࡟㛵ࢃࡿ௚ࠊ๰യ἞⒵ࡸ⣽⬊ቑṪࢆไᚚࡍࡿࠋǃ సື⸆ࡣࠊẼ⟶ᨭࡢ⅖
⑕཯ᛂࢆ࡜ࡶ࡞࠺Ẽ⟶ᨭႍᜥࡢ἞⒪⸆࡜ࡋ࡚౑⏝ࡉࢀࡿࡀࠊ㛗ᮇ㐃⏝ࡣ⅖⑕཯ᛂࡢቑᝏࡸ࣒ࢳࣥࡢ⏘⏕ቑຍ࡜࠸ࡗࡓ๪స⏝ࡶᘬࡁ㉳ࡇࡍࠋࣄ࢔ࣝࣟࣥ㓟ࡣࢧ࢖ࢬ౫Ꮡⓗ࡟⅖⑕཯ᛂࡢㄏᑟࡸᢚไ࡟㛵୚ࡍࡿࡇ࡜࠿ࡽࠊǃ2AR ࢩࢢࢼࣝࡀࣄ࢔ࣝࣟ
ࣥ㓟ࡢྜᡂไᚚ࡟㛵୚ࡍࡿྍ⬟ᛶࢆ᳨ウࡋࡓࠋࡲࡎࠊǃ2AR Ⓨ⌧⣽⬊ࢆ ǃ సື⸆่࡛⃭ࡍࡿ࡜ࠊᇵ㣴ᾮ୰ࡢࣄ࢔ࣝࣟࣥ㓟㔞ࡀୖ᪼ࡋࡓࠋࡇࡢศᏊᶵᵓࢆ᳨ウࡋࡓ࡜ࡇࢁࠊǃ2AR ࡣ cAMP-PKA ࢩࢢࢼࣝࢆ௓ࡋ࡚ STAT3 ࡢ㌿෗άᛶࢆஹ㐍ࡉࡏࠊࣄ࢔ࣝࣟࣥ㓟ྜᡂ㓝⣲ 2㸦HAS2㸧ࡢ㑇ఏᏊⓎ⌧ࢆୖ᪼ࡉࡏ࡚࠸ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ
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Wehrman, T.S., Casipit, C.L., Gewertz, N.M., and Blau, H.M. (2005)
Enzymatic detection of protein translocation. Nat. Methods. 2, 521-527
Hammer, M.M., Wehrman, T.S., and Blau, H.M. (2007) A novel enzyme
complementation-based assay for monitoring G-protein-coupled
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von Degenfeld, G., Wehrman, T.S., Hammer, M.M., and Blau, H.M.
(2007) A universal technology for monitoring G-protein-coupled receptor
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3819-3826
Yashima, S., Shimazaki, A., Mitoma, J., Nakagawa, T., Abe, M., Yamada,H., and Higashi, H. (2015) Close association of B2 bradykinin receptors
with P2Y2 ATP receptors. J. Biochem., Tokyo. 158, 155-163
Abe, M., Watanabe, K., Kuroda, Y., Nakagawa, T., and Higashi, H.
(2018) Homodimer formation by the ATP/UTP receptor P2Y2 via
disulfide bridges. J. Biochem., Tokyo. 163, 475-480
Nakagawa, T., Takahashi, C., Matsuzaki, H., Kuroda, Y., and Higashi,
H. (2018) Regulation of membrane raft recruitment of the bradykinin
B2 receptor by close association with the ATP/UTP receptor P2Y2.
Biochem. Biophys. Res. Commun. 505, 36-39
Nakagawa, T., Takahashi, C., Matsuzaki, H., Takeyama, S., Sato, S.,
Sato, A., Kuroda, Y., and Higashi, H. (2017) N-glycan-dependentcell-surface expression of the P2Y2 receptor and N-glycan-independent
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Shimazaki, A., Nakagawa, T., Mitoma, J., and Higashi, H. (2012)28

9.

Gangliosides and chondroitin sulfate desensitize and internalize B2
bradykinin receptors. Biochem. Biophys. Res. Commun. 420, 193-198㯮⏣႐ᖾ㸪୰ᕝဴே㸪ᮾ ⚽ዲ, ǃ2 ࢔ࢻࣞࢼࣜࣥཷᐜయࢩࢢࢼࣝ࡟ࡼࡿࣄ
࢔ࣝࣟࣥ㓟ྜᡂࡢୖ᪼,10.

➨ 12 ᅇᮾ໭⢾㙐◊✲఍㸪ᘯ๓㸪2018 ᖺ 9 ᭶㯮⏣႐ᖾ㸪୰ᕝဴே㸪ᮾ ⚽ዲ,ศᏊᶵᵓ,ǃ సື⸆࡟ࡼࡿࣄ࢔ࣝࣟࣥ㓟⏘⏕ቑຍࡢ➨ 57 ᅇ᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊఍㸪௝ྎ㸪2018 ᖺ 10 ᭶29⢾㙐࡟ࡼࡿࡀࢇ⣽⬊ࡢᶵ⬟࡜୰ᯡ⚄⤒⣔࡟࠾ࡅࡿ⅖⑕཯ᛂࡢไᚚ
⣽⬊ไᚚ㒊㛛  㢳 ᘓᅜ⢾ࢱࣥࣃࢡ㉁ࡣ⢾㙐࡜⺮ⓑ㉁ࡀඹ᭷⤖ྜࡋࡓ⏕యศᏊ࡛࠶ࡿࠋ⺮ⓑ㉁ୖࡢ⢾㙐ࡢ⤖ྜᵝᘧࡣ୍⯡ⓗ࡟ࡣ N-ᆺ࡜ 0-ᆺ࡜࠸࠺㸰✀㢮ࡀ࠶ࡿࠋ⢾㙐ࡣࢱࣥࣃࢡ㉁ࡢ≀ᛶ࡟ᙳ㡪ࢆ୚࠼ࡿ࡜࡜ࡶ࡟ࢱࣥࣃࢡ㉁ࡢᶵ⬟ࠊࡑࡋ࡚ศᏊ㛫ࡢㄆ㆑ࡸ⣽⬊ෆࢩࢢࢼࣝఏ㐩࡟኱ࡁ࡞ᙺ๭ࢆᣢࡘࠋ⣽⬊⾲㠃ࡢ⢾㙐ࡣࠊ⣽⬊ቑṪࠊ⣽⬊᥋╔ࠊ⛣ື࠾ࡼࡧᾐ₶࡞࡝ከᩘࡢ⏕≀Ꮫⓗ㐣⛬࡟㛵୚ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋ࢖ࣥࢸࢢࣜࣥࡣ
Ș㙐࡜ș㙐࠿ࡽᡂࡿ࣊ࢸࣟ஧㔞య࡛ࠊ⣽⬊እ࣐ࢺࣜࢵࢡࢫ㸦ECM㸧࡬ࡢ⣽⬊ࡢ᥋╔ࢆ፹௓ࡍࡿ୺せ࡞⢾⺮ⓑ㉁࡛ࠊ⣽⬊ෆ⺮ⓑ㉁ࡢࢳࣟࢩࣥࣜࣥ㓟໬ࡸపศᏊ㔞
GTP ⤖ྜ⺮ⓑ㉁ࡢάᛶ໬ࢆ㏻ࡌ࡚⣽⬊ෆ㦵᱁⣔ࡢ෌⦅ᡂࡸቑṪ㺃ศ໬ࡢࢩࢢࢼࣝ
ఏ㐩࡟㛵ࢃࡿࠋࡲࡓࠊ࢖ࣥࢸࢢࣜࣥ࡜⣽⬊ቑṪᅉᏊཷᐜయ࡜ࡢ┦஫స⏝ࡣࠊ⣽⬊ࡢቑṪ࣭⛣ື࣭⏕Ꮡ࡞࡝ࡢ⏕⌮ⓗ࡞㐣⛬ࡢࡳ࡞ࡽࡎࠊࡀࢇ⣽⬊ࡢቑṪ࣭㌿⛣㺃ᾐ₶࡞࡝⑓⌮ⓗ࡞㐣⛬࡟ࡶ㔜せ࡞ᙺ๭ࢆᢸ࠺࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡇࢀࡲ࡛ࠊᡃࠎࡣศ
ᒱᆺ⢾㙐ࡢ⏕ྜᡂ࡟㛵ࢃࡿ N-࢔ࢭࢳࣝࢢࣝࢥࢧ࣑ࣥ㌿⛣㓝⣲ III (GnT-III)ࠊN࢔ࢭࢳࣝࢢࣝࢥࢧ࣑ࣥ㌿⛣㓝⣲ V (GnT-V)ࠊȘ1,6 ࣇࢥ࣮ࢫ(ࢥ࢔ࣇࢥ࣮ࢫ)㌿⛣㓝⣲ (Fut8)࠾ࡼࡧࢩ࢔ࣝ㓟㌿⛣㓝⣲࡞࡝ࡢᶵ⬟࡟ὀ┠ࡋࠊ⣽⬊᥋╔ศᏊ࡛࠶ࡿ࢖ࣥ
ࢸࢢࣜࣥࡀࣔࢹࣝศᏊ࡜ࡋ࡚◊✲ࢆ⾜ࡗ࡚ࡁࡓࠋࡑࡢ⤖ᯝࠊ⢾㙐ᵓ㐀ࡢࡳ࡞ࡽࡎ
⢾㙐௜ຍࢧ࢖ࢺࡶศᏊࡢᶵ⬟࡟㔜せ࡛࠶ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋᮏᖺᗘࡣࠊᡃࠎ
ࡣ⢾㙐⏕ྜᡂࡢ᪂ࡓ࡞ไᚚᶵᵓࡸ⢾㙐ࡢᶵ⬟࡟㛵ࡋ࡚୺࡟௨ୗࡢ㸲Ⅼ࡟ࡘ࠸࡚◊✲ࡋࡓࠋI. ࢖ࣥࢸࢢࣜࣥȘ㸱ș1 ࡜ PI4 ࢟ࢼ࣮ࢮ」ྜయ࡟ࡼࡿࢩ࢔ࣝ㓟௜ຍࡢไᚚ
⣽⬊⭷ࢱࣥࣃࢡ㉁ࡢࢩ࢔ࣝ㓟ቑຍࡣࠊࡀࢇ㌿⛣͌ᾐ₶࡟㛵㐃ࡍࡿ⌧㇟࡜ࡉࢀࠊ⣽⬊⾲㠃ࡢࢩ࢔ࣝ㓟ྵ㔞ࡀ㌿⛣⬟࡜┦㛵ࡍࡿ࡜࠸࠺ሗ࿌ࡶከࡃ࡞ࡉࢀ࡚࠸ࡿࠋࡑࡢከ
ࡃࡣࠊࢩ࢔ࣝ㓟ࢆ௜ຍࡍࡿࢩ࢔ࣝ㓟㌿⛣㓝⣲࡜ࢩ࢔ࣝ㓟ࢆ㝖ཤࡍࡿࢩ࢔ࣜࢲ࣮ࢮ
ࡢⓎ⌧࡟↔Ⅼࢆᙜ࡚࡚◊✲ࡋࡓ⤖ᯝ࡛࠶ࡿࠋGolgi phosphoprotein 3 (GOLPH3)ࡣࠊከࡃࡢᅛᙧ⒴࡟㧗㢖ᗘ࡛ቑᖜࡉࢀࠊࡀࢇࡢணᚋ࡜㈇࡟┦㛵ࡍࡿᙉຊ࡞ࡀࢇ㑇ఏᏊ࡛࠶ࡿࠋᡃࠎࡣࠊ௨๓ GOLPH3 ࡀࢩ࢔ࣝ㓟㌿⛣㓝⣲ࡢⓎ⌧ࡢㄪ⠇࡛ࡣ࡞ࡃࠊࡑࡢ㌿⛣㓝⣲ࡢാࡁຠ⋡ࢆୖ᪼ࡉࡏࠊ࢖ࣥࢸࢢࣜࣥ࡬ࢩ࢔ࣝ㓟ಟ㣭ࡢቑຍࠊࡀࢇ⣽⬊ࡢ㌿⛣͌ᾐ₶࡟ಁ㐍ⓗ࡟ാࡃ࡜࠸࠺඲ࡃ᪂つ࡞ศᏊᶵᵓࢆぢฟࡋ࡚࠸ࡿ㸦Isaji,
et al., JBC. 2014 㸧ࠋ ᭱ ㏆ ࠊ ࡉ ࡽ ࡟ GOLPH3 ࡢ PH ࢻ ࣓ ࢖ ࣥ ࡜ ⤖ ྜ ࡍ ࡿ
Phosphatidylinositol-4-phosphate (PI4P)࡟ὀ┠ࡋࠊPI4 ࢟ࢼ࣮ࢮࡀࢩ࢔ࣝ㓟ಟ㣭࡟ᚲ㡲࡛࠶ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊࡑࡢಟ㣭࡟ࡣ࢖ࣥࢸࢢࣜ
ࣥȘ5ș1 ࡛ࡣ࡞ࡃࠊȘ㸱ș1 ࡜ PI4 ࢟ࢼ࣮ࢮ㛫ࡢ┦஫స⏝ࡀᚲせ࡛࠶ࡿࡇ࡜ࡶ᫂
ࡽ࠿࡟࡞ࡗࡓ (1)ࠋȘ㸱ș1 ࡜ PI4 ࢟ࢼ࣮ࢮࡢ࡝ࡗࡕ࠿Ḟኻࡲࡓࡣࣀࢵࢡࢲ࢘ࣥ
ࡉࡏࡿ࡜ࢩ࢔ࣝ㓟ಟ㣭ࡀⴭࡋࡃపୗࡋࡓࠋ௒ᚋࠊ௚ࡢ⢾㙐ᵓ㐀ࡢ⏕ྜᡂ࡬ࡢᙳ㡪ࢆྵࡵࡓヲ⣽࡞ศᏊᶵᗎࢆゎ᫂ࡋ࡚⾜ࡃࠋ30

II. N-࢔ࢭࢳࣝࢢࣝࢥࢧ࣑ࣥ㌿⛣㓝⣲-I㸦GnT-I㸧ࡣୖ⓶㛫ⴥ㌿᥮(EMT)ࡢࢫ࢖ࢵ
ࢳ࡜ࡋ࡚ാࡃ
N-ᆺ⢾㙐ࡢᵓ㐀ࡢ≉ᚩ࠿ࡽ㧗࣐ࣥࣀ࣮ࢫᆺࠊΰᡂᆺ࠾ࡼࡧ」ྜᆺࡢ㸱✀ࡢ⢾㙐࡟኱ูࡉࢀ࡚࠸ࡿࠋࡇࢀࡲ࡛ࠊ」ྜᆺ N-ᆺ⢾㙐ࡢ࢖ࣥࢸࢢࣜࣥᶵ⬟࡟㛵ࡋ࡚ࡣ◊✲ࡉࢀ࡚ࡁࡓࡀࠊ㧗࣐ࣥࣀ࣮ࢫᆺ⢾㙐ࡋ࠿ᣢࡓ࡞࠸࢖ࣥࢸࢢࣜࣥࡢᶵ⬟࡟㛵ࡋ࡚ࡣ඲ࡃ୙࡛᫂࠶ࡿࠋᡃࠎࡣ㧗࣐ࣥࣀ࣮ࢫᆺ࡜」ྜᆺ࠾ࡼࡧࣁ࢖ࣈࣜࢵࢻᆺࡢ N-ᆺ⢾㙐ࡢᶵ⬟ࢆ᫂☜࡟༊ูࡍࡿࡓࡵ࡟ࠊ㧗࣐ࣥࣀ࣮ࢫᆺࡢ⢾㙐ࡋ࠿సࢀ࡞࠸ N-࢔ࢭࢳࣝࢢࣝࢥࢧ࣑ࣥ㌿⛣㓝⣲-I㸦GnT-I㸧Ḟኻ⣽⬊㸦KO㸧ࢆᶞ❧ࡋࠊゎᯒࡋࡓ(2)ࠋࡑ
ࡢ⤖ᯝ GnT-I KO ⣽⬊࡛ࡣࠊ⣽⬊จ㞟ࡀㄏᑟࡉࢀࠊࡉࡽ࡟⣽⬊⛣ືࡀⴭࡋࡃῶᑡ
ࡍࡿࡇ࡜ࡀศ࠿ࡗࡓࠋࡲࡓ⯆࿡῝࠸ࡇ࡜࡟ࠊ」ྜᆺ⢾㙐ࡢ႙ኻ࡟ࡼࡾ㛫ⴥ⣔⣽⬊࣐࣮࣮࢝ࡢపୗ࠾ࡼࡧୖ⓶⣔࣐࣮࣮࢝ࡢቑຍࡀㄆࡵࡽࢀࡓࡇ࡜࠿ࡽࠊ⣽⬊࡟㛫ⴥୖ⓶㌿᥮(MET)ࡀ⏕ࡌ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ୍᪉ࠊKO ⣽⬊࡟ GnT-I ࢆⓎ⌧ࡉࡏࡓ Rescue ⣽⬊࡛ࡣࠊ⣽⬊จ㞟ࡣᢚไࡉࢀࠊࡲࡓ MET ࡜ࡣ㏫ࡢୖ⓶㛫ⴥ㌿᥮㸦EMT㸧࣐࣮࣮࢝ࡢⓎ⌧࠾ࡼࡧࡀࢇࡢᝏᛶ໬࡟㛵୚ࡍࡿ㠀ཷᐜయᆺࢳࣟࢩࣥ
࢟ࢼ࣮ࢮ࡛࠶ࡿ FAK ࡢάᛶࡀቑᙉࡍࡿࡇ࡜ࢆ☜ㄆࡋࡓ(ᅗ㸯)ࠋࡍ࡞ࢃࡕ GnT-Iࡣࠊࡀࢇ⣽⬊࡟࠾ࡅࡿᙧែࠊ᥋╔⬟࠾ࡼࡧ㐠ື⬟ࢆࡼࡾᝏᛶᗘࡢ㧗࠸≧ែ࡟⥔ᣢࡍࡿࡇ࡜࡟㈉⊩ࡋ࡚࠾ࡾࠊࡇࢀ࡟ࡣ⣽⬊᥋╔ศᏊ࡛࠶ࡿș1 ࢖ࣥࢸࢢࣜࣥࡀࡶࡘ
」ྜᆺ N-ᆺ⢾㙐ࡀ῝ࡃ㛵୚ࡋ࡚࠸ࡿࡇ࡜
ࡀ♧၀ࡉࢀࡓࠋࡉࡽ࡟ GnT-I ࡢⓎ⌧ࡀࠊ⣽⬊⏕Ꮡ࠾ࡼࡧᢠࡀࢇ๣࡟ᑐࡍࡿឤཷᛶ࡟㔜せ࡛࠶ࡿࡇ࡜ࡶศ࠿ࡗࡓࠋᮏ◊✲ࡣࠊ㧗࣐ࣥࣀ࣮ࢫᆺ⢾㙐ࡀ୺࡟⣽⬊㛫᥋╔࡟ᐤ୚ࡋࠊ」ྜᆺ⢾㙐ࡀ ECM ᥋╔ࡸ⒴ࡢ
ᝏᛶ໬ࡸ㌿⛣࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡍ
EMT ࡟ാࡃࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋGnT-Iࡀᢠࡀࢇ἞⒪ࡢ᪂ࡓ࡞ᶆⓗ࡜࡞ࡿྍ⬟ᛶࢆ♧ࡋࡓࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ
III. O-GlcNAc ௜ຍ࡟ࡼࡿ࢖ࣥࢸࢢࣜࣥࡢ inside-out ࢩࢢࢼࣝไᚚ
ࢱࣥࣃࢡ㉁ࡢ୺せ࡞⢾㙐ಟ㣭ࡣ N-ᆺ࡜ O-ᆺ⢾㙐ಟ㣭ࡔࡀࠊࡇࢀࡽ࡜ࡣู࡟᰾ࡸ⣽⬊㉁࡟Ꮡᅾࡍࡿࢱࣥࣃࢡ㉁࡟ N-࢔ࢭࢳࣝࢢࣝࢥࢧ࣑ࣥ(GlcNAc)ࡀ 1 ศᏊ⤖ྜࡍࡿ O-GlcNAc ໬ࡀ࠶ࡿࠋO-GlcNAc ໬ࡣࢱࣥࣃࢡ㉁ࡢಟ㣭ࡉࢀࡿ㒊఩ࡀࢭࣜࣥ
ࡲࡓࡣࢫࣞ࢜ࢽࣥṧᇶࡢỈ㓟ᇶ࡛࠶ࡿࡇ࡜࡟ຍ࠼ࠊO-GlcNAc ㌿⛣㓝⣲(OGT)࡟
ࡼࡾ㌿⛣ࠊO-GlcNAc ษ᩿㓝⣲ O-GlcNAcase(OGA)࡟ࡼࡾຍỈศゎࡉࢀࡿࡓࡵࠊ
ࣜࣥ㓟໬࡜ྠᵝ࡟ࢲ࢖ࢼ࣑ࢵࢡ࡟ኚ໬ࡍࡿࡇ࡜ࡀฟ᮶ࡿࠋᚑࡗ࡚ࠊO-GlcNAc ໬ࡣ⺮ⓑ㉁ࡢ㔜せ࡞ᶵ⬟ไᚚᶵᵓࡢ୍ࡘ࡛࠶ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋᐇࡣࠊO-GlcNAc
ࡣࠊከᩘࡢ⒴⤌⧊࡟࠾࠸࡚ୖ᪼ࡋࠊ⣽⬊ቑṪ࣭⏕Ꮡࡸ㌿⛣࡟㛵ࢃࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࡀࠊ࡝ࡢࡼ࠺࡟⒴⣽⬊ࡢ᥋╔࣭ᾐ₶ࢆㄪ⠇ࡍࡿࡢ࠿୙᫂Ⅼࡀከ࠸ࠋᡃࠎࡣࠊ
OGT ࣀࢵࢡࢲ࢘ࣥ⣽⬊ࢆᶞ❧࣭ゎᯒࡋࠊO-GlcNAc ࡀ⣽⬊᥋╔ࠊ⛣ື࠾ࡼࡧ᥋╔ᩬ㸦FA㸧」ྜయࡢ㔜せ࡞ㄪ⠇ᅉᏊ࡛࠶ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ(3)ࠋ OGT ࡢࣀࢵࢡ31ࢲ࢘ࣥ㸦KD㸧࡟ࡼࡗ࡚ࠊࣇ࢕ࣈࣟࢿࢡࢳࣥୖࡢ⣽⬊᥋╔ࡣ᭷ព࡟ಁ㐍ࡉࢀࡓࡀࠊ
୍᪉࡛ࠊ⣽⬊⛣ືࡣᢚไࡉࢀࡿഴྥࢆ♧ࡋࡓࠋᑐ↷⣽⬊࡜ẚ࡭࡚ KD ⣽⬊࡟࠾ࡅࡿ⣽⬊᥋╔ᩬࡀ᭷ព࡟ቑຍࡋࡓࠋ⣽⬊᥋╔ᩬ࡟ࡣࠊ࢖ࣥࢸࢢࣜࣥ௨እ࡟ Focal
adhesion kinase (FAK)ࠊࣃ࢟ࢩࣜࣥཬࡧࢱ
ࣜࣥ࡞࡝ศᏊࡀ㞟✚ࡋ」ྜయࡀᙧᡂࡉࢀࡿࠋ
KD ⣽⬊࡟ࡣࡑࢀࡽࡢ」ྜయࡢ㐣ᙧᡂࡀほᐹࡉࢀࡓࠋ༶ࡕࠊ᥋╔ᩬࡢ㐣๫࡞ᡂ⇍໬ࡀ
KD ⣽⬊࡛⏕ࡌ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
ࣃ࢟ࢩࣜࣥࡸࢱࣜࣥ࡬ࡢ O-GlcNAc ௜ຍࡀ☜ㄆࡉࢀࡓࠋࡉࡽ࡟ࠊࡇࢀࡲ࡛࡟ሗ࿌ࡀ࡞࠿
ࡗࡓ FAK ࡬ࡢ O-GlcNAc ௜ຍࡶึࡵ࡚ぢฟࡋࡓࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊO-GlcNAc ໬ࡣ᥋╔ᩬࡢ」ྜయᙧᡂ࡟㔜せ࡛࠶ࡾࠊ࢖ࣥࢸࢢࣜࣥ
ࢆ௓ࡋࡓ⣽⬊᥋╔࣭⛣ື࡞࡝ࡢᶵ⬟࡟᪂つ࡞ㄪ⠇ᶵᵓ㸦࢖ࣥࢸࢢࣜࣥࡢ inside-out ࢩࢢࢼࣝ㸧࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡿ㸦ᅗ㸰㸧ࠋFAK ࡟ࡣࣜࣥ㓟໬ࡉࢀࡿࢭࣜࣥࠊࢫ
ࣞ࢜ࢽࣥṧᇶࡀከᩘᏑᅾࡍࡿࡢ࡛ࠊO-GlcNAc ࡀࡇࢀࡽ࡜➇ྜࡍࡿ࠿ࡲࡓࡣ≉␗ⓗ࡟ಟ㣭ࡍࡿ࠿ゎᯒࡍࡿᚲせࡀ࠶ࡿࠋIV. ⚄⤒⅖⑕࡟࠾ࡅࡿࢥ࢔ࣇࢥ࣮ࢫࡢᶵ⬟ࢥ࢔ࣇࢥ࣮ࢫࡣ⏕యෆ࡛⬻⤌⧊࡟᭱ࡶ㧗ࡃⓎ⌧ࡋ࡚࠸ࡿࠋࢥ࢔ࣇࢥ࣮ࢫ㌿⛣㓝⣲(Fut8)ࢆḞᦆࡉࡏࡓ࣐
࢘ࢫࡣࠊ␗ᖖ࡞࣍ࢵࣆࣥࢢࠊㄆ▱ᶵ⬟ཬࡧᾏ㤿ࢽ࣮ࣗࣟࣥࡢ㛗ᮇቑᙉ
(LTP)ࡢῶᙅࢆྵࡵࡓ⤫ྜኻㄪ⑕ᵝ࡞⑕≧ࢆ♧ࡍࠋ᭱㏆ࠊᡃࠎࡣࠊࢥ࢔
ࣇࢥ࣮ࢫ࡜⚄⤒⅖⑕཯ᛂࡢ㛵ಀ࡟ὀ┠ࡋ࡚ゎᯒࢆ⾜ࡗࡓ(4)ࠋFut8 Ḟᦆ࣐࢘ࢫࡢ⬻⤌⧊ࢆࢢࣜ࢔⣽⬊ࡢ୍ࡘ࡛ࠊ୰ᯡࡢච␿ᢸᙜ⣽⬊࡜ࡋ࡚▱ࡽࢀࡿ࣑ࢡࣟࢢࣜ࢔ࡢ࣐࣮࣮࢝ᢠ Iba1 ᢠయ࡛ᰁⰍࡋࡓ࡜ࡇࢁࠊ㔝⏕ᆺࡼࡾ࣑ࢡࣟࢢࣜ࢔ࡢࢧ࢖ࢬ࣭ᩘ࡜ࡶ࡟᭷ព࡟ቑຍࡋ࡚
࠸ࡓࠋࡇࢀࡣࠊFut8 Ḟᦆ࣐࢘ࢫࡢ࣑ࢡࣟࢢࣜ࢔ࡣ่⃭ࢆకࢃ࡞࠸≧ἣ࡛άᛶ໬ࡋ
࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋࡲࡓࠊච␿ᢸᙜ⣽⬊άᛶ໬స⏝ࢆࡶࡘ࣏ࣜከ⢾(LPS)ࢆ⭡⭍ෆ࡟ᢞ୚ࡍࡿ࡜ Fut8 Ḟᦆ࣐࢘ࢫࡣ㔝⏕ᆺ࣐࢘ࢫࡼࡾឤཷᛶࡀ㧗ࡲࡗ࡚࠸ࡓࠋ࣑ࢡࣟࢢࣜ࢔ࡢ⣽⬊ᰴ BV2 ⣽⬊ࢆ⅖⑕ᛶࢧ࢖ࢺ࢝࢖ࣥ IFN-Ț่࡛⃭ࡍࡿ࡜ࢥ࢔ࣇࢥ࣮ࢫࡢⓎ⌧ࡀ⃰ᗘ౫Ꮡⓗ࡟ቑຍࡋࡓࠋࡉࡽ࡟ࠊࢥ࢔ࣇࢥ࣮ࢫ࡜࣑ࢡࣟࢢ
ࣜ࢔άᛶ໬ࡢ㛵㐃ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊFut8 Ḟᦆ BV2 ⣽⬊ࢆసᡂࡋࠊIFN-Ț࡟ࡼࡿ iNOS ࡢⓎ⌧ㄏᑟࢆ᳨ウࡋࡓ࡜ࡇࢁࠊ㔝⏕ᆺࡼࡾ Fut8 Ḟᦆ BV2 ⣽⬊ࡢ࡯࠺32ࡀࡼࡾ㢧ⴭ࡛࠶ࡗࡓࠋࢢࣜ࢔⣽⬊ࡢ୍ࡘ࡛࠶ࡿ࢔ࢫࢺࣟࢧ࢖ࢺࡶ࣑ࢡࣟࢢࣜ࢔࡜
࡜ࡶ࡟ࠊ⅖⑕࣓ࢹ࢕࢚࣮ࢱ࣮ࡸ⅖⑕ಁ㐍ᛶࢧ࢖ࢺ࢝࢖ࣥࢆ⏘⏕ࡍࡿࡢ࡛ࠊC6 ࢢࣜ࢜ࣈࣛࢫࢺ࣮࣐⣽⬊ࢆ⏝࠸᳨࡚ウࡋࡓ࡜ࡇࢁࠊ㔝⏕ᆺࡼࡾ Fut8 Ḟᦆ C6 ⣽⬊ࡢ࡯࠺ࡀ IL-6/STAT3 ࢩࢢࢼࣝఏ㐩⤒㊰ࡀάᛶ໬ࡉࢀࡿࡇ࡜ࡀࢃ࠿ࡗࡓࠋ㔝⏕ᆺࡢึ௦ᇵ㣴࢔ࢫࢺࣟࢧ࢖ࢺ࡟ L-ࣇࢥ࣮ࢫࡢ࢔ࢼࣟࢢ࡛࠶ࡿ 2-fluorofucose (2FF) ࢆῧຍࡍࡿࡇ࡜࡟ࡼࡾࠊࣇࢥࢩࣝ໬ࢆᢚไࡍࡿ࡜ࠊC6 ⣽⬊࡜ྠᵝࡢ⤖ᯝࡀᚓࡽࢀࡓࠋ
ࡇࢀࡽࡢ⤖ᯝࡣࠊࢥ࢔ࣇࢥࢩࣝ໬ࡢḞᦆ࡟ࡼࡾࢢࣜ࢔⣽⬊ࡀᐜ᫆࡟άᛶ໬≧ែ࡟࡞ࡿࡇ࡜ࡍ࡞ࢃࡕࠊࢥ࢔ࣇࢥ࣮ࢫࡣࢢࣜ࢔⣽⬊ࡢάᛶ໬ࡢᢚไ࡟ാࡃࡇ࡜ࢆ♧ࡋ࡚࠾ࡾ㸦ᅗ㸱㸧ࠊLTP ࡢῶᙅ࡞࡝ Fut8 Ḟᦆ࣐࢘ࢫࡢ⬻⚄⤒࡟ぢࡽࢀࡿ㞀ᐖࡣࠊࢽ࣮ࣗࣟࣥࡔࡅ࡛ࡣ࡞ࡃࢥ࢔ࣇࢥ࣮ࢫࡀ↓ࡃ࡞ࡿࡇ࡜࡟ࡼࡾࠊࢢࣜ࢔⣽⬊ࡢάᛶ໬ࡢᢚไࡀゎ㝖ࡉࢀࡓࡇ࡜࡟㉳ᅉࡋࡓࡶࡢ࡛࠶ࡿࡇ࡜ࢆᙉࡃ♧၀ࡋ࡚࠸ࡿࠋ௒ᚋࠊ
Fut8 ࡀᇶ㉁࡜ࡍࡿࢱࣥࣃࢡ㉁ࢆ௓ࡋࡓ⅖⑕཯ᛂ࡟㛵ࡋ࡚ࡼࡾヲ⣽࡞◊✲ࢆ⾜࠺ࡇ࡜࡟ࡼࡾࠊ⤫ྜኻㄪ⑕࡞࡝ࡢ⑓ែゎᯒࡀ኱࠸࡟㐍ᒎࡍࡿ࡛࠶ࢁ࠺ࠋ
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33ࣞࢡࢳࣥࡢᢠ⭘⒆࣓࢝ࢽࢬ࣒ࡢゎ᫂࡜ࡀࢇ⸆≀἞⒪࡬ࡢᛂ⏝ศᏊㄆ㆑㒊㛛  ⣽㔝 㞞♸
㸦㸯㸧Raji ⣽⬊࡟࠾ࡅࡿࢼ࣐ࢬ༸ࣞࢡࢳࣥ㸦SAL㸧ྲྀࡾ㎸ࡳᶵᵓࡢゎ᫂ ࡇࢀࡲ࡛࡟ SAL ࡣ㸪ᾋ㐟⣔ࡢࣄࢺࣂ࣮࢟ࢵࢺࣜࣥࣃ⭘⣽⬊ᰴ Raji ࡢ⣽⬊⭷࡟
㧗Ⓨ⌧ࡋ࡚࠸ࡿࢢࣟ࣎ࢺࣜ࢔࢜ࢩࣝࢭ࣑ࣛࢻ㸦Gb3㸧ࡢ⢾㙐࡟⤖ྜࡍࡿࡇ࡜࡟ࡼ
ࡾ㸪MEK-ERK ⤒㊰ࡢάᛶ໬࡟㉳ᅉࡍࡿ p21 ࡢⓎ⌧ቑຍࢆ௓ࡋ࡚⣽⬊ቑṪᢚไࢆᘬࡁ㉳ࡇࡍࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ㸬ࡋ࠿ࡋ㸪Gb3 ࡟⤖ྜࡋࡓ SAL ࡀ⣽⬊⭷ୖ࡟␃ࡲࡿࡢ࠿㸪࠶ࡿ࠸ࡣ⣽⬊ෆ࡬⛣⾜ࡍࡿ࠿ࡣ᫂ࡽ࠿࡛࡞࠿ࡗࡓ㸬ᮏᖺᗘࡣࡇࡢⅬ࡟╔┠ࡋ㸪HiLyte FluorTM 555 Labeling kit ࢆ⏝࠸࡚⺯ගᶆ㆑ SAL㸦HL-SAL㸧ࢆస〇ࡋ㸪ඹ↔Ⅼ࣮ࣞࢨ࣮㢧ᚤ㙾࡞ࡽࡧḟୡ௦ᆺࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮
ImageStreamX Mark II ࢆ⏝࠸᳨࡚ウࡋࡓ㸬HL-SAL㸦50 μg/mL㸧࡛ 24 ᫬㛫ฎ⌮ࡋࡓ⣽⬊ࢆほᐹࡋࡓ࡜ࡇࢁ㸪Raji ࡢ⣽⬊⭷࠾ࡼࡧ⣽⬊ෆ㸪≉࡟᰾㏆ഐ࡟ SALࡀ㞟✚ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸬⣽⬊ෆࡢ⺯ගࡣ㸪⭷࡟⤖ྜࡋࡓ HL-SAL ࢆࣁࣉࢸࣥ⢾࡟ࡼࡾ㝖ཤࡋࡓᚋࡶṧᏑࡋ࡚࠸ࡓ㸬ࡲࡓ HL-SAL ࡣ㸪ฎ⌮ 10 ศᚋ࡟ࡣ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿࡇ࡜ࡀ♧ࡉࢀ㸪ࡑࡢᚋ 24 ᫬㛫ࡲ࡛᫬㛫౫Ꮡⓗ࡟⣽⬊ෆࡢ⺯ග㔞ࡀቑຍࡋࡓ㸬ࡉࡽ࡟㸪Gold Nanoparticle (20 nm)࡛㔠ᶆ㆑ࡋࡓ SAL ࢆ⏝࠸㸪㉮ᰝᆺ㟁Ꮚ㢧ᚤ㙾࡛ほᐹࡋࡓ⤖ᯝ࠿ࡽ㸪SAL ࡣ⣽⬊㉁ෆ࡛ࡣᾮ⬊ᵝࡢ⭷ᵓ㐀≀ࡢෆ㒊࡟ྲྀࡾ㎸ࡲࢀ࡚࠸ࡿྍ⬟ᛶࡀ♧ࡉࢀࡓ㸬ࡇࡢ⤖ᯝ࠿ࡽ㸪SAL ࡣ Gb3
࡟⤖ྜࡋࡓᚋ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡛ྲྀࡾ㎸ࡲࢀࡿࡢ࡛ࡣ࡞࠸࠿࡜⪃࠼㸪ࢡࣛࢫࣜࣥ௓ᅾᆺ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ㜼ᐖ๣ Pitstop2㸪⬡㉁ࣛࣇࢺ௓ᅾᆺ࢚ࣥࢻࢧ࢖ࢺ࣮
ࢩࢫ㜼ᐖ๣࡛࠶ࡿ࣓ࢳࣝ-E-ࢩࢡࣟࢹ࢟ࢫࢺࣜࣥ㸦MECD㸧࠾ࡼࡧ࢚ࣥࢻࢧ࢖ࢺ࣮
ࢩࢫ᫬࡟⣽⬊⭷࠿ࡽᑠ⬊యࢆศ㞳ࡍࡿ㝿࡟ᶵ⬟ࡍࡿࢲ࢖ࢼ࣑ࣥࢆ㜼ᐖࡍࡿ
Dynago-4a ࠾ࡼࡧ Dynasore ࢆ⏝࠸᳨࡚ウࡋࡓ㸬MECD㸪Dynago-4a ࠾ࡼࡧ
Dynasore ࡛๓ฎ⌮ࡍࡿࡇ࡜࡟ࡼࡾ HL-SAL ࡢྲྀࡾ㎸ࡳࡀ㜼ᐖࡉࢀࡓ୍᪉㸪
Pitstop2 ๓ฎ⌮࡛ࡣ㜼ᐖࡉࢀ࡞࠿ࡗࡓ㸬ࡇࢀࡽࡢ⤖ᯝ࠿ࡽ㸪SAL ࡣ Raji ⣽⬊⭷࡟Ꮡᅾࡍࡿ Gb3 ࡟⤖ྜࡋ㸪⬡㉁ࣛࣇࢺࢆ௓ࡋࡓ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡟ࡼࡾ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ㸬㸦㸰㸧*E ᳨ฟ࡟฼⏝ࡍࡿᢠ *E ᢠయ㸦$㸧ࡢㄪ〇࡟ࡘ࠸࡚
 SAL ࡢ⣽⬊⭷ࣞࢭࣉࢱ࣮࡛࠶ࡿ *E ࡢ᳨ฟ࡟ࡣ㸪⌧ᅾᕷ㈍ࡉࢀ࡚࠸ࡿ࣐࢘ࢫࣔ
ࣀࢡ࣮ࣟࢼࣝᢠయ %*5㸦,J*㸧࠾ࡼࡧࣛࢵࢺࣔࣀࢡ࣮ࣟࢼࣝᢠయ࡛࠶ࡿ 
㸦,J0㸧ࡀỗ⏝ࡉࢀ࡚࠾ࡾ㸪ࡑࢀࡒࢀ *E ࡟ᑐࡍࡿ཯ᛂᛶࡀ␗࡞ࡿ㸬୍᪉࡛㸪⟽Ᏺࡽ࡟ࡼࡾᶞ❧ࡉࢀࡓ࣐࢘ࢫࣔࣀࢡ࣮ࣟࢼࣝᢠయ $㸦,J0㸧ࡶ୍㒊ࡢ◊✲ᐊ࡛฼⏝ࡉࢀ࡚࠸ࡿ㸬௒ᅇ㸪ࡼࡾ཯ᛂᛶࡢ㧗࠸ᢠ *E ᢠయࢆຠ⋡ࡼࡃྲྀᚓࡍࡿࡓࡵ࡟
$ ࢆ⏘⏕ࡍࡿࣁ࢖ࣈࣜࢻ࣮࣐ࢆᘯ๓኱Ꮫ་Ꮫ㒊Ἢᒀჾ⛉Ꮫㅮᗙ࠿ࡽ౪୚࠸ࡓ
ࡔࡁ㸪ᢠయࡢ⢭〇᪉ἲ࡜㈓ἲ࡟ࡘ࠸᳨࡚ウࡋࡘࡘࡋࡓ *E ࡟ᑐࡍࡿ཯ᛂᛶࢆ
34%*5 ࡜ẚ㍑ࡋࡓ㸬 ࣁ࢖ࣈࣜࢻ࣮࣐ࡢᇵ㣴ୖΎࢆ 3URWHLQ /$JDURVH ࣒࢝ࣛࢆ㏻
ࡍࡇ࡜࡟ࡼࡾᢠయࢆ྾╔ࡉࡏ㸪 0 ࢢࣜࢩࣥሷ㓟ሷ㸦S+ 㸧࡟ࡼࡾ⁐ฟࡉࡏࡓ㸬ᢠయࡢ཯ᛂᛶࡣ㸪*E 㝧ᛶ⣽⬊࡜ࡋ࡚ 5DML ࠾ࡼࡧ⭈ࡀࢇ⣽⬊ᰴ 726 ࢆ㸪ྠ㝜ᛶ⣽⬊࡜ࡋ࡚៏ᛶ㦵㧊ᛶⓑ⾑⑓⣽⬊ᰴ . ࢆ⏝࠸࡚ࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜἲ
࡟ࡼࡾ᳨ウࡋࡓ㸬ࡣࡌࡵ࡟ . ࡟ࡣ཯ᛂࢆ♧ࡉࡎ㸪5DML ࡟ᑐࡋ࡚཯ᛂࡍࡿ $ࡢ⃰ᗘ࡞ࡽࡧ࡟⺯ගᶆ㆑㸰ḟᢠయࡢ᭱㐺࡞᮲௳ࢆ᳨ウࡋࡓ㸬⺯ගᙉᗘࡢศ㞳ࡢᗘྜ࠸࠿ࡽ㸪$ ࡢ⃰ᗘࢆ  JP/ ࡜ࡋ㸪⺯ගᶆ㆑㸰ḟᢠయࢆ  ಸᕼ㔘࡜ࡍࡿ࡜㸪*E ࢆ᳨ฟࡍࡿ㝿ࡢឤᗘ࡜ࡋ࡚㐺ษ࡞᮲௳࡛࠶ࡿࡇ࡜ࡀศ࠿ࡗࡓ㸬5DML ࡟ᑐࡋ $  JP/ ࡛ᚓࡽࢀࡿ⺯ගᙉᗘࡣ㸪%*5 ࡛ࡣ  JP/ ࡟┦ᙜࡋ㸪㔜㔞ẚ࡜ࡋ࡚ $ ࡣ࠾ࡼࡑ  ಸࡢ཯ᛂᛶࢆ♧ࡋࡓ㸬*E ࡢⓎ⌧㔞ࡀ 5DML ࡼࡾప࠸ 726࡟ᑐࡋ࡚ࡣ㸪$㸦JP/㸧࡜ྠ⛬ᗘࡢ⺯ගᙉᗘࢆᚓࡿࡢ࡟ %*5 ࡣ JP/
ࢆせࡋࡓ㸬㈓ⶶ᪉ἲ࡟㛵ࡋ࡚ࡣ㸪෾⤖ಖᏑࡋ࡞࠸ሙྜ㸪 ࢝᭶⛬ᗘ࡛࠶ࢀࡤ㸪ࢫࣆ࣒ࣥ࢝ࣛ⢭〇ᚋ୰࿴ฎ⌮ࢆ⾜࠸㸪ࡑࡢࡲࡲ㸦㧗ሷ⃰ᗘ⦆⾪ᾮ୰㸧ಖᏑࡋࡓ᪉ࡀ㸪཯ᛂᛶࡀపୗࡋ࡞࠸ࡇ࡜ࡀศ࠿ࡗࡓ㸬 㸦㸱㸧࢘ࢩ࢚࢞ࣝ༸⏤᮶ࢩ࢔ࣝ㓟⤖ྜᛶࣞࢡࢳࣥ (cSBL)ࡢஙࡀࢇ⣽⬊࡟ᑐࡍࡿᢠ⭘⒆స⏝cSBL ࡣ㸪ࣞࢡࢳࣥάᛶ࠾ࡼࡧࣜ࣎ࢾࢡࣞ࢔࣮ࢮάᛶࢆ᭷ࡍࡿከᶵ⬟ࢱࣥࣃࢡ㉁࡛࠶ࡾ㸪ࡀࢇ⣽⬊࡟⣽⬊Ṛࢆㄏᑟࡍࡿ㸬ᡃࠎࡣࡇࢀࡲ࡛㸪cSBL ࡢᝏᛶ⬚⭷୰⓶⭘㸦MPM㸧࡟ᑐࡍࡿ௚๣࡜ࡢే⏝ຠᯝ࡟ࡘ࠸࡚᫂ࡽ࠿࡟ࡋ࡚ࡁࡓ㸬ᮏᖺᗘࡣ㸪✀ࠎࡢஙࡀࢇ⣽⬊࡟ᑐࡍࡿᢠ⭘⒆ຠᯝ࡜ HER ࣇ࢓࣑࣮ࣜศᏊࡢⓎ⌧࡟ཬࡰࡍຠᯝ࡟࠾࠸࡚ᚓࡓ᪂ࡋ࠸▱ぢࢆㄽᩥⓎ⾲ࡋࡓ㸬ஙࡀࢇࡣ㸪ศᏊⓎ⌧࡟ከᵝᛶࢆ♧ࡍ⑌ᝈ࡛࠶ࡾ㸪୺࡟࢚ࢫࢺࣟࢤࣥཷᐜయ㸦ER㸧㸪
ࣉࣟࢤࢫࢸࣟࣥཷᐜయ (PgR)㸪ࣄࢺୖ⓶ᡂ㛗ᅉᏊཷᐜయ 2㸦HER2㸧ࡢⓎ⌧࡟ᇶ࡙࠸࡚ศ㢮ࡉࢀ࡚࠸ࡿࡀ㸪ࡇࢀࡽཷᐜయࡢ㐣๫Ⓨ⌧ࡲࡓࡣ㑇ఏᏊቑᖜࡢ᭷↓࡟ࡼࡗ࡚ணᚋࡸ㑅ᢥ἞⒪ࡀኚࢃࡗ࡚ࡃࡿ㸬ࡇࢀࡲ࡛࡟ሗ࿌ࡋ࡚ࡁࡓ ER ࡸ Bcl-2 ࠾ࡼࡧ MAPK ࡢⓎ⌧࣭άᛶ໬࡟ᑐࡍࡿ cSBL ࡢຠᯝ࡟╔┠ࡋ㸪cSBL ฎ⌮ஙࡀࢇ⣽⬊࡟࠾ࡅࡿ㘽ศᏊ㸦ER㸪PgR ࠾ࡼࡧ HER ࣇ࢓࣑࣮ࣜศᏊ㸧ࡸ Bcl-2 ࣇ࢓࣑ࣜ ࣮ ศ Ꮚ ࠾ ࡼ ࡧ MAPK ࡢ Ⓨ⌧࣭άᛶ໬ࡢㄪᰝࢆ⾜ࡗࡓ㸬ࡑࡢ⤖ᯝ㸪p38 ࡢάᛶ໬ࡣ㸪cSBL
ࢆฎ⌮ࡋࡓஙࡀࢇ⣽⬊࡛ᙉࡃほᐹࡉࢀ㸪cSBL ࡢᢠ⭘⒆స⏝࡟῝ࡃ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡓ㸬Bcl-2 ࣇ࢓࣑࣮ࣜศᏊ࡟
ᑐࡍࡿ cSBL ࡢᙳ㡪ࡣ⣽⬊ࡢ✀㢮࡟ࡼࡗ࡚␗࡞ࡿྍ⬟ᛶࡀ࠶
35ࡿ࡜⪃࠼ࡽࢀࡓ㸬୍᪉㸪ஙࡀࢇ㛵㐃ศᏊ㸦ER㸪PgR ࠾ࡼࡧ HER2㸧ࡢⓎ⌧ࡣ㸪࠸ࡎࢀࡶ cSBL ฎ⌮࡟ࡼࡾῶᑡࡋࡓ㸬ࡉࡽ࡟㸪ከᩘࡢ⣽⬊࡟࠾࠸࡚㸪ࡑࢀࡒࢀ࡟
Ⓨ⌧ࡍࡿ඲࡚ࡢ HER ࣇ࢓࣑࣮ࣜศᏊࡀῶᑡࡋ㸪cSBL ࡣ⣽⬊✀࡟㛵ࢃࡽࡎ
HER ࣇ࢓࣑࣮ࣜศᏊࡢⓎ⌧ࢆ඲⯡ⓗ࡟పୗࡉࡏࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓ㸬 HERࣇ࢓࣑࣮ࣜࢆᶆⓗ࡜ࡋࡓศᏊᶆⓗ⸆࡛ࡣ㸪EGFR ࡢኚ␗࡟ࡼࡗ࡚⪏ᛶࢆ⋓ᚓࡋࡓࡾ㸪HER1 ࠾ࡼࡧ HER3 ࢆάᛶ໬ࡋࡓࡾࡍࡿࡇ࡜ࡀၥ㢟࡜࡞ࡗ࡚࠸ࡿ㸬ࡋࡓࡀࡗ࡚㸪ࡑࢀࡽࡢ⸆๣࡜ cSBL ࡜ࡢే⏝ࡣ㸪ஙࡀࢇࡢ⸆≀἞⒪࡟࠾ࡅࡿ᭷ຠ࡞㑅ᢥ⫥࡟࡞ࡾᚓࡿ࡜⪃࠼࡚࠸ࡿ㸬2018 ᖺ࡟Ⓨ⾲ࡋࡓㄽᩥ
6LDOLFDFLGELQGLQJOHFWLQIURPEXOOIURJHJJVLQKLELWVKXPDQPDOLJQDQWPHVRWKHOLRPDFHOOJURZWKLQYLWURDQGLQYLYR7DWVXWD76DWRK76XJDZDUD6+DUD$+RVRQR0
PLoS One  H  
6LDOLF$FLG%LQGLQJ/HFWLQIURP%XOOIURJ(JJV([KLELWVDQ$QWL7XPRU(IIHFW$JDLQVW%UHDVW &DQFHU &HOOV ,QFOXGLQJ 7ULSOH1HJDWLYH 3KHQRW\SH &HOOV 7DWVXWD 7 6DWR 7
6DWR76XJDZDUD66X]XNL7+DUD$+RVRQR0Molecules    ࢼ࣐ࢬ༸ࣞࢡࢳࣥࡢࡀࢇ἞⒪࡬ࡢᛂ⏝ࢆ┠ᣦࡋࡓᇶ♏◊✲㸪Ⳣཎᰤ⣖㸪⸆Ꮫ㞧ㄅ    *E ᳨ฟ࡟฼⏝ࡍࡿᢠ *E ᢠయ㸦$㸧ࡢ᭷⏝ᛶࡢ᳨ウ Ⳣཎᰤ⣖㸪㮚れ⚈Ꮚ㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪ఀ⸨῟㸪బ⸨ಙ㸪኱ᒣຊ㸪⣽㔝㞞♸ ᮾ໭་⛉⸆⛉኱Ꮫ◊✲ㄅ  
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㝣㟂㸪⚟⏣཭ᙪ㸪ᗉᏊ㞙ே㸪ẁ⛬೧㸪ᙇ෤ᱵ㸪ᚎᚿ೧㸪ᙇᅜ೧㸪ఀᕥ἞▱ᘺ㸪㢳ᘓᅜ➨  ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪௝ྎ㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3
y *OF1$F ಟ㣭࡟ࡼࡿ⣽⬊᥋╔ᩬ࡜⣽⬊⛣ືࡢไᚚ࡟㛵ࡍࡿ◊✲ఀᕥ἞▱ᘺ㸪ᚎᚿ೧㸪⚟⏣཭ᙪ㸪㢳ᘓᅜ➨  ᅇᮾ໭⢾㙐◊✲఍㸪ᘯ๓㸪 ᖺ  ᭶㸪₇㢟␒ྕ 2y ࢢࣜ࢔⣽⬊࡟࠾ࡅࡿࢥ࢔ࣇࢥ࣮ࢫࡢᙺ๭࡟㛵ࡍࡿ◊✲39⚟⏣཭ᙪ㸪㝣㟂㸪ᗉᏊ㞙ே㸪ᙇ෤ᱵ㸪ఀᕥ἞▱ᘺ㸪㢳ᘓᅜ➨  ᅇᮾ໭⢾㙐◊✲఍㸪ᘯ๓㸪 ᖺ  ᭶㸪₇㢟␒ྕ 2y )XW Ḟᦆ࡟ࡼࡿࢢࣜ࢔⣽⬊ࡢάᛶ໬࡜ࡑࡢᶵᗎ࡟㛵ࡍࡿ◊✲ᗉᏊ㞙ே㸪⚟⏣཭ᙪ㸪㝣㟂㸪ᙇ෤ᱵ㸪ఀᕥ἞▱ᘺ㸪㢳ᘓᅜ➨  ᅇᮾ໭⢾㙐◊✲఍㸪ᘯ๓㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3y *OF1DF ಟ㣭࡟ࡼࡿ⣽⬊᥋╔࡜⣽⬊⛣ືࡢไᚚ
ఀᕥ἞▱ᘺ㸪ᚎᚿ೧㸪⚟⏣཭ᙪ㸪㢳ᘓᅜ➨  ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍㸪ி㒔㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3y ࢢࣜ࢔⣽⬊࡟࠾ࡅࡿ Į ࣇࢥ࣮ࢫࡢᙺ๭࡟㛵ࡍࡿ◊✲⚟⏣཭ᙪ㸪㝣㟂㸪ᗉᏊ㞙ே㸪ᙇ෤ᱵ㸪ఀᕥ἞▱ᘺ㸪㢳ᘓᅜ➨  ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍㸪ி㒔㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3
y *OF1$F ಟ㣭࡟ࡼࡿ⣽⬊᥋╔ᩬ࡜⣽⬊⛣ືࡢไᚚ
ఀᕥ἞▱ᘺ㸪ᚎᚿ೧㸪⚟⏣཭ᙪ㸪㢳ᘓᅜ➨  ᅇ᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊఍㸪௝ྎ㸪 ᖺ  ᭶㸪₇㢟␒ྕ 2%y ⢾㌿⛣㓝⣲ *Q7, ࡣୖ⓶㛫ⴥ㌿᥮࡜㛫ⴥୖ⓶㌿᥮ࢆไᚚࡍࡿ
ᙇᅜ೧㸪ఀᕥ἞▱ᘺ㸪⚟⏣཭ᙪ㸪㢳ᘓᅜ➨  ᅇ᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊఍㸪௝ྎ㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3%y ࢢࣜ࢔⣽⬊ࡢάᛶ໬࡟ཬࡰࡍࢥ࢔ࣇࢥ࣮ࢫࡢᶵ⬟ᗉᏊ㞙ே㸪⚟⏣཭ᙪ㸪㝣㟂㸪ᙇ෤ᱵ㸪ఀᕥ἞▱ᘺ㸪㢳ᘓᅜ➨  ᅇ᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊఍㸪௝ྎ㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3%y ,PSRUWDQFHRIJO\FRV\ODWLRQDQGLWVUHODWLRQVKLSVZLWKGLVHDVHV㢳ᘓᅜ኱㐃⌮ᕤ኱ᏛᏛ⾡ㅮᗙ㸦Ꮫ⏕ᩍ⫱ࢭ࣑ࢼ࣮㸧㸪㑈ᑀ┬┙㘊ᕷ㸪 ᖺ  ᭶y *O\FRV\ODWLRQLQFDQFHUFHOOVDQGWRZDUGVFOLQLFDODSSOLFDWLRQ㢳ᘓᅜ኱㐃་⛉኱ᏛᏛ⾡ࢭ࣑ࢼ࣮㸪኱㐃ᕷ㸪 ᖺ  ᭶y )XQFWLRQVRI1JO\FDQLQ(07DQGFDQFHUFHOOVVZLWFKLQJRI(07E\1DFHW\OJOXFRVDPLQ\OWUDQVIHUDVH, *Q7, 㢳ᘓᅜ➨  ᅇ༡ி⭘⒆デ᩿἞⒪ᅜ㝿఍㆟㸪༡ிᕷ㸪 ᖺ  ᭶㸪せ᪨㞟 Sy )XQFWLRQVRIJO\FRV\ODWLRQLQFHOODGKHVLRQDQG(07RIFDQFHUFHOOV㢳ᘓᅜ୰ᅜ⛉Ꮫ㝔㐣⛬ᕤ⛬◊✲ᡤ࣭⏕໬ᕤ⛬ᅜᐙ㔜Ⅼᐇ㦂ᣐⅬ◊✲ࢭ࣑ࢼ࣮㸪໭ிᕷ㸪 ᖺ  ᭶y 370RIJO\FRV\ODWLRQLQFHOODGKHVLRQDQG(07RIFDQFHUFHOOVDQGGLVHDVHV㢳ᘓᅜ໭ி኱Ꮫ࣭⏕࿨⛉ᏛᏛ㝔Ꮫ⾡ࢭ࣑ࢼ࣮㸪໭ிᕷ㸪 ᖺ  ᭶y *O\FRV\ODWLRQLQFDQFHUFHOOVDQGGLVHDVHV

40

㢳ᘓᅜ
ኳὠ་⛉኱Ꮫ࣭ᇶ♏་Ꮫ㝔Ꮫ⾡ࢭ࣑ࢼ࣮㸪ኳὠᕷ㸪 ᖺ  ᭶㸺ศᏊㄆ㆑㒊㛛㸼y ࢼ࣐ࢬ༸ࣞࢡࢳࣥࡣ⭈⒴⣽⬊࡟࠾࠸࡚ 6XQLWLQLE ࡢຠᯝࢆቑᙉࡍࡿఀ⸨῟㸪Ⳣཎᰤ⣖㸪ᒾᮧ኱ᚄ㸪㜿༡๛㸪ᾏἲᗣ⿱㸪❧⏣ᓅ⏕㸪⣽㔝㞞♸㸪బ⸨ಙ㸪➨  ᅇ᪥ᮏἪᒀჾ⛉Ꮫ఍⥲఍㸪ி㒔㸪 ᖺ  ᭶㸪₇㢟␒ྕ 33y ᪂つᝏᛶ୰⓶⭘἞⒪⸆ࡢ㛤Ⓨࢆ┠ᣦࡋࡓ࢘ࢩ࢚࢞ࣝ༸⏤᮶ࢩ࢔ࣝ㓟⤖ྜᛶࣞࢡࢳࣥ F6%/ ࡢᛂ⏝◊✲
❧⏣ᓅ⏕㸪బ⸨ ⛱அ㸪Ⳣཎ ᰤ⣖㸪⣽㔝 㞞♸᪥ᮏ⏕໬Ꮫ఍ᮾ໭ᨭ㒊➨  ᅇ౛఍ ▮ᕵ ᖺ  ᭶ ₇㢟␒ྕ 2y ࡀࢇ⣽⬊࡟ࡼࡗ࡚␗࡞ࡿࢼ࣐ࢬ༸ࣞࢡࢳࣥࡢຠᯝࡢẚ㍑
Ⳣཎᰤ⣖㸪▼ᕝᗂᑜ㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪ఀ⸨῟㸪బ⸨ಙ㸪⣽㔝㞞♸➨  ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪௝ྎ㸪 ᖺ  ᭶㸪せ᪨㞟 Sy ࢼ࣐ࢬ༸ࣞࢡࢳࣥࡢࣄࢺࢭ࣑ࣀ࣮࣐⣽⬊ᰴ -.7 ࡟ᑐࡍࡿస⏝Ⳣཎᰤ⣖㸪▼ᕝᗂᑜ㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪ఀ⸨῟㸪బ⸨ಙ㸪኱ᒣຊ㸪⣽㔝㞞♸➨  ᅇᮾ໭⢾㙐◊✲఍㸪ᘯ๓㸪 ᖺ  ᭶㸪せ᪨㞟 S
y 6LDOLF $FLG%LQGLQJ /HFWLQIURP %XOOIURJ(JJV ([KLELWVDQ$QWL7XPRU (IIHFW $JDLQVW %UHDVW &DQFHU&HOOV
7DWVXWD76DWR66DWR76XJDZDUD66X]XNL7+RVRQR0➨  ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍㸪ி㒔㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3y ࢼ࣐ࢬ༸ࣞࢡࢳࣥࡢ⣽⬊ෆ㍺㏦ᶵᵓࡢゎ᫂ᮏ⏣ᤩኴ㸪Ⳣཎᰤ⣖㸪▼ᕝᗂᑜ㸪❧⏣ᓅ⏕㸪⣽㔝 㞞♸➨  ᅇ᪥ᮏ⏕໬Ꮫ኱఍㸪ி㒔㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3y ࢼ࣐ࢬ༸ࣞࢡࢳࣥࡣ⭈⒴⣽⬊࡟࠾࠸࡚ 6XQLWLQLE ࡢຠᯝࢆቑᙉࡍࡿఀ⸨῟㸪Ⳣཎᰤ⣖㸪❧⏣ᓅ⏕㸪⣽㔝㞞♸㸪బ⸨ಙ➨  ᅇ᪥ᮏ⒴Ꮫ఍Ꮫ⾡⥲఍㸪኱㜰㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3
y +H/D ⣽⬊࡟࠾ࡅࡿ 6$/ ྲྀࡾ㎸ࡳᶵᵓࡢゎ᫂ᫍࡦ࠿ࡾ㸪Ⳣཎᰤ⣖㸪▼ᕝᗂᑜ㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪⣽㔝㞞♸➨  ᅇ᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊኱఍㸪௝ྎ㸪 ᖺ  ᭶㸪せ᪨㞟 Sy ᢠ *E ᢠయ࡛࠶ࡿ %*5 ࠾ࡼࡧ $ ࡢ *E ࡟ᑐࡍࡿ཯ᛂᛶࡢẚ㍑
㮚れ⚈Ꮚ㸪Ⳣཎᰤ⣖㸪▼ᕝᗂᑜ㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪⣽㔝㞞♸➨  ᅇ᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊኱఍㸪௝ྎ㸪 ᖺ  ᭶㸪せ᪨㞟 Sy ࣂ࣮࢟ࢵࢺࣜࣥࣃ⭘⣽⬊ᰴ 5DML ࡟࠾ࡅࡿࢼ࣐ࢬ༸ࣞࢡࢳࣥࡢྲྀࡾ㎸ࡳᶵᵓࡢゎ᫂Ⳣཎᰤ⣖㸪▼ᕝᗂᑜ㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪⣽㔝㞞♸➨  ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍㸪ᶓ὾㸪 ᖺ  ᭶㸪₇㢟␒ྕ 3



41

CHAPTER FIVE

Biology of GM3 Ganglioside
Jin-ichi Inokuchi*,1, Kei-ichiro Inamori*, Kazuya Kabayama†,
Masakazu Nagafuku*, Satoshi Uemura‡, Shinji Go*, Akemi Suzuki*,
Isao Ohno§, Hirotaka Kanoh*, Fumi Shishido**

Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University,
Sendai, Miyagi, Japan†
Osaka University, Toyonaka, Osaka, Japan‡
Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, Japan§
Center for Medical Education, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, Japan
1

Corresponding author. E-mail address: jin@tohoku-mpu.ac.jp

Contents
1. Introduction
2. Molecular Biology of GM3S/ST3GAL5
2.1 Tissue Distribution of GM3S mRNATranscriptional Variants
2.2 Translation of GM3S Isoforms by Leaky Scanning System
2.3 Regulation of ER Export of GM3S by R/K-Based Motif
2.4 Regulation of Retrograde Transport of M1-GM3S Isoform by R-Based Motif
2.5 Golgi Retention and Maturation of N-Glycans in GM3S
2.6 Perspectives
3. Insulin Resistance in Adipocytes and Macrophages
3.1 GM3 is an Inducer of Insulin Resistance
3.2 Insulin Resistance as a Membrane Microdomain Disorder
3.3 GM3 Functions as a Physiological Regulator for Insulin Signaling andAdipogenesis
3.4 Serum GM3 as a New Biomarker of Metabolic Syndrome
3.5 Pathogenic Control of Adipocytes by Increased Expression of GM3
3.6 Perspectives
4. Leptin Signaling and Resistance in Hypothalamus
4.1 Leptin Receptor Signaling
4.2 Leptin Resistance
4.3 Leptin Signaling in Ganglioside-Deficient Mice
4.4 Perspectives
5. T-Cell Immunity and Immune Disorders
5.1 Ganglioside Expression in CD4+ and CD8+ T-Cell Subsets
5.2 Ganglioside Expression in TCR-Mediated T Cell Activation
5.3 T Cell Gangliosides in Autoimmune and Allergic Diseases
5.4 Perspectives
Progress in Molecular BiologyandTranslational Science, Volume 156
ISSN 1877-1173
http://dx.doi.org/10.1016/bs.pmbts.2017.10.004© 2018 Elsevier Inc.
All rights reserved.

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Jin-ichi Inokuchi et al

6. GM3S Deficiency and Hearing Loss
6.1 GM3S Deficiency in Humans
6.2 Significant Increase of GSLs During Postnatal Maturation of Murine Cochlea
6.3 Glycocalyx Integrity and Membrane Cycling in GM3S-Deficient Mice
6.4 GM3-Enriched Membrane Organization, PTPRQ-Myosin VI Complex
Localization and Hair Cell Morphology
6.5 Perspectives
7. Conclusions and Future Directions
Acknowledgments
References

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Abstract
Since the successful molecular cloning in 1998 of GM3 synthase (GM3S, ST3GAL5), theenzyme responsible for initiating biosynthesis of all complex gangliosides, the effortsof our research group have been focused on clarifying the physiological and pathological implications of gangliosides, particularly GM3. We have identified isoforms of
GM3S proteins having distinctive lengths of N-terminal cytoplasmic tails, and foundthat these cytoplasmic tails define subcellular localization, stability, and in vivo activityof GM3S isoforms. Our studies of the molecular pathogenesis of type 2 diabetes,
focused on interaction between insulin receptor and GM3 in membrane microdomains, led to a novel concept: type 2 diabetes and certain other lifestyle-relateddiseases are membrane microdomain disorders resulting from aberrant expressionof gangliosides. This concept has enhanced our understanding of the pathophysiological roles of GM3 and related gangliosides in various diseases involving chronic
inflammation, such as insulin resistance, leptin resistance, and T-cell function andimmune disorders (e.g., allergic asthma). We also demonstrated an essential role of
GM3 in murine and human auditory systems; a common pathological feature of GM3S
deficiency is deafness. This is the first direct link reported between gangliosides andauditory functions.

1. INTRODUCTION
The presence of sialic acid at the surface of animal cell membranes
was first demonstrated in 1951 by Tamio Yamakawa.1 He isolated a sialic
acid-containing glycolipid, which he called “hematoside,” from 100 g of dried
ghosts obtained from 10 L of packed horse erythrocytes.1,2 Hematoside(now termed “GM3 ganglioside”) is the first product in the biosynthetic
pathway of ganglio-series gangliosides, and the common precursor of complex
gangliosides, such as a- and b-series gangliosides (Fig. 1). Gangliosides are
expressed in a cell type-specific manner on the outer leaflet of cell membranes,
and interact with various molecules on plasma membranes and certain intracellular membranes based on potentials of noncovalent bondings, for example,

153

Biology of GM3 Ganglioside[(Fig._1)TD$IG]

Cer
GD1
GlcCer

GalGalNAcGM1b

GalNAcGM1b

Cer

Cer

Cer

Cer
o-Series
LacCer

GA2

Cer

GA1

Cer
B4galnt1

GD1c

GM1b

Cer

Cer

Cer

B3galt4

St3gal5(GM3S)

GM3

GM2

Cer

GM1

Cer
B4galnt1(GM2S)

GD1a

Cer

GT1a

Cer

Cer
St8sia5St3gal2/3

B3galt4

a-Series

St8sia1(GD3S)

GD3

GD2

Cer

GD1b

Cer

B4galnt1

GT1b

Cer

b-Series

St8sia1

GT3

Cer

St8sia5St3gal2/3

B3galt4

GQ1b

Cer

GT2

Cer
B4galnt1

GT1c

Cer

GQ1c

Cer

B3galt4

St3gal2/3

GP1c

Cer

Cer

St8sia5c-Series

Fig. 1 Biosynthetic pathway of gangliosides.

hydrogen bonding and electrostatic and hydrophobic interactions. Membersof the various ganglioside families participate in numerous cellular activities byforming dynamic functional complexes termed “membrane microdomains”
or “lipid rafts.”3,4 Expression levels of cellular gangliosides are affected byvarious extracellular stimuli, including inflammatory cytokines. The presenceof gangliosides in membrane microdomains thus reflects characteristics ofindividual cells under various physiological and pathological environments.
Since molecular cloning of GM3S was achieved in 1988,5 the activity of ourresearch group has been focused on elucidating the biological functionsof GM3 and related gangliosides. Here, we review our recent discoveries
and advances regarding the molecular biology of GM3S, and the pathophysiological significance of GM3 and related gangliosides.6–8

2. MOLECULAR BIOLOGY OF GM3S/ST3GAL5
GM3S is a type II membrane protein having a catalytic domain in theluminal side (Fig. 2). Human and mouse GM3Ss initially undergo modification with two or three N-glycans, respectively, in the endoplasmic

154

Jin-ichi Inokuchi et al[(Fig._2)TD$IG]
Retrograde
transport
NH2
R-based motif

-COP
-COP
?

NH2

Golgi retention
NH2

GTP

R/K-based motif

LacCer

GM3
NH2

CMP-Sia

Sar1

N94
N126

D198

GM3S

ER exporting
N-GlycanIsoform
names

M1

M2

M3

Localization

ER

Golgi

Golgi

Stability

HigherLower

Higher

Structure of
N-glycans

Highmannose
type

Highmannose
typeComplex
type

Fig. 2 Regulation of intracellular dynamics of GM3S isoforms by R-based andR/K-based motifs located within cytoplasmic tails. All isoforms have an R/K-based
motif, which functions as an endoplasmic reticulum (ER) export signal byinteracting with Sar1 protein. The R/K-based motif also functions as a Golgi
retention signal in M3 isoform only, leading to stabilization of this isoform in Golgi.
R/K-based motifs of M1 and M2 isoforms are presumably unable to function as Golgi
retention signals because of their long cytoplasmic tails. Only M3 carries highly
matured N-glycans. M1, which has R-based motif in its cytoplasmic tail, is targeted
for ER by interaction with β-COP-δ-COP complex. The significance of ER targeting of
M1 is unclear.

reticulum (ER).9 N-glycans are further modified in the Golgi apparatus,and glycan structures are converted from high-mannose type (immatureform) to complex type (mature form). The presence of high-mannose
type sugar chains is essential for enzyme activity.9 Lactosylceramide(LacCer), the acceptor substrate for GM3S, is synthesized in the luminalside of Golgi.10 CMP-sialic acid, a donor substrate for GM3 synthesis, issynthesized in cytosol and transported to the luminal side by CMP-sialic
acid transporter localized in Golgi.11 GM3 synthesis occurs subsequently
in the luminal side of trans-Golgi.12 This section describes regulationof intracellular dynamics of three GM3S isoforms (M1, M2, and M3)
we discovered.

Biology of GM3 Ganglioside155

2.1 Tissue Distribution of GM3S mRNATranscriptional Variants
Human GM3S has five mRNA variants,5,13–15 which are categorized intoa-, b-, and c-type based on differences in transcription start sites (TSSs).16
a-Type and b-type are further subdivided into a1- (type 1), a2- (type 2),
b1- (type 3), and b2-type (type 4) based on differences in splicing. a2-Type isexpressed in a variety of tissues.16 c-Type (type 5) is expressed specifically andexclusively in pancreas.15
Mouse GM3S has two transcriptional variants: a- and b-type.13,17 The
TSSs of these variants are very close, and their transcriptional regulation istherefore considered to be similar. Tissue distribution of the two variants isnearly identical.16 We recently discovered a liver-specific transcriptionalvariant, c-type.18 The c-type TSS is located downstream and far away fromthe TSSs of a- and b-type. We are currently investigating the liver-specificexpression mechanism of c-type.

2.2 Translation of GM3S Isoforms by Leaky Scanning System
In general, the first AUG codon is recognized by ribosomes as an initiation
codon (first-AUG rule).19 In mammals, the optimal context for recognitionof the AUG start codon is GCCRCCAUGG (underline: initiation codon;
R: A or G). When the first AUG residue is presented in a weak context, inparticular, lacking both R in position -3 and G in position +4, the first AUG
codon is sometimes skipped and a downstream AUG is recognized as an
initiation codon. This process is termed a leaky scanning system.19 By using
this system, GM3S isoforms (M1, M2, M20 , and M3 in human; M1, M2, andM3 in mice), each having a distinctive length of N-terminal cytoplasmic tail,are translated from each mRNA variant. The lengths of isoforms M1, M2,
M20 , and M3 are respectively 69, 42, 47, and 14 aa.
Three isoforms (M1, M2, M3) or two isoforms (M2, M3) were detected,
respectively, in cells expressing mouse a- or b-type variant. In contrast, one
isoform (M3) or two isoforms (M1 and M3) were detected respectively incells expressing human a1- or a2-type variant.16 M20 and M3 isoforms weredetected in cells expressing human c-type variant.20 Thus, the leaky scanningsystem seems to elicit coexistence of various GM3S isoforms in cells.

2.3 Regulation of ER Export of GM3S by R/K-Based Motif
Transport of GM3S from ER to Golgi is the first regulatory step for GM3
synthesis. Glycosyltransferases are loaded efficiently into COPII vesicles
through interaction between [R/K](X)[R/K] motifs in the cytoplasmic tail

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and small GTPase Sar1, a COPII coated-protein.21,22 All GM3S isoforms
carry RR sequence just above the transmembrane domain. However, twolysine residues located downstream of the RR sequence are also involved in
ER export signal [2R3R(X)59K(X)313K] in M3-GM3S.23 We therefore
propose this “R/K-based motif” as a new ER export signal (Fig. 2).
Formation of COPII-coated vesicles begins with conversion from Sar1GDP to Sar1-GTP by the guanine nucleotide exchange factor, Sec12.24
Sar1-GTP is anchored to ER membrane and recruits Sec23–Sec24 complex
by interacting with Sec23.25 The Sar1-Sec23-Sec24 complex recruits
many cargo proteins containing ER export signals (DXE and FF sequences)through interaction with Sec23 or Sec24 to generate a prebudding
complex.26–30 Sec23 also exhibits GTPase-activating protein activity forSar1-GTP. Once Sar1 forms a prebudding complex, Sar1-GTP is hydrolyzed
by Sec23 and is dissociated from prebudding complex.31 Prebudding complex without Sar1 is unstable, but Sar1 is immediately reactivated by Sec12and incorporated into the complex. When prebudding complex without
Sar1 contains unfolded proteins, it breaks before reassembly of Sar1-GTP
because of weak interaction between the proteins and Sec23/Sec24. The
GTP/GDP cycle of Sar1 thus functions as a selective sorting system for cargo
proteins.31 If R/K-based motif interacts only with Sar1, the cargo proteinsdissociate from prebudding complex when Sar1-GTP is converted to Sar1GDP. The interaction by itself is therefore insufficient for glycosyltransferases
to be efficiently concentrated in COPII-coated vesicles. Our current
hypothesis is that R/K-based motif functions as a guide to prebudding
complex through interaction with Sar1.23 Glycosyltransferases carrying
R/K-based motif may be transferred to other cargo proteins (receptors) in
prebudding complex.

2.4 Regulation of Retrograde Transport of M1-GM3S Isoform
by R-Based MotifAmong three isoforms, only M1-GM3S has a retrograde transport signal (Rbased motif) in its N-terminal cytoplasmic tail (Fig. 2).16 Accordingly, most
M1-GM3S is transported from Golgi to ER by R-based motif, and localizedin ER. As R-based motifs interact with an acidic pocket of β-COP-δ-COP
complex in COPI subunits, proteins carrying R-based motif are efficiently
concentrated in COPI-coated vesicles involved in retrograde transport.32
R-based motifs must be positioned at a certain distance from the lipid bilayer
to function,33 indicating that R/K-based motif (ER export signal) just abovetransmembrane domain cannot function as a retrograde transport signal. In

Biology of GM3 Ganglioside157

vivo enzymatic activity of M1-GM3S is lower than those of M2-GM3S and
M3-GM3S. However, GM3 synthesis is not completely eliminated when
GM3S-KO cells express only M1-GM3S, suggesting that some M1-GM3S
is sorted to trans-Golgi by escaping retrograde transport.
What is the biological significance of ER targeting of M1-GM3S? One
possibility is that M1-GM3S has functions other than GM3 synthesis in
ER. For instance, protein O-fucosyltransferase (OFUT1) is known as a glycosyltransferase localized in ER, similar to M1-GM3S.34 OFUT1 has an ER
targeting signal (KDEL-like motif) in its C-terminus and is localized in ER.
OFUT1 glycosylates the epidermal growth factor (EGF)-like domain
of Notch in Golgi, but displays Notch chaperone activity in ER.34
Glycosylation activity of OFUT1 is not necessary for chaperone activity.
The strong protein-protein interaction between OFUT1 and EGF-likedomain of Notch presumably allows the unusual function. In contrast,
GM3S recognizes the glycosphingolipids (GSLs), LacCer and galactosylceramide (GalCer) as substrates. GM3S does not display sialylation activity toward
lactose and N-glycans of glycoproteins,17 so the active site of GM3S presumably recognizes both carbohydrate (hydrophilic) and ceramide (hydrophobic)
portions. The partial hydrophobic pocket may interact with unfolded proteins,
but the folding site is limited within the lipid bilayer. It therefore seems
unlikely that M1-GM3S is involved in protein folding, in contrast to OFUT1.
Another possibility is that M1-GM3S regulates expression levels of
M2-GM3S and M3-GM3S in Golgi. As indicated by expression patternsof mRNA variants in tissues, three GM3S isoforms usually coexist within asingle cell. All GM3S isoforms are capable of interacting with each other(unpublished data); therefore, M2-GM3S and M3-GM3S may partiallylocalize in ER by coexpression with M1-GM3S. Total amounts of GM3S
isoforms in Golgi are consequently maintained at low level. We were able toestablish stable clones expressing M2-GM3S or M3-GM3S only at low level,
but easily established stable clones expressing M1-GM3S at high level. Thesefindings suggest that excessive amounts of GM3S in Golgi are cytotoxic.
M1-GM3S may promote cell survival by suppressing accumulation of
M2-GM3S and M3-GM3S in Golgi.

2.5 Golgi Retention and Maturation of N-Glycans in GM3S
Golgi cisternae (cis, medial, and trans) are characterized by Golgi-resident
enzymes. In the cisternal maturation model, the Golgi apparatus is regarded
as a dynamic structure.35,36 According to this model, a new cis cisterna is
continually formed by vesicles supplied from ER, and progressively matures

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to a medial cisterna and then a trans cisterna. Retrograde transport of Golgiresident enzymes by COPI-coated vesicles explains their distinctive distribution and Golgi retention. The (F/L)(L/I)XX(R/K) motif of Kre6, whichis involved synthesis of β1-6 glycans synthesis, functions as a Golgi retention
signal in Saccharomycescerevisiae.37 This motif interacts with Vps74 in COPIcoated protein complex. Golph3 and Golph3L, the mammalian homologs of
Vps47, cannot function complementarily in vps47Δ strain and (F/L)(L/I)
XX(R/K) motif is not conserved in mammalian glycosyltransferases. Golgi
retention of enzymes in mammals therefore presumably occurs by othermechanisms.
The half-life of M3-GM3S is longer than that of M2-GM3S, althoughboth are localized in Golgi.16 We recently found that partial mutations of R/
K-based motif (R2A, R3A, R2A/R3A, K9A/K13A) in M3-GM3S lead toaltered localization from Golgi to endosomes and to rapid degradation,23
suggesting that R/K-based motif also functions as a Golgi retention
signal (Fig. 2). The motif is presumably involved in retrograde transport
from Golgi. Masking of R/K-based motif by the long cytoplasmic tail of
M2-GM3S may contribute to its low stability.
The differences of N-glycan structures among GM3S isoforms are noteworthy. The N-glycan structure of M1-GM3S is high-mannose type (immature form), as evidenced by the immediate relocation of M1-GM3S from Golgi
to ER by R-based motif. The N-glycan structure of M2-GM3S localized in
Golgi is also high-mannose type, whereas that of M3-GM3S is complex type(mature form).16 The proportion of mature N-glycans in M3-GM3S is reducedby mutations (R2A, R3A, R2A/R3A, K9A/K13A) of Golgi retention signal(R/K-based motif).23 Our findings, taken together, suggest that Golgi-retention time of glycoprotein determines maturation level of N-glycans. For fullmaturation of N-glycans, glycoproteins must undergo modification by several
glycosyltransferases in dynamic Golgi cisternae. A single passage through Golgi
is not sufficient to convert all glycosyltransferases to mature forms.

2.6 Perspectives
Our studies of GM3S isoforms help clarify the regulatory mechanism of
ganglioside synthesis in dynamic Golgi cisternae. Issues to be addressed in the
future studies include:
1. Determination of three-dimensional structure of GM3S by X-ray
crystallography.
2. Identification of molecules involved in ER export and Golgi retention of
GM3S.

Biology of GM3 Ganglioside

159

3. Examination of the heterooligomer formation by GM3S isoforms, andcytotoxic effects of GM3S by the accumulation in Golgi.
4. Elucidation of N-glycan maturation mechanism through analysis of
N-glycan structures of GM3S isoforms and their mutants, which cannot
be tethered to Golgi.
Elucidation of the posttranslational regulation of GM3S will contributeto more effective therapeutic strategies for pathological conditions resulting
from abnormal GM3 synthesis.

3. INSULIN RESISTANCE IN ADIPOCYTES AND
MACROPHAGES
Visceral adipose tissues are comprised of not only adipocytes; but also
immune cells, including resident macrophages, T lymphocytes, and othertypes of cells.38–41 It remains unclear how interactions between adipocytes
and resident macrophages are affected by expression of specific GSLs. We
have shown that expression of GSLs and their corresponding synthasegenes in adipocytes is maintained by soluble factors secreted from resident
macrophages under not only inflammatory states, but also steady-statephysiological conditions. Obese GM3S-deficient mice fed a high-fat dietwere resistant to development of proinflammatory states in adipose tissues.
This section focuses on association of GM3 with metabolic syndrome, andthe physiological and pathological implications of GM3 present in adipose
tissues.

3.1 GM3 is an Inducer of Insulin Resistance
Insulin elicits a wide variety of biological activities, which can be categorized
into metabolic and mitogenic actions. Binding of insulin-to-insulin receptor(IR) activates IR internal-tyrosine (Tyr) kinase activity. Tyr-phosphorylated
and activated IR is able to recruit and phosphorylate adaptor proteins, such as
IR substrate (IRS). Phosphorylated IRS activates PI3-kinase (PI3K).
Activated PI3K is translocated to lipid rafts and converts PIP2 to PIP3, andPIP3 then recruits PDK1 to phosphorylate Akt. Full activation of Akt may
be required for signaling by phosphorylation of the other site by mTORC2(mTOR complex 2).42 This IR-IRS-PI3K-Akt signaling cascade is a
representative metabolic pathway triggered by insulin, and results in translocation of glucose transporter 4 (GLUT-4) to plasma membrane to facilitate
glucose uptake.

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When mouse adipocytes were cultured in low concentrations of TNFαthat did not cause generalized suppression of expression of adipocyte genes(e.g., IRS-1, GLUT-4), TNFα interfered with insulin action.43 Prolonged
treatment (≥72 h) was required for this effect, in contrast to many acuteeffects of this cytokine. The slowness of the effect suggests that insulin
resistance in adipocytes treated with 0.1 nM TNFα was associated with
progressive increases in cellular GM3 content, GM3S activity, and GM3SmRNA content, and that such treatment upregulates GM3 synthesis at thetranscriptional level in cultured adipocytes.44,45 In contrast, ceramide levels
under TNFα treatment increased transiently up to 6 h, and returned tonormal by 24 h. These observations reflect the distinct and independentroles of GM3 and ceramides in development of insulin resistance in adipocytes.44,45 To determine whether elevated GM3 in 3T3-L1 adipocytes
treated with TNFα is involved in insulin resistance, we used D-threo-1phenyl-2-decanoylamino-3-morpholino-1propanol (D-PDMP), an inhibitor of glucosylceramide synthase,46 to deplete cellular GSLs derived from
GlcCer. D-PDMP treatment counteracted TNFα-induced increase of GM3
content in adipocytes, and reverted to normal the TNFα-induced defect in
Tyr phosphorylation of IRS-1 in response to insulin stimulation.7 Thesefindings are consistent with the observation that insulin signaling wasenhanced in GM3S-knockout mice.47 It has been reported that TNFαtreatment of adipocytes induces an increase in serine phosphorylation of
IRS-1.48 Such phosphorylation is important because immunoprecipitated
IRS-1 that has been serine phosphorylated in response to TNFα is a direct
inhibitor of IR Tyr kinase activity. We found that TNFα-induced serine
phosphorylation of IRS-1 in adipocytes was completely suppressed by DPDMP inhibition of GM3 biosynthesis, suggesting that TNFα-induced
increase of GM3 synthesis causes upregulation of serine phosphorylationof IRS-1.45 An improved PDMP analog and a different type of GlcCer
synthase inhibitor,49,50 had therapeutic value when orally administered indiabetic rodent models.
TNFα levels were fairly high in two obese diabetic rodent models(Zucker fa/fa rats and ob/ob mice),48 but much lower in adipose tissues fromlean control animals. We, therefore, examined GM3S mRNA expression in
epididymal fat of the diabetic models. Northern blot analysis showed that
GM3S mRNA content in adipose tissues of the two models was significantly
higher than in lean controls.45 TLC comparison of GM3 band mobility
revealed the appearance of GM3 species with lower mobility (i.e., morehydrophilic) in the models.

Biology of GM3 Ganglioside161

We also examined GM3 expression in diet-induced obese (DIO) mice.51
Following 10 weeks of feeding, mean body weight of standard diet (SD) andhigh-fat diet (HFD) groups were 31.0 ± 0.6 g and 46.0 ± 0.8 g and fasted
blood glucose levels were 137 and 203 mg/dL, respectively.51 GM3 level in
epididymal fat was threefold higher in HFD than in SD group. mRNA levelof GM3S gene was also threefold higher in HFD group.51
These findings strongly indicate that increased expression of GM3 inabdominal adipose tissues tends to induce malfunctions of adipose tissue, forexample, chronic low-grade inflammatory states in obesity. In recent studiesof omental adipose tissues in obese and insulin-resistant women, adipocyte
hypertrophy, and macrophage infiltration were associated with increases ofGM3 content and GM3S gene expression.52 Therefore, drugs that target
GM3 biosynthesis may be useful for treating insulin resistance in obesity.

3.2 Insulin Resistance as a Membrane Microdomain Disorder
In studies of TNFα-induced insulin resistance in adipocytes, we showedthat transformation to a resistant state might result from increased GM3
biosynthesis following upregulation of GM3S gene expression. Increased
GM3 level during chronic exposure to TNFα may therefore suppress insulin
signaling.45 GSLs, including GM3, are important components of lipid rafts. We
considered the possibility that increased GM3 levels in lipid rafts confer insulin
resistance in TNFα-treated adipocytes. We examined GM3-protein interactions within plasma membranes of living cells by performing cross-linking assay
with a photoactivatable, radioactive GM3 derivative. Adipocytes were preincubated with [3H]GM3(N3) and then irradiated to induce crosslinkingof GM3. Target proteins were separated by SDS-PAGE and visualized byautoradiography. A specific radioactive band corresponding to the 90-kDa
IRβ-subunit was immunoprecipitated with anti-IRβ antibodies, indicating
direct association of GM3 with IR. We concluded that IR forms complexes
with caveolin-1 and GM3 independently in 3T3-L1 adipocytes.53
Lipids are asymmetrically distributed in the outer and inner leaflets ofplasma membranes. In typical mammalian cells, most acidic phospholipids
are located in the inner leaflet, and acidic GSLs, such as sulfatides andgangliosides are in the outer leaflet. Binding of proteins to lipid membranes
is often mediated by electrostatic interactions between protein basic domains
and acidic lipids. Gangliosides, which bear sialic acid residues, are ubiquitously present in the outer leaflet of vertebrate plasma membranes. GM3 isthe most abundant ganglioside, and the primary ganglioside found in
adipocytes.54 Glycan chains of GSLs, including gangliosides are oriented

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at a defined angle to the axis of ceramide.55 GM3s spontaneously form
clusters via hydrophobic interaction between their own saturated acyl chains,regardless of any repulsion between negatively charged sialic acids in theglycan chains.56 GM3 clusters, together with other cell surface gangliosides,
thus form GSL-enriched microdomains (GEM) and produce a negatively
charged environment just above the plasma membrane. On the other hand,
IR has a sequence in its transmembrane domain, that is, homologous(conserved) in mammals and presents the basic amino acid lysine (IR944)
just above the transmembrane domain. During lateral diffusion, electrostaticinteraction may occur between the lysine residue at IR944 and the negative
charge of GM3 cluster, because of their close topological proximity on theplasma membrane. In a study using FRAP technique in living cells, we foundthat the mechanism for dissociation of IR-caveolin-1 complex is based oninteraction of a lysine residue localized just above the transmembrane domain
of IRβ-subunit with GM3 clusters at the cell surface.53 Based on theseobservations, we proposed a mechanism that determines insulin resistancestate through localization shift of IR from caveolae to GEM in adipocytes.
Insulin signaling in skeletal muscle was shown to be higher in GM3S
knockout (GM3S-null) mice than in wild-type (WT) C57BL/6 mice.47
On the other hand, inhibition of insulin signaling by exogenous additionof saturated fatty acids into C2C12 myotubes was not eliminated by treatment with a GlcCer synthase inhibitor (D-PDMP analog).57 The involvement of GM3 in pathophysiology of insulin resistance in mammalian skeletal
muscle requires further study.
We explored the role of acyl chain length of ceramides in insulin signaling
using ceramide synthase 2 (CerS2)-null mice that lack the ability to synthesize very long acyl chain (C22-C24) ceramides.58 These mice did not display
IR or Akt phosphorylation in liver in response to insulin. The absence of IR
phosphorylation was correlated with its inability to translocate into detergent-resistant membranes (DRMs). The properties of DRMs in the CerS2null mice differed significantly from those in WT mice, indicating thataltered sphingolipid acyl chain length directly affects IR translocation tolipid rafts and subsequent signaling.

3.3 GM3 Functions as a Physiological Regulator for Insulin
Signaling and Adipogenesis
Visceral adipose tissues, particularly mesenteric adipose tissue, play key
roles in the pathogenesis of metabolic syndrome.38–40,59–63 To investigate
the fundamental characteristics of mesenteric adipocytes, we established aBiology of GM3 Ganglioside163

physiologically relevant differentiation system in which rat mesentericstromal vascular cells (mSVC) were induced to differentiate into mesenteric-visceral adipocytes (mVAC).64,65 We optimized the concentrations of
insulin and insulin-like growth factor (IGF-1) at levels comparable to thosein vivo: 0.85 and 200 ng/mL. IGF-1 and insulin were shown to functionsynergistically; IGF-1 by itself enhanced CCAAT/enhancer binding protein alpha (C/EBPα) and adipocyte lipid binding protein (aP2) mRNA
expression, but in the absence of insulin at physiological concentration
IGF-1 did not induce lipid droplet accumulation associated with adipocyte
maturation.
We used the same differentiation system to elucidate the role of resident
macrophages in mesenteric adipose tissue in physiological adipogenesis.
Adipogenesis of mSVCs in the system was increased by removal of resident
macrophages, along with enhanced insulin signaling and concomitantdecrease of GSLs, including GlcCer, LacCer, and GM3. Phosphorylationlevels of IR and IRS-1 after insulin stimulation were enhanced by depletion of macrophages, and protein level of IR per se was increased in
mSVCs.51
Levels of GSLs (particularly GM3) in preadipocytes and mature adipocytes are thus precisely maintained by soluble factors secreted from resident
macrophages to control physiological adipogenesis (Fig. 3). Adipogenesis of
MEFs prepared from GM3S-null mice was accelerated, in combination withenhanced insulin signaling.51 GM3 is directly involved in insulin signaling
and adipogenesis.

3.4 Serum GM3 as a New Biomarker of Metabolic Syndrome
GM3 is the major ganglioside in serum and is associated with serum lipoproteins.66 We examined the relationships of serum GM3 levels with: (i)adiposity indices, and (ii) metabolic risk variables.66 Serum GM3 levels weresignificantly elevated in type 2 diabetes patients with severe obesity (visceralfat area > 200 cm2, BMI > 30). GM3 level was positively correlated with
LDL-c (0.403, P = 0.012) in type 2 diabetes mellitus, but not related toblood pressure. High levels of small dense LDL (>10 mg/dL) were associatedwith increased GM3. Serum GM3 levels were affected by abnormalities of
glucose and lipid metabolism, and by visceral obesity. Small dense LDL levels
are associated with atherosclerosis development,67,68 and GM3 has been
detected in atherosclerotic lesions.67,68 Thus, GM3 is a potentially usefulmarker for management of metabolic syndrome, including insulin resistance,
and for early diagnosis of atherosclerosis.

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Adipocyte phenotypes regulated by GM3
High☆ Insulin resistance☆ Decreased adipogenesis
Adipose tissue hypertrophy

GM3synthesis
Soluble factors:
TNF , IL-6, IL1- , etc.

☆ Homeostatic regulationof membrane receptors(IR, IGF-1R, TCR, MC4R)
Macrophages☆ Enhanced adipogenesis withupregulation of insulin signaling
Adipose tissue hyperplasia

Low

Fig. 3 GM3 as homeostatic and pathogenic mediator in adipogenesis and insulin
signaling. Interactions between macrophages and adipose progenitor cells may
influence the number of preadipocytes and/or their differentiation capacity, andinduce adipose tissue dysfunction by inhibiting overall adipogenic capacity. Controlof GM3 levels is a potential therapeutic intervention strategy for restoring healthyadipose tissue function in obese individuals, distinct from weight-reduction strategies.

The structural diversity of ceramide species arises from several types ofmodification in N-acyl chains [chain length (C16-24); alpha hydroxylation; desaturation] and in sphingoid bases (d18:1, d18:0; hydroxylation at
C4), resulting in a large number of possible molecular species. We performed LC-MS/MS analysis to identify serum GM3 molecular species in
125 Japanese residents.69 Individuals were categorized based on presence
versus absence of metabolic disease risk factors, including visceral fat
accumulation, hyperglycemia, and dyslipidemia. A total of 23 GM3molecular species were identified, of which 8 were significantly elevatedin individuals with visceral fat accumulation and metabolic disease(defined as the presence of hyperglycemia and dyslipidemia). All of theGM3 molecular species were composed of sphingenine [d18:1(Δ4)], andsix of the eight elevated species contained α-hydroxylated acyl chains.
Hydroxylated GM3 species were, in order of decreasing abundance,
d18:1-h24:0 = d18:1-h24:1 > d18:1-h22:0 >> d18:1-h20:0 > d18:1-h21:0> d18:1-h18:1. We conducted univariate and multiple linear regression

Biology of GM3 Ganglioside165

analyses using numerous clinical health variables associated with obesity,
type 2 diabetes, metabolic disease, atherosclerosis, and hypertension. GM3(d18:1-h24:1) was selected as the best candidate for metabolic screening. This
molecular species showed significant correlation with intima media thickness(a criterion for diagnosis of atherosclerotic disease in humans) and othermetabolic disease risk factors, including autotaxin, LDL-c, and homeostatic
model assessment insulin resistance (HOMA-IR).69

3.5 Pathogenic Control of Adipocytes by Increased Expressionof GM3
Adipose tissue macrophages are classified into two major subtypes: M1 andM2. A concept of M1/M2 polarization has been developed based on expression patterns of cytokines, surface markers, and metabolic enzymes.70 M1
macrophages are potent effector cells that produce inflammatory cytokines,
such as TNF-α, IL1-β, and IL-6. M2 macrophages are present as resident
cells in nearly all organs under physiological conditions, and help maintaintissue homeostasis.71,72 They exert antiinflammatory effects by producing
IL-10 and arginase I enzyme (Arg1). IL-10 potentiates insulin signaling in
adipocytes,72,73 and Arg1 reduces nitric oxide synthesis and inflammation bymetabolizing arginine to ornithine.71 Consumption of HFD shifts cytokine
expression of murine adipose tissue macrophages from M2- to M1-likepatterns by decreasing expression of IL-10 and Arg1 and increasing expression of TNFα and iNOS.73
We analyzed gene expression profiles in epididymal adipose tissue of
GM3S-null mice fed with HFD, but not with SD.51 In comparison withexpression levels in GM3S+/ mice, proinflammatory cytokine TNFα wassignificantly decreased, whereas antiinflammatory cytokine IL-10 wasincreased. Levels of adiponectin were increased, while those of atherogenic
genes, such as PAI-1 and iNOS were reduced. M2 signature genes, such as
MGL1 and Arg1 showed increased expression in the GM3S-null mice.
Glucose tolerance test (GTT) and insulin tolerance test (ITT) both revealed
significant improvement of insulin resistance under HFD condition. Thus,
GM3 plays key roles in development of obesity-induced chronic low-grade
inflammatory states and of insulin resistance (Fig. 4).51

3.6 Perspectives
Adipocytes are more than inert energy depots; adipose tissue is a biologically
active organ responsible for important physiological processes, including
energy homeostasis and whole-body insulin sensitivity. Dynamic remodeling166[(Fig._4)TD$IG]

Jin-ichi Inokuchi et al

Obese wild C57/BL6 mice

Obese GM3S-deﬁcient mice

Chronic low-grade inﬂammatory states

Antiinﬂammatory states

Development of
adipose tissue dysfunction andinsulin resistanceMetabolicphenotype

Preservation of
adipose tissue function andinsulin sensitivity

GM3S ↑
Adipose tissue
expansion

Hypertrophy

Hyperplasia

GM3S ↑

Reducedadipogenesis

Preadipocyte
reservoir

TNF ↑IL1- ↑iNOS ↑
PAI-1 ↑

Macrophage
phenotype

M1 > M2 like
NF-κB activation
by Gangliosides

Adequate
adipogenesis
IL-10 ↑
Adiponectin ↑Leptin ↑Mgl1 ↑
Arg1 ↑

M2 > M1 like
GM3S ↑

Chronic positive energy imbalance

Fig. 4 Regulation of adipose tissue remodeling by GM3 during chronic positive energy
imbalance. Chronic positive energy imbalance [high fat diet (HFD)] leads to obesity.
Macrophage phenotype may affect the mechanism whereby adipose tissue expands. In
the obese state, an adipose tissue mass increase through hyperplasia and hypertrophy,
and the latter is associated with activation of stress signaling. When proinflammatory M1
macrophages are predominant, the preadipocyte reservoir may be inadequate becauseof reduced preadipocyte survival, proliferation, and/or adipogenic capacity. Energy
storage occurs via exaggerated adipose hypertrophy, resulting in dysfunctional
adipose tissue and contributing to an inflamed insulin-resistant state. Chronic
increase of GM3 through upregulation of St3gal5 gene by proinflammatory cytokines,
such as TNFα and IL-β may promote development of insulin resistance.45,53 In obese
St3gal5-deficient mice, insulin action was enhanced without any significant impact on
diet-induced obesity.51 These mice showed anti-inflammatory M2-like phenotypes invisceral adipose tissue (epididymal fat),51 and significant increases of adiponectin andinterleukin-10 (IL-10) in comparison with obese WT mice. IL-10 and adiponectin playessential roles in maintaining insulin sensitivity of adipocytes.72,73

of adipose tissue architecture occurs during its expansion. Under positive
caloric balance, development of metabolic disease is more closely related to
how fat is stored (through adipocyte hypertrophy vs. hyperplasia) than simply
to the amount of stored fat. Inhibitors of adipogenesis were frequently
proposed in the past as potential antiobesity drugs. However, results from avariety of recent mouse and human experiments indicate that adipogenesis
inhibitors are poor candidates for amelioration of metabolic disease states,
because limitation of fat cell expansion is associated with insulin resistance. ItBiology of GM3 Ganglioside167

was proposed over a decade ago that type 2 diabetes might result fromfailure of adipocyte differentiation.74 This hypothesis is now generally
accepted, and has been supported by several independent lines of investigation in adipocyte biology.
Interactions between macrophages and adipose progenitor cells must be
considered, because macrophages affect numbers of preadipocytes and/or
their differentiation capacity, and induce adipose tissue dysfunction by inhibiting overall adipogenic capacity. The existence of resident and infiltrating
macrophages is well documented, and studies during the past decade suggestthat such macrophages are modulated in obesity and type 2 diabetes.
Proinflammatory TNFα75 and IL1-β76 are potent inhibitors of adipocyte
differentiation, and both of these cytokines induce insulin resistance inadipocytes.77,78 TNFα expression is induced in adipose tissue of obese
diabetic rodents48 and humans.79 TNFα detected in adipose tissue has beenshown to be secreted from macrophages.80 These studies, taken together,indicate that macrophages in adipose tissue produce TNFα and IL1-β, which
can inhibit differentiation of preadipocytes and induce insulin resistance inmature adipocytes. Less attention has been paid to the ability of these cytokines to induce insulin resistance by inhibiting adipogenesis. Recent studies
using new model systems demonstrate that limitation of adipose tissue
expansion is associated with insulin resistance. Mice that are very obese
but have unlimited adipose tissue expansion are metabolically healthy andinsulin-sensitive.81 Overall, these studies support the concept that adequate
numbers of differentiation-competent preadipocytes allow for hyperplastic
growth by preserving metabolic function in obesity.
As described earlier, our studies using GM3S gene and GM3S-knockout
mice have shown that GM3 is crucially involved in both homeostatic adipogenesis (by controlling insulin signaling) and development of obesityinduced chronic low-grade inflammatory states and insulin resistance inadipose tissue. GM3 expression in adipocytes is controlled by soluble factors
secreted from resident macrophages, to execute physiological adipogenesis.
GM3 expression in adipose tissue is further increased under obesity-induced
proinflammatory conditions, and GM3S-deficient mice fed a high-fat
diet are less likely to develop insulin resistance or chronic low-grade inflammatory states (Fig. 4).51 GM3 thus functions as a novel homeostatic andpathogenic mediator in adipose tissue (Fig. 3).
Our findings provide a rationale for design of novel therapies against
metabolic syndrome (including type 2 diabetes) through inhibition of GM3
biosynthesis to maintain homeostatic insulin signaling. Extensive reduction

168

Jin-ichi Inokuchi et alof all complex gangliosides by inhibition of GM3 biosynthesis produces
widespread physical and chemical modifications of all cellular plasma membranes, particularly of lipid microdomains, and is therefore drastic and of little
therapeutic value. Such extensive ganglioside depletion is not necessary
for treatment of metabolic disorders. We have demonstrated the effectivenessof D-PDMP on impaired insulin signaling in TNFα-treated 3T3-L1 adipocytes, and that normalization of elevated GM3 levels was sufficient toameliorate the state of insulin resistance.45
In humans, GM3 is expressed predominantly in insulin-responsive organs(skeletal muscle, liver, adipose tissue) and lymphocytes. The presence ofGM3-enriched membrane microdomains (lipid rafts) reflects the characteristics of individual cells. For accumulation of gangliosides in lipid
rafts, critical roles are played by hydrogen donors and acceptors; and theabundance of (relative to phospholipids) long, saturated acyl chains in theceramide backbone of gangliosides. Structural diversity of sphingoid bases
and N-acyl chains in the ceramide moiety is essential in determining
the behavior of gangliosides in cell membranes, and the localization of lipid
rafts. A “sphingolipidomics” approach must be used to precisely characterize
the ceramide structures actually present in cell membranes.

4. LEPTIN SIGNALING AND RESISTANCE IN
HYPOTHALAMUS
Leptin is a 16-kDa protein required for maintenance of energy
homeostasis and body weight.82 White adipose tissue produces and secretes
leptin into the bloodstream, and circulating levels are correlated with body
fat content.83 Leptin signals adiposity status to the brain, particularly to themediobasal hypothalamus (MBH), which plays a key role in control of
feeding, body weight, and energy expenditure. Within the MBH, the arcuate nucleus (ARC) is an important site of leptin action. The ARC contains
two interconnected groups of neurons. One group is orexigenic and releases
agouti-related peptide (AgRP) and neuropeptide Y (NPY). The other groupis anorexigenic and releases α-melanocyte-stimulating hormone (α-MSH),
generated from proopiomelanocortin (POMC) precursors, which binds tomelanocortin receptor 4 (MC4R) in the paraventricular nucleus (PVH) andother hypothalamic regions, and exerts anorectic effects. NPY binds to its
cognate receptor, thereby stimulating feeding and reducing basal energy
expenditure. AgRP acts as an inverse agonist of MC4R and inhibits

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α-MSH activity.84 AgRP/NPY neurons and POMC neurons thereby
regulate melanocortin signaling in a coordinated manner to maintain energybalance.
Elevated levels of leptin reduce food intake and body weight through thelong form of the leptin receptor, LepRb,85 which is highly expressed incertain hypothalamic nuclei, including the ARC and ventromedial hypothalamus (VMH). LepRb is expressed in both AgRP/NPY neurons and
POMC neurons. Leptin regulates energy balance by inhibiting neuronal
activity and expression of NPY and AgRP in AgRP/NPY neurons, andpromoting synthesis of α-MSH in POMC neurons.86 Leptin increase cangenerate a strong, beneficial signal to prevent or reduce obesity. However,leptin activity may be disrupted by development of leptin resistance, aphenomenon defined by reduced ability to suppress food intake and bodyweight gain.87
Gangliosides play important roles in neural development and functions.88,89 Recent studies using ganglioside-deficient mouse models havehelped elucidate the functions of specific gangliosides in hypothalamic leptin
signaling.

4.1 Leptin Receptor Signaling
Leptin action is transmitted by long form receptor LepRb, which is highly
expressed in brain regions involved in control of feeding and energy expenditure.90 LepRb is a transmembrane receptor and a member of the class I
cytokine receptor family.85 Six LepR isoforms (LepRa, b, c, d, e, f) have been
identified in mice; they arise from the single Lepr gene by alternative mRNA
splicing,91,92 and have been categorized into three classes: secreted (LepRe),
short (LepRa, LepRc, LepRd, LepRf), and long (LepRb) forms. The six
isoforms share an N-terminal extracellular domain with the ability to bind
leptin, but differ in their C-terminal cytoplasmic domains. Only long-form
LepRb is capable of mediating leptin signaling. LepRb-deficient db/db mice
have an obese phenotype similar to that of leptin-deficient ob/ob mice.93
LepRb has no inherent enzymatic activity, but produces a signal throughbinding to a cytoplasmic Tyr kinase, Janus kinase 2 (JAK2).94 Leptin binding
to the extracellular domain of LepRb results in conformational change of thereceptor and JAK2 activation, and JAK2 subsequently phosphorylates otherTyr residues within LepRb and JAK2 itself.95 Three tyrosine residues
in LepRb (Tyr985, Tyr1077, Tyr1138) undergo phosphorylation, and recruit
Src homology 2 (SH2) domain-containing proteins to the LepRb-JAK2
complex, thereby enabling JAK2 to phosphorylate these signaling proteins.96

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In addition to JAK2, certain members of the Src Tyr kinase family are involvedin control of leptin signaling. Phospho-Tyr1138 binds to the SH2 domainof signal transducer and activator of transcription 3 (STAT3). STAT3 is subsequently phosphorylated by JAK2, resulting in nuclear translocation andtranscriptional initiation of target genes including POMC and suppressor ofcytokine signaling 3 (SOCS3), a negative-feedback regulator of leptin signaling.97 Numerous studies have demonstrated the essential role of the JAK2/
STAT3 pathway in antiobesity action of leptin in the brain. STAT5 is similarly
recruited to phospho-Tyr1077 in response to leptin, and phosphorylated andactivated by JAK2. Phospho-Tyr985 serves as a binding site for the SH2 domainof protein Tyr phosphatase 2 (SHP2), which mediates leptin-dependent
activation of the extracellular signal-regulated kinase (ERK) pathway. The
SHP2/ERK pathway is also involved in the antiobesity effect of leptin.98,99

4.2 Leptin Resistance
Leptin resistance is a key risk factor for obesity. Diet-induced obese (DIO)
mice and obese human patients typically show high circulating leptin levels
and reduced responsiveness to leptin’s antiobesity effects.87 Even after exogenous leptin administration, obese subjects do not show reductions offood intake or body weight.100 Various molecular mechanisms have been
proposed to explain leptin resistance; these include impairment of leptintransport across the blood–brain barrier (BBB), suppressed leptin signaling,
and impaired leptin-targeted neural circuits.96,101
4.2.1 The BBB and Leptin Resistance
Circulating leptin, as an energy regulator, must across the BBB to reach theneurons responsible for suppressing food intake and increasing energy
expenditure. Impaired leptin transport across the BBB has been observedin obese mice,102,103 suggesting a possible cause of leptin resistance in obese
humans and rodents.104 The median eminence (ME), located on the floor of
the third ventricle, forms a BBB structure that regulates passage of metabolic
hormones and nutrients from blood to cerebrospinal fluid and energysensing MBH neurons.105 Tanycytes, which are specialized radial glial cells
that line the walls of the third ventricle, have been identified as gatekeepers
controlling entry of leptin into the hypothalamus.106 Tanycytes express all six
LepR isoforms, activate STAT3 and ERK in response to leptin, and thereby
take up leptin through clathrin-coated vesicles. Internalized leptin is thenreleased into the MBH under the control of LepRb-ERK signaling. This
pathway is disrupted in DIO mice.

Biology of GM3 Ganglioside171

4.2.2 Suppression of LepRb Signaling
In obese rodents and humans, chronically elevated levels of circulating leptin
activate an inhibitory negative feedback mechanism, resulting in impairmentof LepRb signaling. SOCS3 is a member of a large family of cytokineinducible inhibitors of signaling. Its gene expression is induced by leptin,
and also by cytokines, such as interleukin-6.97,107 SOCS3 binds to JAK2 andinhibits its kinase activity,108 and also inhibits LepRb signaling throughbinding to phospho-Tyr985 of LepRb.109 Peripheral leptin administration
rapidly induces SOCS3 mRNA in the hypothalamus of leptin-deficient
ob/ob mice, but not of LepRb-deficient db/db mice.110 In leptin-resistantobese mouse model Ay/a, SOCS3 expression in the ARC and dorsomedial
hypothalamic nuclei is elevated, suggesting that SOCS3 is a mediator of
leptin resistance in obesity. Mice with heterozygous Socs3 deficiency orneuron-specific Socs3 knockout showed increased leptin sensitivity and resistance to DIO,111,112 supporting the concept that SOCS3 is a physiologicalnegative regulator of LepRb signaling.
LepRb signaling is also suppressed by many other molecules, including
protein Tyr phosphatases (PTPs). PTP1B, a nonreceptor Tyr phosphataseknown to inhibit insulin signaling, suppresses LepRb signaling in vivo bydephosphorylating and thereby inhibiting JAK2.113 PTP1B-null mice are
hypersensitive to leptin and resistant to DIO.113,114 Mice with neuronaldeletion or POMC-neuron-specific deletion of PTP1B show increased
leptin sensitivity and energy expenditure, and are less susceptible to
DIO.115,116 These findings suggest that increased PTP1B expression mediates leptin resistance in obesity.
4.2.3 Impairment of Downstream Signaling of LepRb
Melanocortin receptor 4 (MC4R) plays a central role as a leptin-targetedneural circuit in energy homeostasis. MC4R gene mutations with associatedloss of function are the most common monogenic form of obesity inhumans.117 Mc4r-deficient mice develop obesity associated with hyperphagia,118 and do not respond to the anorectic action of MTII, a synthetic
analogue of α-MSH.119 In Ay/a mice, melanocortin signaling is systemicallydisrupted by ectopic expression of agouti signaling protein (ASP), a homologue of AgRP.120 Constitutive expression of ASP in the Ay/a hypothalamus
blocks the effect of α-MSH and inhibits activation of MC4R, resulting in
disruption of hypothalamic control of feeding and energy expenditure, andin development of leptin resistance.100 Downstream of MC4R, expression ofbrain-derived neurotrophic factor (BDNF) in the VMH is regulated by the

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leptin-MC4R axis, and inhibition of the BDNF/TrkB pathway results in
leptin resistance.121,122

4.3 Leptin Signaling in Ganglioside-Deficient Mice
Adult mammalian brain gangliosides consist mainly of a- and b-series gangliosides, such as GM1, GD1a, GD1b, and GT1b (Fig. 1).89 GM3S-deficient
mice that lack these ganglioside species have enhanced insulin signaling andare less susceptible to DIO.47,51 On the other hand, GD1a has an enhancing
effect on EGFR activation.123 In a 2013 study of mice with deletion of
inducible neuron-specific Ugcg (the gene encoding GlcCer synthase),
Nordstrom et al. reported possible functions of neuronal ganglioside species
in LepR.124 These mice lacked gangliosides, as well as GlcCer and LacCer indistinct populations of forebrain neurons following administration of tamoxifen. Loss of GlcCer synthase in hypothalamic neurons inhibited LepR
signaling, and the mice developed progressive obesity. The authors demonstrated that GM1 and GD1a in hypothalamic neuronal cell line N-41 interact
closely with LepR, suggesting that these a-series gangliosides enhance theaction of leptin in hypothalamic neurons. Ji et al., in 2015 and 2016 studies,
showed that GD3 synthase (GD3S)-null mice, which lack all b-series gangliosides, show reduced leptin secretion from adipocytes and increased leptin
sensitivity.125,126 Serum leptin levels in the GD3S-null mice were strongly
reduced and leptin was accumulated in adipose tissues, indicating impaired
leptin secretion. Addition of exogenous b-series gangliosides into medium of
primary adipocyte culture restored leptin secretion.125 These findings
suggest that b-series gangliosides are involved in leptin secretion from adipocytes, although the mechanism is unknown. GD3S-null mice did not
develop obesity, even though levels of circulating leptin were greatly
reduced. Leptin signaling was altered in the hypothalamus of these mice.
LepRb signaling in the hypothalamus, as measured by phosphorylated
STAT3 levels, was enhanced in the mice, and GD3S overexpression in
N-41 cells reduced responsiveness to leptin.126 Increased levels of a-series
gangliosides in GD3S-null mice enhance leptin sensitivity, which may
explain the apparently normal energy expenditure observed.
An obese mouse model, KK-Ay, was established by introducing Ay
mutation into KK mice, resulting in more severe and earlier onset of obesityand diabetic pathology.127 Based on our finding that GM3S-null mutation in
C57BL/6 mice resulted in no overt difference in obesity phenotype relative
to WT,47,51 we introduced GM3S-null mutation in KK-Ay mice to evaluate
the pathophysiological roles of GM3 and related gangliosides in obesity.

Biology of GM3 Ganglioside173

Whereas KK-Ay mice were hyperphagic and developed morbid obesity, theKK-Ay/GM3S-null mice had much lower body weight and food intake,comparable to values in control KK mice (unpublished data). The KK-Ay/
GM3S-null mice were less susceptible to central leptin resistance, suggestingthat GM3 and/or related ganglioside species negatively regulate hypothalamic leptin signaling.

4.4 Perspectives
Numerous recent studies indicate that GM3 and related gangliosides
play essential roles in hypothalamic control of energy homeostasis. GM1
and GD1a have been implicated as positive regulators of leptin signaling.
KK-Ay/GM3S-null mice, which do not express a- or b-series gangliosides, are less susceptible to leptin resistance. We observed enhancement of
leptin-dependent ERK phosphorylation in GM3S-deficient hypothalamic
neuronal cells (unpublished data), suggesting differential roles of specificgangliosides in LepR signaling (Fig. 5). Altered expression of gangliosides
may also affect other signaling pathways (e.g., insulin signaling) involved
in hypothalamic control of feeding and energy balance.

[(Fig._5)TD$IG]

Fig. 5 Leptin receptor-signaling pathway, and alterations in signals observed inganglioside-deficient models. Increased or decreased activation of each pathway
component is indicated by an arrow with respective color.

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There is increasing evidence for association of hypothalamic inflammation with development of leptin resistance and obesity.128 Activation of
inflammatory pathways in the hypothalamus is increased by DIO, even
without overt weight gain. GM3 levels are elevated in visceral fat of obese
model animals and in sera of obese patients.45,64,69 It is therefore conceivablethat inflammation resulting from DIO alters ganglioside expression in the
hypothalamus. Such alteration could affect interactions between neurons andnonneuronal cell populations and play a pathophysiological role in regulationof energy homeostasis.

5. T-CELL IMMUNITY AND IMMUNE DISORDERS
T cells are lymphocytes that have differentiated under the influence ofthe thymus and are responsible for cell-mediated immunity. T-cell activation,
mediated by T cell antigen receptors (TCRs) in combination with costimulatory receptors, requires recruitment of both extracellular and intracellularmolecules into specific cell membrane regions termed “lipid rafts.”129 T cells
are functionally subdivided into T helper (Th) cells and T cytotoxic (Tc)cells, which typically express specific cell surface molecules CD4 and CD8,
respectively. CD4+ Th cells and CD8+ Tc cells have distinctive immunological functions, but are similar in terms of mechanisms of TCR-mediatedsignaling. Th and Tc cells have differing ganglioside levels and gangliosidespecies composition, providing distinctive intracellular signaling events fortheir respective immune functions.130 This section summarizes gangliosidespecies-specific functions in activation of the two T cell subsets, and roles ofgangliosides in immune diseases.

5.1 Ganglioside Expression in CD4+ and CD8+ T-Cell Subsets
Gangliosides are classified into several series based on the absence (o-series)
or presence of one (a-series) or two (b-series) sialic acid residues linked
to the galactose residue in the second position from ceramide backbone(Fig. 1).131,132 GM3, the simplest member of the a-series, is synthesized by
GM3S. GM3 can be altered by GM2/GD2 synthase (GM2/GD2S, also
termed B4galnt1) to form GM2 (a downstream a-series ganglioside), or by
GD3S to form GD3 (the simplest b-series ganglioside). GM2/GD2S also
elongates LacCer to form GA2, the simplest precursor of the o-series gangliosides. At each branching point in the various ganglioside biosynthetic

Biology of GM3 Ganglioside175

pathways, competition among responsible enzymes determines the relative
expression levels of o-, a-, and b-series gangliosides.
T cells interact with and help activate antigen-presenting cells (APCs)
through a unique immunological synapse, and lipid rafts are involved in thisinteraction.133–137 GM1 (a downstream a-series ganglioside) accumulates atthe site of contact between TCRs and beads coated with TCR-stimulating
antibodies in CD4+ T cells, but not in CD8+ T cytotoxic cells.138 GM1 andGM3 define different types of raft membrane domains that segregate respectively to the leading pole or the trailing uropod of polarized human
T cells.139 Expression of GM1 varies depending on cell type and developmental stage, and staining with cholera toxin B subunit (CTx-B) is commonly used for visualization of rafts. CTx-B also reacts with an o-series
ganglioside termed extended-GM1b (Fig. 1). Elucidating the gangliosidecompositions of CD4+ and CD8+ T cells is essential for understanding
the role of rafts in differentiation, maturation, and activation processes ofthese two T-cell subsets. Differences have been reported in gangliosidecomposition of primary T-cell subsets freshly isolated from lymphoid organs
versus cultured T cells, including cloned cell lines and blasted cells stimulated
by concanavalin A.
Biochemical analyses (TLC, HPLC) of whole T cell populations havetraditionally been used to evaluate ganglioside expression patterns in primary
T cells. However, whole T cell populations (i.e., all cells that express TCRs)
are highly mixed populations, and such results are therefore of limited value
for studies of specific T cell subsets. Flow cytometric techniques allow
multiparameter analysis of target cells at the single-cell level, and therefore
provide a valuable tool for determining levels of specific gangliosides in each
T cell subset. Flow cytometric analyses using mAbs specific for various
gangliosides revealed differential ganglioside expression by mature CD4+
and CD8+ T cells.140,141 We used LC-MS/MS for structural analysis
of gangliosides in immature thymocytes, and in CD4+ and CD8+ T cells
isolated from mouse lymphoid organs.130 The two T cell subsets both express
six ganglioside species (GM1a, GM1b, GD1b, GD1c, GalNAcGM1b,
extended-GM1b), but differ markedly in their expression levels of thesesix species. Expression of o-series gangliosides (GalNAcGM1b, extendedGM1b) increases greatly following differentiation of thymocytes to CD4+
and CD8+ T cells. Most of the gangliosides expressed in CD8+ T cells areo-series. GM1b is expressed strongly in both subsets, whereas GM1a isexpressed strongly in thymocytes and CD4+ T cells; but at only trace levels
in CD8+ T cells.

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GM2/GD2S gene expression is markedly higher in CD4+ and CD8+ T
cells than in thymocytes. GM3S gene expression is higher in CD4+ T cells
and lower in CD8+ T cells relative to thymocytes.130 These gene expression
patterns account in part for the differential ganglioside expression profiles
during the differentiation processes from immature thymocytes to mature
CD4+ and CD8+ T cells. In regard to GD1c, CD4+ T cells can be separated
into GD1c-positive IL-2-producing Th1-like cells and GD1c-negative
IL-4-producing Th2-like cells.141 Thus, it is possible to classify T cell subsets
into distinctive functional subpopulations based on differences in gangliosideexpression profiles.

5.2 Ganglioside Expression in TCR-Mediated T Cell Activation
T cell activation is initiated and maintained by engagement of TCRs withmajor histocompatibility complex (MHC)-peptide complex on APCs, andby engagement of costimulatory molecules. CD4 and CD8 are coreceptors
that bind to nonpolymorphic regions of MHC and facilitate signaling by
TCRs during T cell activation. T cell activation requires recruitment of both
extracellular and intracellular molecules into specific cell membrane regions(lipid rafts).
Cholesterol, a major raft lipid component, plays a key role in maintaining
raft structure. It fills the space between hydrocarbon chains of sphingolipids,
and functions as a “glue” holding the raft assembly together. Treatment withdrugs (e.g., methyl-β-cyclodextrin) that deplete cholesterol from cell membranes typically results in disruption of raft-mediated cellular functions.142 In
spite of some concerns regarding side effects of the drug on signaling
events,143 experimental approaches involving methyl-β-cyclodextrin havebeen widely used to document the important functions of cholesterol in rafts.
Many studies have been based on manipulation of cholesterol level in lipid
rafts, but a much smaller number have addressed the role of gangliosides inraft functions.
We highlighted one important aspect of ganglioside expression in T cells
in our 2012 study of two strains of mice with distinctive genetic characteristics.130 One strain, carrying a defective GM3S enzyme, expressed o-series,
but not a- or b-series gangliosides. The other strain had an altered form of
GM2/GD2S enzyme, and expressed GM3 and GD3; but no other gangliosides (Fig. 1). In WT mice, expression of a-series GM1a is higher in CD4+
than in CD8+ T cells. In GM3S-null mice, CD4+ T cells show severe
impairment of TCR-mediated proliferation and cytokine production.
These defects are reversed by preincubation of cells with a-series177

Biology of GM3 Ganglioside(GM3, GM1a), but not with b-series gangliosides. Most gangliosides
expressed in WT CD8+ T cells are o-series (GA1, GM1b, GalNAcGM1b,
extended-GM1b). T cells from GM2/GD2S-null mice also show severe
impairment of TCR-mediated proliferation and cytokine production.
These defects are reversed by preincubation of cells with o-series GA1 andGM1b, but not with a- or b-series gangliosides. These findings indicate thatthe two T cell subsets have specific raft microenvironments involving different gangliosides, and that these raft microenvironments are the basis of
distinctive functions following TCR stimulation (Fig. 6). The two T cellsubsets, although they have common mechanisms of TCR-mediated signaling, have differing cellular and molecular modifications. CD4 and CD8 are[(Fig._6)TD$IG]
T helper cell

: a-Series
CD4 TCR: o-Series
St3gal5B4galnt1

Full activation

Thymocyte
TCR
CD4

CD8

Lineagecommitment

T cytotoxic cell
St3gal5

TCR

CD8

B4galnt1

Full activation

Fig. 6 Distinct expression signatures of ganglioside species during T-cell development.
Lineage commitment from thymocytes (CD4/CD8 double positive) to mature single
positive T cell subsets is accompanied by selective ganglioside expression. CD4+ T
helper (Th) cells express predominantly a-series gangliosides because of upregulationof St3gal5 gene, while CD8+ T cytotoxic (Tc) cells carry o-series gangliosides because of
downregulation of St3gal5 and upregulation of B4galnt1 genes. Evidently, each T cellsubset has unique lipid rafts composed of differing ganglioside species, and these rafts
are the basis for distinctive functions in intracellular events following TCR-mediatedstimulation. The ganglioside selection process is thus essential for formation of distinct,
functional lipid rafts in mature T cells.

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localized to lipid rafts by palmitoylation (a process whereby lipids are attached
to other molecules), but raft localization is not determined solely by thisprocess.144,145 To ensure movement of CD4 and CD8 to specific, correct
locations on the membrane, it may be essential that these molecules interact
with rafts carrying specific ganglioside species. Specific roles of the various
gangliosides in regulation of membrane microenvironments remain to beelucidated.

5.3 T Cell Gangliosides in Autoimmune and Allergic Diseases
Almost all T cells that recognize self or harmless antigens are killed orinactivated in normal individuals. Failure of normal self-tolerance mechanisms leads to development of autoimmune and allergic diseases. The four
T helper cell subpopulations (Th1, Th2, Th17, Treg) produce different
cytokines and have distinctive effector functions, presumably based on
differing organization of TCR signaling complexes in lipid rafts.146 Lipid
composition of lipid rafts and TCR signaling ability differ in Th1 versus Th2
cells.147–150 Studies of clinical cases and mouse models indicate that some ofthese disease processes are related to ganglioside composition of T cells; wewill review three of these diseases in this section.
Allergic asthma, a type 1 hypersensitivity reaction, is a manifestation of
immediate and late-phase reactions in the lung. Innate immune cells, such as
mast cells, basophils, and eosinophils are the effector cells of airway inflammation.151,152 Adaptive immunity contributes to initiation of allergic
reactions. CD4+ T cells play a crucial role in production of Th2 cytokine(IL-4 and IL-13), which then help B cells produce IgE antibodies specific forthe “harmless” antigens.153,154 We examined mechanisms of allergic airway
inflammation and airway hyperresponsiveness triggered by inhalation ofovalbumin (OVA) antigen in OVA-sensitized mice. GM3S-null mice
showed striking reduction of allergic airway responses normally induced
by OVA inhalation, that is, extensive mucus hypersecretion (characteristicof asthma), airway infiltration of inflammatory cells, OVA-specific IgE production, and increased Th2 cytokine levels in serum.130 CD4+ T cells fromthese mutant mice lack GM1a, and self-reactive CD4+ T cells display
enhanced GM1a expression resulting in persistence of abnormal cell activation.155,156 Karman et al. demonstrated suppression of airway inflammation
by administration of antisense oligonucleotides against GM3S gene.157
These findings indicate that a-series ganglioside GM1a is essential for
T helper cell function. The novel CD4+ T cell subsets Th17 and Treg were
described recently. In allergic airway inflammation, the balance between

Biology of GM3 Ganglioside179

effector Th2 cells and suppressive Treg cells is skewed toward Th2 predominance.158 It has been suggested that Th17 cells contribute to neutrophilic,steroid-resistant severe asthma and enhance Th2-mediated airway inflammation, although the precise role of Th17 cells in asthma remains unclear.159
GM3S-null mice displayed a reduction of Th17 cell number induced by invitro culture.160
Systemic lupus erythematosus (SLE), one of the most common autoimmune diseases, presents with multisystem clinical manifestations, including
rashes, arthritis, glomerulonephritis, hemolytic anemia, thrombocytopenia,
and central nervous system involvement.161,162 A variety of autoantibodies
are typically found in sera of SLE patients, and autoreactive T and B cells are
involved in pathogenesis of this disease. T cells from SLE patients have intrinsic
alterations of lipid components of lipid rafts.155,163,164 GM1a expression levels
in CD4+ T cells (but not CD8+ T cells) from SLE patients, in comparison withhealthy controls, are significantly higher. LacCer, GA2, Gb3, GM1a, and GD1a
are also upregulated in SLE CD4+ T cells.165 Increased GM1a expression ismore marked in CD45RO-positive CD4+ T cells (memory T cells) from active
SLE patients than from inactive SLE patients.155 Activated human T cells show
upregulation of GM1a levels.155,164,166,167 GM1a expression is also enhanced inself-reactive CD4+ T cells, resulting in persistence of abnormal cell activation.155,156 These findings, taken together, clearly indicate that gangliosides are
key factors in SLE pathogenesis and pathology. In a 2014 study by McDonald
etal., SLE CD4+ T cells were stimulated for 72 h with a combination of antiCD3 mAb, anti-CD28 mAb, and GlcCer synthase inhibitor NB-DNJ. In thetreated cells, expression levels of GM1a and LacCer were similar to thoseobserved in healthy controls, and defects in TCR signaling were partiallyreversed.165 Expression levels of cellular GSLs are determined by the coordinated effects of de novo synthesis, turnover, and recycling.168 The increased
GSL expression in SLE CD4+ T cells was associated with accelerated internalization of GSLs from plasma membrane into intracellular compartments andrapid recycling of GSLs back to plasma membrane, resulting in a net increase inplasma membrane expression. SLE T cells display both increased GSL biosynthesis and accelerated trafficking to and from plasma membrane, which
presumably lead to aberrant accumulation of gangliosides in lipid rafts.169
Rheumatoid arthritis (RA), another common autoimmune disease, ischaracterized by synovial inflammation associated with destruction of joint
cartilage and bone.170 Both cell-mediated and humoral immune responses
evidently contribute to RA development. Fluid around inflamed synovia
and joints contains numerous types of inflammatory cells (Th1 cells, Th17

180

Jin-ichi Inokuchi et alcells, activated B cells, macrophages) and cytokines (IL-1, IL-8, TNFα, IL-6,
IL-17, IFNγ).171,172 GM3 and GM3S gene expression levels were higher insynovia of RA patients than in those of patients with osteoarthritis, which is
not an autoimmune disease.173 Progression of collagen-induced inflammatory arthritis, a mouse model of RA, was accelerated in GM3S-null mice,and induction of IL-17-producing cells in regional lymph nodes wasenhanced by collagen immunization.173 In another report, on the other
hand, GM3S-null mice exhibit a decreased number of Th17 cells induced
by in vitro culture.160 Future studies will clarify whether a-series gangliosides
expressed in Th17 cells are involved in RA development. CD4+ T cells
can be separated into GD1c-positive IL-2 producing Th1-like cells and
GD1c-nagative IL-4 producing Th2-like cells.141 It appears likely that each
subpopulation of CD4+ T cells has a unique, characteristic gangliosideexpression pattern in its lipid rafts, and these patterns are responsible for
specific functions of Th effector cells.

5.4 Perspectives
Results of numerous recent studies indicate that pathogenesis of allergic andautoimmune diseases are controlled in part by a variety of lipid rafts withdiffering ganglioside profiles in individual CD4+ T cell subsets. Differentiation
events of T cells in thymus are associated with differential ganglioside expression patterns in individual T cell subsets. The various subpopulations ofeffector CD4+ T cells in peripheral lymphoid organs appear to have unique
ganglioside expression patterns in their rafts, which are responsible for specificTh functions. Thus, the ganglioside selection process is essential for formationof distinct, functional lipid rafts in mature T cells. Although allergic reactions
share certain common features, they differ greatly in the types of antigens
involved. Reduction of a-series gangliosides by GM3S inhibition, andconsequent modulation of ganglioside expression in lipid rafts, is potentiallya powerful technique for therapy of immune system disorders.

6. GM3S DEFICIENCY AND HEARING LOSS
6.1 GM3S Deficiency in Humans
GM3S deficiency is a rare neurological disorder in which mutationsof this enzyme result in a complete lack of GM3 and its downstream
biosynthetic derivatives. The clinical manifestations of GM3S

Biology of GM3 Ganglioside181

deficiency include infantile-onset epilepsy, severe intellectual disability,
irritability, failure to thrive, blindness, choreoathetosis, and cutaneous
dyspigmentation. The disorder was first reported in Old Order
Amish174,175 and more recently in French and Korean families orindividuals.176,177
Infants with GM3S deficiency display feeding difficulties and vomiting
within the first few weeks after birth, which prevent normal growth andweight gain. Seizures begin to occur within the first year and worsen overtime. Multiple types of seizures have been reported, and include symptoms,
such as muscle rigidity, convulsions, and loss of consciousness. Affectedchildren typically have severe intellectual disability and do not develop skills,
such as speaking, unsupported sitting, walking, and reaching for objects.
Vision and hearing abilities decline over time, although they are present
at birth.174–182 In some cases, there are alterations of skin coloring on thearms, legs, and face (e.g., dark freckle-like spots or light patches),178 or
respiratory chain dysfunction.
Two homozygous mutations have been reported: c.862C>T(p.Arg288*) and c.994G>A (p.Glu332Lys). The nonsense mutationc.862C>T in Old Order Amish patients results in protein truncation
between sialyl motif L and S. The c.994G>A mutation in an
African–American family, within sialyl motif S, results in disruption of
sialyl motif S function (interaction with LacCer acceptor and a sugar
donor). The reported Korean patients had compound heterozygous
and missense mutations in sialyl motif L (c.584G>C/p.Cys195Ser, andc.601G>A/p.Gly201Arg). These patients displayed various Rett-like phenotypes, but not epilepsy or blindness. These differences in symptoms may
reflect differences in the mutation region of ST3GAL5. Humans who lack
all a- and b-series gangliosides because of ST3GAL5 c.694C>T mutations
display major disruptions of auditory function, neonatal-onset hearing
impairment, weak or absent middle ear muscle reflexes (MEMRs) anddistortion product otoacoustic emissions (DPOAEs), abnormal auditory
brainstem response (ABR) morphology and thresholds, absent cochlear
microphonics (CMs), and delayed cortical auditory evoked potentials(CAEPs). Absent DPOAEs and CMs indicate severe outer hair cell(OHC) impairment. The presence of ABRs in seven of eight patients inour 2015 study suggested at least partial inner hair cell (IHC) function.
However, numerous abnormalities revealed by MEMR and ABR testing
indicated significant dysfunction of IHCs and/or central auditory
pathways.181

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6.2 Significant Increase of GSLs During Postnatal Maturation of
Murine Cochlea
We investigated GSL expression in WT murine cochlea during the postnatal
period of auditory system maturation. Mice began to recognize sound by
postnatal day 12 (P12), designated the “onset of hearing.” On P1, GM3 was
the dominant cochlear GSL. After P3, there were marked increases ofcochlear GM3 GlcCer, complex gangliosides (GM1, GD1a, GD3, GD1b,
GT1b), and sulfatides (SM3, SM4). In contrast, cerebral gangliosides
achieved stable, mature expression as early as P3.181
Significant alterations of ganglioside expression in murine cochlea afterP3 reflect their essential role in hearing onset.181 Following auditory maturation, GM3 and GM1 are distributed discretely among IHCs, OHCs,
Deiters cells, and pillar cells of the organ of Corti (Table 1). Both IHCs
and OHCs express GM3, which is localized on the apical surface, cuticularplate, and stereocilia. GM1 is the primary IHC ganglioside. It is also thepredominant ganglioside on the apical surface of supporting cells, but islargely absent from OHCs.181

6.3 Glycocalyx Integrity and Membrane Cycling in
GM3S-Deficient Mice
Apical membranes of stereocilia are covered with a glycocalyx composed ofsialic acid-containing glycoproteins and glycolipids (including gangliosides),183 creating a dense negative charge field that normally prevents fusionof adjacent stereocilia. In animal models, aminoglycoside administration
reduced expression of sialoglycoconjugates in the OHC glycocalyx andenhanced fusion of stereocili.184,185
To elucidate the roles of various ganglioside species in hearing, we
compared auditory function in GM3S-null versus GM2/GD2S-null mice,
which have distinctive GSL expression patterns within the organ of Corti.
GM2/GD2S-null cochlea had high GM3 content and was associated with
Table 1 Comparison of GM3 and GM1 Expression of Specific Regions in the Organof Corti at P14.
OHC
IHC
DC
PC
Stereocilia Cuticular Cell Stereocilia Cuticular Cell Cell
Cell Cell
Plate
Body
Plate
Body Surface Body Body

GM3 ++
GM1 

+





++
++

+
+


+

DC, Deiters cell; IHC, inner hair cell; OHC, outer hair cell; PC, pillar cell.


++

+
+


+

Biology of GM3 Ganglioside183

intact hair bundles and normal auditory function; whereas GM3S-nullcochlea had no GM3 and was associated with deafness. Confocal
laser microscopy with phalloidin staining revealed normal IHC and OHC
morphology in 4-week-old GM2/GD2S-null mice. In contrast, OHCs in
GM3S-null mice had blebs and intracellular vesicles composed of membranous structures, indicative of lack of balance between endocytosis andexocytosis.181 GM3 is expressed in IHCs and OHCs of WT mice, whereas
only IHCs express GM1. This may account for the observation that OHCs
in GM3S-null mice degenerate prior to IHCs.

6.4 GM3-Enriched Membrane Organization, PTPRQ-Myosin
VI Complex Localization and Hair Cell Morphology
Cochlear hair cells are specialized for auditory and vestibular transduction.
Projecting from their apical surface are filopodial processes (stereocilia) that
contain hundreds of cross-linked actin filaments. Proteins, such as unconventional myosins, Usher proteins, and deafness-related gene products[protein Tyr phosphatase receptor Q (PTPRQ), cadherin23, protocadherin15, usherin, VLGR1] are expressed predominantly or exclusively instereocilia, forming a structured interactive network for mechanoelectricaltransduction.186
In the normal organ of Corti, functional PTPRQ-myosin VI complexes
are essential to maintain integrity of this network.187 PTPRQ, a shaft
connector at the tapered base of stereocilia, consists of an extracellulardomain with 18 fibronectin III (FNIII) repeats, a membrane spanningdomain, and a cytoplasmic domain having phosphatidylinositol and Tyr
phosphatase activities.187,188 Myosin VI is an actin-based motor protein
whose complex with PTPRQ mediates interactions between plasma membrane and cytoskeleton.189 PTPRQ-deficient mice are deaf and have fused
stereocilia that do not taper at the base.190 Myosin VI-deficient mice arealso deaf and have comparable structural changes of hair cells, accompanied
by maldistribution of PTPRQ along the stereocilia.190–192
In bullfrogs (Rana catesbeiana), PTPRQ is colocalized with gangliosiderich membrane domains in basal stereocilia.193 We examined the relationshipbetween gangliosides and basal PTPRQ-myosin VI complex by immunostaining (Fig. 7). PTPRQ is localized exclusively in bases of stereocilia in
WT murine IHCs. In contrast, in GM3S-null mice PTPRQ is maldistributed along shafts of fused stereocilia (Fig. 7B), and myosin VI is present frombase to midshaft but absent from distal regions (Fig. 7A). These structural
disruptions presumably result in loss of normal ciliary motor action. In

184

Jin-ichi Inokuchi et al[(Fig._7)TD$IG]

IHC

(A)
Myosin VI

OHC
Merge

Myosin VI Merge

+/+

–/–
5 m

5 m

(B)

PTPRQ

Merge

PTPRQ

Merge

+/+
–/–
5 m

5 m

(C)
St3gal5 –/– mice

WT mice

Myosin VI
PTPRQFig. 7 Maldistribution of protein Tyr phosphatase receptor Q (PTPRQ) and myosin VI in
stereocilia of GM3S-null mice. (A) Confocal images show stereocilia of inner hair cells(IHCs) and outer hair cells (OHCs) from WT and GM3S-null mice stained for myosin VI,(B) PTPRQ (green), and F-actin (phalloidin; magenta). (C) Schematic depiction of
maldistributed PTPRQ and myosin VI in GM3S-null IHC stereocilia.181

OHCs of GM3S-null mice, myosin VI is expressed mostly at the cuticularplate surface, close to vestigial kinocilia.
These findings, taken together, indicate that GM3-enriched membrane
microdomains are essential for formation and proper localization ofBiology of GM3 Ganglioside

185

PTPRQ-myosin VI complexes in hair cells. Aberrant expressionof PTPRQ-myosin VI complexes resulting from absence of GM3 alters
the structure of stereocilia and impairs their ability to transduce auditory
signals (Fig. 7C).

6.5 Perspectives
GM3 plays a specific and essential role in development, function, andviability of cochlear hair cells. In humans with GM3S deficiency, hearing
is already impaired at birth. However, studies of auditory phenotype in
GM3S-null mice suggest that ganglioside addition may partially restore
auditory function during a critical postnatal “window.” These findings are
relevant to our understanding of hearing loss in humans with GM3S deficiency, but do not explain the broader neurological phenotype associatedwith this condition. Further studies are needed to more precisely characterize the role of lipid rafts generally, and GM3 specifically, in development andfunction of the central nervous system.
New concepts and techniques are giving rise to novel strategies to reverse
hearing impairment. Oshima et al. generated functional hair cell-like cells
from embryonic and induced pluripotent stem cells.194 Such strategies will
help promote research on the auditory functions of GSL-enriched microdomains, and lead to new, more effective therapies to overcome hearing loss
in humans.

7. CONCLUSIONS AND FUTURE DIRECTIONSFunctional roles of GM3 have been investigated indirectly based on
addition of exogenous GM3 to culture medium, or on depletion of precursor
GSLs by GlcCer synthase inhibitors.195 Results of these studies suggested
that GM3 is involved in transmembrane signaling through regulation of
growth factor receptor activities, and in cell adhesion and motility throughinteraction with integrins and/or integrin-related molecule(s). However, it
was not possible to definitively conclude that the effects of exogenously
added or pharmacologically depleted gangliosides reflected the physiological
functions of endogenous GM3.
Our studies during the past decade using GM3S-null mice havehelped demonstrate the crucial roles of GM3 in diabetes, insulin resistance, leptin resistance, helper T cell function, and hearing function. Ourfindings provide a foundation for novel therapies against metabolic

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Jin-ichi Inokuchi et al

syndrome, type 2 diabetes, and immunological disorders (e.g., allergies),
based on inhibition of GM3 biosynthesis. Extensive depletion of all
gangliosides by GM3 biosynthesis inhibition results in excessive physicaland chemical modifications of all plasma membranes, and particularlyof lipid microdomains in membranes. However, effective treatmentof metabolic disorders will not require such extensive ganglioside depletion. In our study of D-PDMP effect on impaired insulin signaling in
TNFα-treated 3T3-L1 adipocytes, normalization of elevated GM3 levels
was sufficient to restore proper insulin resistance status.130 We expectthat comprehensive studies of functional suprabiomolecular complexes
consisting of gangliosides and functional proteins in microdomains
will lead to a novel concept and strategy of “membrane microdomain
orthosignaling therapy.”
Our studies have shown that GM3 functions as a physiological regulatory
factor to maintain homeostasis in adipocytes by modulating insulin signaling,45,51,53,130 and that serum GM3 concentration is elevated in patients withmetabolic syndrome.69 Further analysis of these sera by LC-MS/MS revealed
increased levels of GM3 containing very long acyl chains with or without
α-hydroxy modification (C24:0, αOH-C24:0), and reduced levels of GM3
carrying long acyl chains (C16:0, C18:0).69 Inflammatory cytokines, hypoxic
conditions, and ER stress increase the biosynthesis of GM3 in the adipocytes,
and inflammatory reactions in GM3S null mice are suppressed when thesemice are fed with high fat diets.51 Our ongoing studies are focused on
the molecular mechanisms underlying these responses. We have observedthat C24:0-GM3 and αOH-C24:0-GM3 induce secretion of inflammatory
cytokines when monocytes or macrophages are stimulated by TLR4 ligand,
LPS, or HMGB1; whereas C16:0-GM3, 18:0-GM3, and 24:1-GM3 suppress
secretion of inflammatory cytokines. The effects are triggered by inductionof TLR4/MD2 supramolecular complex formation and upregulation of
signal transduction. Based on these findings, we propose a novel inflammation
loop triggered by specific GM3 molecular species (MS submitted).

ACKNOWLEDGMENTS
The authors’ studies described in this review were supported by the Core Research for
Evolutional Science and Technology division of the Japan Science and Technology Agency(to J.I.), a Grant-in-Aid for Scientific Research (B) from MEXT (J.I.) (16H04767), Mizutani
Foundation for Glycoscience (J.I.), Naito Foundation (J.I.), ONO Medical Research
Foundation (J.I.), Uehara Memorial Foundation (J.I.), Yamada Science Foundation (J.I.), andthe MEXT-Supported Program for the Strategic Research Foundation at Private Universities(S1201031). The authors are grateful to Dr. S. Anderson for English editing of the manuscript.

Biology of GM3 Ganglioside187

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Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2018/06/07/jlr.M085753.DC1.html

Deﬁcient ganglioside synthesis restores responsivenessto leptin and melanocortin signaling in obese KKAy miceKei-ichiro Inamori,* Hideki Ito,* Yumi Tamura,* Takahiro Nitta,* Xiaohua Yang,* Wataru Nihei,*†§§§
Fumi Shishido,* Susumu Imazu, Sohei Tsukita, Tetsuya Yamada, Hideki Katagiri, and1,
Jin-ichi Inokuchi *Abstract GM3, a precursor for synthesis of a- and b-series
gangliosides, is elevated in adipocytes of obese model animals and in sera of obese human patients with type 2 diabetes
and/or dyslipidemia. GM3 synthase (GM3S)-KO C57BL/6
mice display enhanced insulin sensitivity and reduced development of high-fat diet-induced insulin resistance. However,the pathophysiological roles of GM3 and related gangliosides in the central control of feeding and metabolism remain unclear. We found that a mouse model (KKAy GM3S
KO) generated by KO of the GM3S gene in the yellow obese
strain, KKAy, displayed signiﬁcant amelioration of obese
phenotype. Whereas KKAy mice were hyperphagic and developed severe obesity, KKAy GM3S KO mice had signiﬁcantly lower body weight and food intake, and greater glucose
and insulin tolerance. The hypothalamic response to intraperitoneal administration of leptin was greatly reduced in
KKAy mice, but was retained in KKAy GM3S KO mice. In
studies of a cultured mouse hypothalamic neuronal cell line,
enhanced leptin-dependent phosphorylation of ERK was observed in GM3S-deﬁcient cells. Furthermore, KKAy GM3S
KO mice did show altered coat color, suggesting that GM3S
is also involved in melanocortin signaling. Our ﬁndings,
taken together, indicate that GM3-related gangliosides play
key roles in leptin and melanocortin signaling.—Inamori,
K-i., H. Ito, Y. Tamura, T. Nitta, X. Yang, W. Nihei, F. Shishido,
S. Imazu, S. Tsukita, T. Yamada, H. Katagiri, and J-i. Inokuchi.
Deﬁcient ganglioside synthesis restores responsiveness toleptin and melanocortin signaling in obese KKAy mice.
J. Lipid Res. 2018. 59: 1472–1481.
Supplementary key words ANIMAL MODELS s DIABETES s BRAIN s RECEPTORSHORMONE s '- s HYPOTHALAMUS s LEPTIN RESISTANCE s '- SYNthase knockout

This study was supported by Ministry of Education, Culture, Sports, Scienceand Technology Grants-in-Aid for Scientiﬁc Research 26440061 (K-i.I.); and
16H04767 ( J-i.I.), the Uehara Memorial Foundation (K-i.I.), the Kato Memorial Bioscience Foundation (K-i.I.), the Novartis Foundation (Japan) for the
Promotion of Science (K-i.I.), and the Suzuken Memorial Foundation (K-i.I.).
Manuscript received 5 April 2018 and in revised form 22 May 2018.
Published, JLR Papers in Press, June 7, 2018
DOI https://doi.org/10.1194/jlr.M085753

Gangliosides [glycosphingolipids (GSLs) that containsialic acid] are key components of membrane microdomains, where they participate in a variety of biologicalprocesses, including cell growth, adhesion, and signaltransduction (1). GM3, a precursor molecule in synthesisof a- and b-series gangliosides, shows increased levels in association with obesity, inﬂammation, and certain metabolic
diseases (2–4). GM3 synthase (GM3S)-null mice display enhanced insulin sensitivity and reduced development ofhigh-fat diet (HFD)-induced insulin resistance and chronic
low-grade inﬂammatory states (4, 5). Our group observed
associations between numerous metabolic disease risk factors and GM3 molecular species in human serum (6).
The mediobasal hypothalamus, which includes the arcuate nucleus (ARC) and paraventricular nucleus (PVH),
plays essential roles in control of feeding, body weight, andenergy expenditure. ARC contains two interconnectedgroups of neurons; one is orexigenic (appetite-stimulating)and releases agouti-related peptide (AgRP) and neuropeptide Y (NPY), while the other is anorexigenic (appetite-suppressing) and releases ␣-melanocyte-stimulating hormone(␣-MSH) produced from proopiomelanocortin (POMC)
precursors. ␣-MSH binds to melanocortin receptor 4(MC4R) in PVH and other hypothalamic areas, and exerts
anorectic effects. NPY binds to its cognate receptor, stimulates feeding, and reduces basal energy expenditure. AgRP
acts on MC4R as an inverse agonist and inhibits ␣-MSH (7).
AgRP/NPY neurons and POMC neurons thus regulate

Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus;
ASP, agouti signaling protein; ␣-MSH, ␣-melanocyte-stimulating hormone; GM3S, GM3 synthase; GSL, glycosphingolipid; HFD, high-fat
diet; JAK2-STAT3, Janus-activated kinase 2/signal transducers and activators of transcription 3; LepRb, leptin receptor long form; MC1R,
melanocortin receptor 1; MC4R, melanocortin receptor 4; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVH, paraventricular nucleus;TG, triglyceride.
1
To whom correspondence should be addressed.
e-mail: jin@tohoku-mpu.ac.jp
The online version of this article (available at http://www.jlr.org)
contains a supplement.
Copyright © 2018 Inamori et al. Published under exclusive license by The American
Society for Biochemistry and Molecular Biology, Inc.

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Division of Glycopathology,* Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, Sendai, Miyagi 981-8558, Japan; Nippon Boehringer Ingelheim Company, Ltd.,†
Shinagawa-Ku, Tokyo 141-6017, Japan; and Department of Metabolism and Diabetes,§ Tohoku University
Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2018/06/07/jlr.M085753.DC1.htmlMA). Phospho-ERK (E-4) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant mouse leptin was fromR&D Systems (Minneapolis, MN).

Generation of GM3S-deﬁcient mice in KK/KKAy
background
KK (a/a) and KKAy (Ay/a) mice were from CLEA Japan. GM3S
(St3gal5)-deﬁcient mice were generated from C57BL/6 mice as
described previously (16). These mice were backcrossed overeight times into the KK background, and KK/GM3S+/⫺ or KK/⫺/⫺
GM3S
females were crossed with KKAy males to obtain KKAy/
+/⫺
y
GM3S
mice. Genotyping for the a/A alleles was performed by
PCR of ear DNA using primers 5′-GGCCACACTTAGGGAGTTCA-3′y(forward primer for a ‘nonagouti’ and A ‘lethal yellow’ alleles),5′-TGCCTTATTCTGTTTTTGTTCC-3′ (reverse primer for aallele), and 5′-GGTTGGCCACCATGTCTAGT-3′ (reverse primeryfor A allele), according to the Jackson Laboratory website (KK.
yCg-A /J 002468). Genotyping PCR for the GM3S gene was performed using the following primers: 5′-GGAATCCATCCCTTTTCTCACAGAG-3′ and 5′-TGAACTCACTTGGCATTGCTGG-3′ for WT
allele, and 5′-ACTGGGCACAACAGACAATCGG-3′ and 5′-TGGATACTTTCTCGGCAGGAGC-3′ for the KO allele (17). KK and
KKAy mice with WT GM3S alleles were used as control for eachexperiment. Body weight and food consumption were measured
once per week.

GSL analysis
Lyophilized brain tissue was extracted with chloroform/methanol 2:1, 1:1, 1:2 (v/v), in succession. Total lipids were puriﬁed,
separated into acidic and neutral fractions, deesteriﬁed by methanolysis, and analyzed by HPTLC as described previously (18).

RNA isolation, reverse transcription, and quantitative PCR
Total RNA was extracted using the RNeasy Lipid Tissue Minikit (Qiagen). cDNA was generated from 500 ng of RNA, andquantitative PCR was performed on a StepOnePlus real-time PCRsystem (Applied Biosystems) using SYBR Premix Ex Taq II(TaKaRa Shuzo, Kyoto, Japan). Relative mRNA levels were calculated either by a standard curve and normalized to peptidylprolyl–⌬⌬±CT
isomerase B (Ppib) expression or as 2
with glyceraldehyde3-phosphate dehydrogenase.

Glucose and insulin tolerance tests
Mice were either fasted for 16 h and administered glucose solution (1.5 g/kg body weight) orally or fasted for 6 h and injected
intraperitoneally with insulin solution (0.75 U/kg body weight).
Glucose levels in blood (from tail vein) at various time points before and after these treatments were measured with an Accu-Chek
Aviva glucometer (Roche Applied Science).

Plasma parameters
Plasma levels of triglycerides (TGs), NEFAs, total cholesterol,and HDL cholesterol were determined using (respectively) the
Triglyceride E-test kit, the NEFA C-test kit, the Cholesterol E-test
kit, and the HDL-Cholesterol E-test kit (Wako Pure Chemical,
Japan). Serum insulin and leptin levels were determined using
ELISA kits (Morinaga Institute of Biological Science, Yokohama,Japan).

Generation of GM3S-deﬁcient N41 cells

MATERIALS AND METHODS
Reagents
Phospho-STAT3 (Tyr705, D3A7), STAT3 (79D7), ERK1/2,
and c-fos antibodies were from Cell Signaling Technology (Beverly,

Exon 5 of the mouse St3gal5 (GM3S) gene, which contains thecoding sequence for sialyl motif L, was chosen to design targeting
guide RNAs using the online CRISPR Design Tool (19). A primer
pair (5′-CACCGCAGGTACGCGAAGACGGCTA-3′ and 5′-AAACTAGCCGTCTTCGCGTACCTGC-3′) was cloned into plasmid

GM3S KO ameliorates hyperphagia and obese phenotype

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melanocortin signaling in a coordinated manner to maintain energy balance.
Leptin, an adipocyte-derived circulating hormone whose
expression is correlated with adipose mass, is essential for
maintenance of energy homeostasis and body weight. Increased levels of leptin lead to reduction of food intake andbody weight through the long form of its receptor, LepRb(8), which is highly expressed in ARC, ventromedial hypothalamic nucleus, and other hypothalamic nuclei. LepRb isexpressed in both AgRP/NPY neurons and POMC neurons.
Leptin inhibits AgRP neuronal activity and NPY and AgRP
expression in AgRP/NPY neurons, and stimulates POMC
synthesis in POMC neurons (9).
Diet-induced obesity leads to a state of low-grade inﬂammation in peripheral insulin target tissues that is associatedwith development of insulin resistance and type 2 diabetes(10). Diet-induced activation of inﬂammatory pathways inthe mediobasal hypothalamus has been reported (11).
There is increasing evidence for an association between hypothalamic inﬂammation and development of leptin resistance and obesity (12), but the detailed molecular basis forsuch an association is unclear.
An obese diabetic mouse model, KKAy, was establishedby introducing Ay mutation into strain KK, resulting in ectopic expression of agouti signaling protein (ASP), which
is homologous to AgRP. ASP acts as an antagonist at themelanocortin 1 receptor (MC1R) on hair follicular melanocytes to block ␣-MSH-induced eumelanin production,
resulting in the subterminal band of pheomelanin (13). In
the KKAy mouse, it is believed that the ectopic expressionof ASP causes obesity due to disruption of melanocortin
signaling outside the hair follicle. Onset of obesity and diabetic pathology appear earlier and are more severe in
KKAy mice than in KK mice (14, 15). We and Proia’s groupobserved previously that body weight following a HFD feeding regime did not differ signiﬁcantly between GM3S-null
C57BL/6 mice and WT mice (4, 5). In the present study,
we generated GM3S-null mice in a KKAy genetic background (KKAy GM3S KO) to evaluate the pathophysiological roles of GM3 and related gangliosides in obesity. KKAy
mice are hyperphagic and typically develop leptin resistance and morbid obesity. In contrast, KKAy GM3S KO
mice were less likely to develop leptin resistance and hadsigniﬁcantly lower body weight and food intake, comparable to values in control KK mice. The coat color was grayish
in homozygous KKAy GM3S KO mice, but yellow in heterozygotes (similar to KKAy mice), suggesting that GM3 isinvolved in melanin synthesis, possibly through MC1R signaling. Our ﬁndings indicate that GM3 and/or related
ganglioside species play an essential role in leptin and melanocortin signaling.

Supplemental Material can be found at:
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pSpCas9(BB)-2A-GFP (Addgene; #48138). The plasmid was introduced into embryonic mouse hypothalamic N41 cells (CELLutions
Biosystems, Ontario, Canada) using a Nucleofector device with
Cell Line Nucleofector Kit V (Amaxa), as per the manufacturer’s
protocol. The above plasmid without targeting sequence served ascontrol. GFP+ cells were isolated using a FACSAria II ﬂow cytometer(BD Biosciences); clonal cell lines were obtained by limiting dilution; and expression levels of gangliosides were evaluated by TLC.

Leptin administration and immunohistochemistry

Lentiviral construct and establishment of stably
LepRb-expressing cells
To generate a lentiviral construct for stable expression of fulllength mouse leptin receptor (mLepRb), a 3.5 kb mLepRb cDNA
fragment was subcloned into CSII-EF-RfA vector (developed by
Dr. H. Miyoshi, Keio University, Tokyo, Japan and provided by
RIKEN BioResource Research Center, Tsukuba, Japan). HEK293T
cells cultured in DMEM supplemented with 10% FBS were transfected with mLepRb/CSII-EF-RfA, pCAG-HIVgp, and pCMV-VSVG-RSV-Rev plasmids using Lipofectamine 2000 (Invitrogen), andincubated for 24 h at 37°C. Medium was replaced by fresh medium
containing 10 ␮M forskolin, and cells were cultured for another24 h at 32°C. Lentivirus-containing supernatant was harvested bycentrifugation at 200 g for 3 min. For lentiviral transduction ofcultured N41 cells, the supernatant was added; the cells were incubated for 24 h at 32°C; the temperature was increased to 37°C; theincubation was continued for 48 h; the medium was replaced; andthe culture was continued for 1 week prior to experiments. Cells
established by this procedure were designated as N41-LepRb cells.

Leptin treatment of N41 cells and immunoblotting
Phosphorylation assays of ERK and STAT3 were performed using
N41 and N41-LepRb cells, respectively. Near-conﬂuent cells were
washed twice with DMEM, incubated for 2 h with DMEM containing
0.5% FBS, stimulated for various durations with 0.5 ␮g/ml leptin,
washed twice with ice-cold PBS, lysed, and analyzed by Western
blotting with appropriate antibodies, as described previously (2).

Statistical analysis
Data were analyzed by Student’s t-test and ANOVA followed by
Tukey’s post hoc test, using the GraphPad Prism software program.

RESULTSy

Mouse strain KKAy was established in 1969 by A mutation of strain KK, resulting in ectopic expression of ASP
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Intraperitoneal injection of leptin and immunohistochemical
detection of c-fos protein were performed as described previously(20, 21). In brief, mice were fasted for 24 h, intraperitoneally injected with either leptin (4 mg/kg body weight) or saline, anesthetized 2 h later with pentobarbital, and perfused transcardiallywith saline followed by 4% paraformaldehyde in PBS. Brains were
removed, postﬁxed in the same ﬁxative overnight, embedded
in OCT compound (Sakura Finetek, Tokyo, Japan), frozen, andsectioned coronally (thickness 40 ␮m). Floating sections werepretreated for 10 min with 3% hydrogen peroxide, blocked with
3% BSA for 1 h, incubated in c-fos antibody (dilution 1:2,000) for
72 h at 4°C, washed, incubated with Simple Stain MAX-PO (Nichirei
Biosciences, Tokyo, Japan) for 1 h, washed, incubated in 0.05%
DAB solution for 10 min (for visualization of c-fos protein),stained with hematoxylin, dehydrated, and mounted on coverslips. The c-fos-positive cells in ARC were counted at regular intervals in three sections for each animal.

and associated hyperphagia, obesity, and hyperglycemia(22). To investigate the roles of GM3 and/or related gangliosides in the pathogenesis of obesity, we knocked out
the GM3S gene in KKAy to generate strain KKAy GM3S
KO. These mice showed notable amelioration of obese
phenotype in terms of reduced body weight, daily food intake, and blood glucose level (Fig. 1A–C). mRNA expression levels of ASP, Pomc, and Mc4r did not differ signiﬁcantlyin the hypothalami of KKAy versus KKAy GM3S KO mice
(Fig. 1D). The coat color was grayish for KKAy GM3S KO
homozygotes, but yellow (similar to KKAy) for heterozygotes (Fig. 1E). The a/Ay genotype was conﬁrmed by genotyping PCR (Fig. 1F) and it was found that whereas all the
KKAy GM3S+/+ or GM3S+/⫺ mice had yellow coat color, only
6.1% of KKAy GM3S⫺/⫺ mice had yellow and all of the rest
had gray coat color. Brain ganglioside expression patterns
for KKAy GM3S KO were similar to those reported previously for GM3S-deﬁcient mice in a C57BL/6 background(5, 17). Brains of KKAy GM3S KO animals lacked a- andb-series gangliosides (GM1, GD1a, GD1b, and GT1b), themajor ganglioside species in WT brain; rather, they expressed predominantly o-series gangliosides, such as GM1band GD1␣ (Fig. 1H, I).
At age 14 weeks, the glucose tolerance of KKAy GM3S
KO mice was signiﬁcantly greater than that of KKAy mice
(Fig. 2A). Even in the KK background, GM3S KO mice
showed a slight improvement of glucose tolerance at age
24 weeks (Fig. 2B), similarly to our previous ﬁndings for
HFD-fed C57BL/6 GM3S KO mice (4). KKAy GM3S KO
mice also showed greatly increased insulin tolerance at age
24 weeks (Fig. 2C), and reduced expression levels of the
gluconeogenesis genes, G6pc and Pepck, in liver (Fig. 2D).
Plasma insulin levels at ages 16 and 24 weeks were low in
KKAy mice, but signiﬁcantly higher in KKAy GM3S KO
mice, probably due to preserved insulin secretion (Fig. 3A).
Plasma TG and non-HDL cholesterol levels at ages 10 and
16 weeks in KKAy mice were higher than in KK mice, whilethese levels in KKAy GM3S KO mice were lower than in
KKAy mice (similar to those in KK) (Fig. 3B, D). Plasma
leptin levels at age 8 weeks in KKAy GM3S KO mice weresigniﬁcantly higher than in KKAy mice (Fig. 3E). HDL cholesterol and NEFA levels did not differ notably between
KKAy and KKAy GM3S KO mice (Fig. 3C, F).
KKAy mice develop obesity (associated with hyperglycemia and hyperinsulinemia), hyperleptinemia, and leptin
resistance (23). We examined possible changes in central
responsiveness to leptin in KKAy GM3S KO mice. In KKAy
mice, intraperitoneal injection of leptin had only a slighteffect on neuron activation, as assayed by c-fos expressionin ARC (Fig. 4A), supporting the concept that development of obesity in these mice induces increased leptin resistance. In contrast, injection of leptin in KKAy GM3S KO
mice caused a notable increase of c-fos expression (Fig. 4A, B),
indicating that responsiveness to leptin is maintained inthese mice.
To examine the possible effects of loss of GM3S activity on leptin receptor signaling, we established a GM3Sdeﬁcient mutant of mouse hypothalamic neuronal N41
cells. We used the CRISPR/Cas9 system to target exon 5,

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Fig. 1. GM3S KO prevented development of hyperphagia and obesity in KKAy mice. A–C: Body weight (A), food intake (B), and nonfasting blood glucose levels (C) of KKAy and KK mice with or without deletion of the GM3S gene (GM3S KO) (n ⭓ 5 for each group). *P < 0.05,**P < 0.01 versus KKAy GM3S KO. D: Quantitative RT-PCR analysis of mRNA for ASP, Pomc, and Mc4r in KKAy and KKAy GM3S KO hypothalami. White bar, KKAy; black bar, KKAy GM3S KO (n = 3 for each group). *P < 0.05, **P < 0.01 versus KKAy GM3S KO. E: Coat color
phenotype of GM3S KO (GM3S⫺/⫺) observed in the KKAy (a/Ay) background. F: Representative genotyping PCR results for KKAy GM3S KO
mice. Top panel: Genotyping results for the a/Ay allele. The a and Ay alleles generate PCR products of 839 bp and 163 bp, respectively. Coatcolor: B, black; Y, yellow; G, gray. Bottom panel: Genotyping results for GM3S KO. The WT and mutant alleles generate PCR products of 276
bp and 120 bp, respectively. G: The percentage of yellow- or gray-coat-colored offspring compared with the total number of Ay/a offspringborn with each GM3S genotype. The number of each group is indicated on the top of each bar. Because of the infertility of KKAy females,
KKAy males (Ay/a, GM3S+/⫺ or ⫺/⫺) and KK females (a/a, GM3S+/⫺ or ⫺/⫺) were used for breeding. Data of the a/a offspring (black coat

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which contains sialyl motif L, a conserved sequence motif
essential for catalytic activity of mammalian sialyltransferases (24). GSL analysis showed that several obtained clones
lacked GM3 and related gangliosides (Fig. 5A, left), but
contained LacCer (Fig. 5A, right). A major GM1-like band
(Fig. 5A, left) was found to be GM1 by LC/MS/MS multiple reaction monitoring (supplemental data), and most
likely GM1b, an o-series ganglioside also observed in KKAy
GM3S KO brain (Fig. 1D). LepRb is highly expressed in the
mediobasal hypothalamus and mediates anti-obesity signaling by leptin via the Janus-activated kinase 2/signaltransducers and activators of transcription 3 (JAK2-STAT3)
pathway. The hypothalamic ERK pathway plays a key rolein mediating control of food intake and thermogenesis byleptin (25). Leptin, at a concentration 0.5 ␮g/ml, greatly
enhanced ERK phosphorylation in GM3S-deﬁcient N41

cells (Fig. 5B). In contrast, we could not detect STAT3
phosphorylation by leptin, even at 1 ␮g/ml, in either parental or GM3S-deﬁcient N41 cells (data not shown). To
evaluate leptin-induced phosphorylation of STAT3, we established stable expression of LepRb by lentiviral transferof the mouse LepRb gene. LepRb-expressing cells responded
to leptin at 0.5 ␮g/ml, and STAT3 phosphorylation wasslightly reduced in GM3S-deﬁcient cells (Fig. 5C). Theseﬁndings indicate that the leptin-dependent ERK pathway
was enhanced by deﬁcient ganglioside synthesis in KKAy
GM3S KO.
The observed coat color change and reduction of foodintake and body weight in KKAy GM3S KO mice suggested
that GM3 and related gangliosides play an important rolein melanocortin receptor signaling. We examined possible
effects of GM3 deﬁciency on MC4R signaling in HEK293T

color) were excluded. H: TLC of acidic (left) and neutral (right) GSLs from KKAy (+/+) and KKAy GM3S KO (⫺/⫺) brains. GSLs weredetected using orcinol-sulfuric acid reagent. The asterisks indicate o-series gangliosides whose biosynthesis is independent of GM3S [see text
and references (5) and (17) for details]. (I): Biosynthetic pathway of ganglio-series gangliosides. GM3S is a sialyltransferase required for
initiation of synthesis of a- and b-series gangliosides.

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Fig. 2. GM3S KO-enhanced glucose and insulin tolerance in KKAy mice. A, B: Glucose tolerance tests at age 14 weeks (A) and 24 weeks(B). C: Insulin tolerance tests at age 24 weeks. D: Quantitative analysis of mRNA for G6pc and Pepck in liver at age 24 week (n ⭓ 4 foreach group). *P < 0.05, **P < 0.01 vs. KKAy GM3S KO.

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Fig. 3. Blood parameters in KKAy GM3S KO and control mice. Blood plasma or serum samples were collected from mice fed ad libitum at
the indicated times.Insulin (A), TG (B), HDL cholesterol (HDL Chol) (C), non-HDL Chol (D), leptin (E), and NEFAs (F) (n ⭓ 4 for eachgroup). *P < 0.05, **P < 0.01.

cells. Parental and GM3-deﬁcient cells were transfectedwith the mouse Mc4r gene; intracellular cAMP production
was evaluated; and G protein-coupled receptor activation was assayed by TGF-␣ shedding assay, as described
previously (26), using ␣-MSH with or without synthetic
mouse AgRP or ASP. No clear difference was observed between parental and GM3-deﬁcient cells (data not shown;see Discussion).

DISCUSSION
Previous reports by our group and others have demonstrated the increased insulin sensitivity of GM3S KO micein a C57BL/6 background relative to WT mice. KO micedeveloped HFD-induced obesity (as did WT mice), but
exhibited reduced development of insulin resistanceand chronic low-grade inﬂammatory states (4, 5). In the
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present study, we used a GM3S KO strain of obese mousemodel KKAy to evaluate the pathophysiological effects of
GM3 or related gangliosides in hyperphagic and obese phenotypes. The coat color was grayish in KKAy GM3S KO homozygotes, but yellow (similar to KKAy) in heterozygotes.
The yellow Ay mutation (in the agouti gene) causes ectopic
expression of the gene product, ASP, resulting in a switch offollicular melanocytes from synthesis of eumelanin (black)to phaeomelanin (yellow) through inhibition of MC1R signaling as an endogenous antagonist, by blocking ␣-MSH(13). The observed coat color change of KKAy GM3S KO
mice may suggest involvement of GM3/related gangliosides in MC1R signaling in melanocytes, possibly throughdirect effect on MC1R function (e.g., cell surface expression or ␣-MSH binding) or direct or indirect modulationof ASP binding to MC1R, although functional analysis will
be required to establish this point. Mutations of the GM3S
(ST3GAL5) gene were identiﬁed in patients with salt-andpepper syndrome, an autosomal recessive neurocutaneous
disorder characterized by altered skin pigmentation (27).
In contrast to the previous results from GM3S KO
C57BL/6 mice, the obese model KKAy mice that have perturbed melanocortin signaling that thereby cause defects
in the central control of feeding behavior, showed remarkable reductions of body weight and food intake by GM3S
KO, suggesting that the anti-obese effects observed in thepresent study are mediated at least in part by the central
defects in the synthesis of GM3/ related gangliosides.
Given that the reduced body weight and food intake in the
KKAy GM3S KO mice were observed as early as 6 weeks ofage, we believe that the food intake effects are central inorigin. The observed food intake effects suggest that hypothalamic MC4R function may also be modulated by GM3/
related gangliosides (Fig. 6, a schematic illustration of ourcurrent model). We used GM3-deﬁcient HEK293T cells to
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examine ␣-MSH-dependent MC4R activation in the presence/absence of ASP and AgRP. For this assay, we usedcommercially available synthetic mouse ASP(93-132)-NH2
and AgRP(82-131)-NH2, which are conserved Cys-rich C-terminal domains sufﬁcient for melanocortin receptor antagonism (28). The synthetic ASP and AgRP both strongly
inhibited MC4R activation, but the inhibitory effect did not
differ notably in parental versus GM3S-deﬁcient cells.
(data not shown). However, we cannot exclude a possibility
that the cell system may not reﬂect the conditions in neurons (e.g., intrinsic properties and ganglioside expression
pattern) and thus, further investigation will be needed toconclude whether gangliosides are involved in the MC4R
function.
Body weight and food intake were notably lower in KKAy
GM3S KO mice than in KKAy mice, but no striking difference was observed between KK GM3S KO and KK mice
(Fig. 1A, B). These ﬁndings suggest a function of GM3/
related gangliosides in hyperphagia and obesity development, perhaps through altered ASP expression or function
in KKAy mice. No signiﬁcant changes were observed in Asp,
Pomc, or Mc4r gene expression in KKAy GM3S KO hypothalamus at age 6 weeks (Fig. 1D).
In the leptin administration experiments, KKAy GM3S
KO mice were less susceptible to central leptin resistancethan were KKAy mice. In GM3S-deﬁcient hypothalamic
neuronal cells, leptin-dependent STAT3 phosphorylation
was slightly reduced, whereas leptin-dependent ERK phosphorylation was greatly enhanced, suggesting differential
roles of speciﬁc ganglioside(s) in LepRb signaling. Numerous studies have demonstrated an essential role of the
JAK2-STAT3 pathway in the anorectic and metabolic effects of leptin. Leptin binding to LepRb activates JAK2, and
JAK2 subsequently phosphorylates other Tyr residues within
LepRb and JAK2 itself (29, 30). Phospho-Tyr1138 serves as a

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Fig. 4. KKAy GM3S KO mice retained leptin responsiveness in ARC. A: Representative images of basal (saline) and leptin-induced c-fos
immunoreactivity in ARC of KKAy and KKAy GM3S KO mice (male, age 10 weeks). Fasted mice were injected intraperitoneally with either
saline or leptin. ARC sections were stained with c-fos antibody. Scale bars: 300 ␮m. B: The c-fos positive cells in ARC were counted (n = 3 foreach group). *P < 0.05.

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Fig. 5. Leptin-induced ERK phosphorylation was greatly enhanced in GM3S-deﬁcient N41 cells. A: TLC of acidic (left) and neutral (right)
GSLs from control and GM3S-deﬁcient cell clones generated from N41 cells. Std, standard; Pa, parental cells; Cnt, control cells transfectedwith vector without targeting sequence. An asterisk indicates a GM1-like band, which is most likely GM1b, an o-series ganglioside. B, C: WTand GM3S-deﬁcient cells were treated with leptin and then lysed at the indicated times. For pSTAT3 assay (C), the LepRb gene was introduced
for stable expression (see text). The blots are representative of three independent experiments. Bottom: quantiﬁcation of immunoblots(n = 3). *P < 0.05, **P < 0.01. D: Leptin receptor signaling pathway. Thick arrows: increased and decreased phosphorylation of ERK and
STAT3 (respectively) observed in GM3S-deﬁcient cells.

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Fig. 6. A working hypothesis of the involvement of GM3/ related
gangliosides in leptin and melanocortin receptor signaling. Elimination of GM3/related gangliosides ameliorated the obese phenotype of KKAy mice. Ectopic expression of ASP caused alteration of
MC4R signaling and leptin resistance in hypothalamic neurons, resulting in dysregulated feeding and energy expenditure. Elimination of GM3/related gangliosides restored leptin sensitivity. Theseﬁndings and the observed coat color change suggest that GM3/
related gangliosides are involved in MC1R/MC4R signaling (seeDiscussion).

binding site for STAT3, and STAT3 is subsequently phosphorylated by JAK2 and translocated to the nucleus tomodulate expression of its target genes, which include
POMC and suppressor of cytokine signaling 3 (SOCS3) (31).
Protein tyrosine phosphatase 2 (SHP2) is recruited tophospho-Tyr985 in response to leptin, and phosphorylated
by JAK2. Phosphorylated SHP2 can bind the adaptor protein, growth factor receptor-bound protein 2 (Grb2),
which facilitates ERK activation. The SHP2/ERK pathway is
involved in the anti-obesity effect of leptin (25, 32).
Nordstrom et al. (33) reported that loss of GlcCer synthase in hypothalamic neurons inhibited LepRb signaling,using mice with deletion of inducible neuron-speciﬁc Ugcg,
the gene encoding GlcCer synthase. These mice developed
progressive obesity, and the obese phenotype was signiﬁcantly ameliorated by adeno-associated virus-mediated Ugcg
delivery to ARC. GM1 and GD1a in N41 cells interactedwith LepRb, suggesting that these a-series gangliosides enhance leptin signaling in hypothalamic neurons. In a studyof GD3S-null mice, which lack all b-series gangliosides, Ji
et al. (34) observed enhanced LepRb signaling in the hypothalamus, as indicated by phosphorylated STAT3 levels.
Overexpression of GD3S in N41 cells reduced responsiveness to leptin, supporting the concept that certain a-series
gangliosides positively regulate LepRb signaling. The
GM3S KO mice used in the present study lacked all a-series
gangliosides (Fig. 1F, G), but still retained hypothalamic
responsiveness to leptin. Our ﬁndings in GM3S-deﬁcient
N41 cells (Fig. 5B, D) suggest that GM3S KO mice may beprotected from central leptin resistance by enhancementof leptin-dependent ERK phosphorylation. We speculate
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The authors thank all members of the Inokuchi laboratory for
fruitful discussions and technical assistance, Dr. S. Anderson for
helpful comments and English editing of the manuscript, andTohoku Medical and Pharmaceutical University Center for
Laboratory Animal Science for their services.

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Glucose metabolism and insulin sensitivity in transgenic mice overexpressing leptin with lethal yellow agouti mutation: usefulness of

NPC1L1-dependent intestinal cholesterol absorption
requires ganglioside GM3 in membrane microdomains
Wataru Nihei,* Masakazu Nagafuku,* Hirotaka Hayamizu,* Yuta Odagiri,* Yumi Tamura,*,†§
Yui Kikuchi,* Lucas Veillon,* Hirotaka Kanoh,* Kei-ichiro Inamori,* Kenta Arai,§§
1,
Kazuya Kabayama, Koichi Fukase, and Jin-ichi Inokuchi *Abstract Intestinal cholesterol absorption is a key regulatorof systemic cholesterol homeostasis. Excessive dietary cholesterol and its intestinal uptake lead to hypercholesterolemia, a major risk factor for cardiovascular disease. Intestinal
cholesterol uptake is mediated by Niemann-Pick C1-like 1(NPC1L1), a transmembrane protein localized in membrane
microdomains (lipid rafts) enriched in gangliosides and cholesterol. The roles of gangliosides, such as monosialodihexosylganglioside (GM3) and its synthesizing enzyme GM3
synthase (GM3S), in NPC1L1-dependent cholesterol uptake
have not been examined previously. Here, we examined
NPC1L1-dependent cholesterol uptake in a cell model aswell as in wild-type and apoE-deﬁcient mice fed normal or
high-cholesterol diets. We showed that NPC1L1-dependentcholesterol uptake was impaired in GM3S-deﬁcient cells andthat GM3S deﬁciency promoted resistance to hypercholesterolemia in both wild-type and apoE-deﬁcient mice fed thehigh-cholesterol but not the normal diet. Our ﬁndings suggest that GM3 and related gangliosides are essential for
NPC1L1-mediated intestinal cholesterol absorption and arepotential targets for hypercholesterolemia therapy.—Nihei,
W., M. Nagafuku, H. Hayamizu, Y. Odagiri, Y. Tamura, Y.
Kikuchi, L. Veillon, H. Kanoh, K-i. Inamori, K. Arai, K.
Kabayama, K. Fukase, and J-i. Inokuchi. NPC1L1-dependent
intestinal cholesterol absorption requires gangliosideGM3 in membrane microdomains. J. Lipid Res. 2018. 59:
2181–2187.
Supplementary key words GANGLIOSIDES s MONOSIALODIHEXOSYLGANGLIOSIDE s CHOLESTEROL ABSORPTION s HYPERCHOLESTEROLEMIA s LIPID TRANSPORT s
Niemann-Pick C1-like 1

This study was supported by Ministry of Education, Culture, Sports, Science andTechnology Research Grant-in-Aid 16H04767 (J.I.); grants-in-aid from theMizutani Foundation for Glycoscience (J.I.), Takeda Science Foundation (J.I.),
Fugaku Trust for Medicinal Research (J.I.), Naito Foundation (J.I.), Ono Medical Research Foundation (J.I.), and Uehara Memorial Foundation (J.I.); and theMinistry of Education, Culture, Sports, Science and Technology-supported Program for the Strategic Research Foundation at Private Universities.
Manuscript received 11 August 2018 and in revised form 14 September 2018.
Published, JLR Papers in Press, September 21, 2018
DOI https://doi.org/10.1194/jlr.M089201

Cholesterol is an important component of cell membranes. It is a precursor for biosynthesis of steroid hormones and bile acids and is present in the circulatory
system. Lowering cholesterol levels in plasma reduces therisk of coronary heart disease, a major cause of death indeveloped countries (1, 2). The transmembrane protein
Niemann-Pick C1-like 1 (NPC1L1) plays an essential rolein dietary cholesterol absorption and biliary cholesterol reabsorption (3–5). NPC1L1 mediates cellular cholesterol
uptake through vesicular endocytosis and is a target of thecholesterol absorption inhibitor ezetimibe (6–10). NPC1L1
is localized in ganglioside-enriched membrane domains
and requires lipid raft proteins ﬂotillin-1 and -2 to createcholesterol-enriched membrane microdomains for efﬁcient cholesterol uptake (11, 12).
Gangliosides (glycosphingolipids [GSLs] that contain atleast one sialic acid) are enriched in the outer leaﬂet ofplasma membranes and concentrated in specialized membrane microdomains, termed lipid rafts, that function as
platforms for cell-cell interaction and cell signaling (13,14). The ganglioside monosialodihexosylganglioside (GM3),
synthesized by GM3 synthase (GM3S), is a precursor of a-, b-,
and c-series gangliosides, interacts with transmembrane
receptors such as the epidermal growth factor and insulin
receptors, and regulates receptor functions by creating a
specialized lipid environment (15, 16). There is often aclose functional relationship between gangliosides andﬂotillins in membrane microdomain organization (17, 18);
however, no study to date has addressed the role of gangliosides in NPC1L1-mediated cholesterol absorption. We
demonstrate in the present study that i) NPC1L1-dependent
cellular cholesterol uptake is inhibited in GM3S-deﬁcient
cells and ii) genetic hypercholesterolemia, diet-induced

Abbreviations: GM3, monosialodihexosylganglioside; GM3S, GM3
synthase; GSL, glycosphingolipid; NPC1L1, Niemann-Pick C1-like 1;
M␤CD, methyl-␤-cyclodextrin.
1
To whom correspondence should be addressed.
e-mail: jin@tohoku-mpu.ac.jp

Copyright © 2018 Nihei et al. Published under exclusive license by The American
Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org

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Division of Glycopathology,* Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, Sendai, Japan; Department of Bioinformatics and Computational Biology,† The
University of Texas MD Anderson Cancer Center, Houston, TX; and Laboratory of Natural ProductChemistry,§ Department of Chemistry, Osaka University, Toyonaka, Japanhypercholesterolemia, and the intestinal cholesterol absorption rate are reduced in GM3S-deﬁcient mice. Ourﬁndings suggest that gangliosides, particularly GM3, arepotential targets for hypercholesterolemia therapy.

and washed with PBS, and total cellular lipids were extracted as
described by Bligh and Dyer (24). Lipid extracts were dried by an
N2 stream and resuspended in 1 ml 1% Triton X-100 in chloroform, chloroform was evaporated by an N2 stream, and detergentsolubilized lipids were resuspended in 1 ml distilled water. Totalcholesterol and phospholipid concentrations were determinedTMusing LabAssay cholesterol and phospholipid test kits (Wako
Pure Chemical Industries, Osaka, Japan).

MATERIALS AND METHODSVisualization of living cells and ﬂuorescencequantiﬁcation

C57BL/6 mice and apoE-deﬁcient mice (B6.KOR/stm Slc-Apoeshl)
were from Japan SLC, Inc. (Hamamatsu, Japan). GM3S (St3gal5)deﬁcient mice were generated in our lab as described previously(19). To generate ApoEshl/GM3S+/⫺ mice, ApoEshl mice were crossed⫺/⫺with GM3S
mice.
shl
⫺/⫺
ApoE /GM3S
mice and littermate controls were generated
by heterozygous mating. Mice were analyzed for the GM3S genotype by PCR and for apoE protein expression by immunoblotting
as described previously (20, 21). Mice were fed a regular chowdiet (CE-2; CLEA Japan, Tokyo, Japan) or high-cholesterol diet
(Research Diets; New Brunswick, NJ) ad libitum. All animal experiments were approved by appropriate institutional review board
committees at Tohoku Medical and Pharmaceutical University.

For time-lapse ﬂuorescence imaging, cells were seeded onto
35-mm glass-bottom dishes (Greiner Bio-One, Frickenhausen,
Germany) coated with poly-L-lysine (Sigma-Aldrich) and transfected at 24 h. The medium was replaced by cholesterol-depleting
medium at 48 h, and cells were cultured overnight. CholesterolM␤CD complex was added to the medium the next day with orwithout 30 μM ezetimibe. Living cells were visualized by confocal
laser scanning microscopy (FluoView FV1000; Olympus, Tokyo,
Japan) for 60 min. The relative intensity of plasma membranelocalized NPC1L1-GFPturbo was quantiﬁed as described previously(6). The intensity at various time points was normalized relative
to the intensity at time zero (deﬁned as 100%). Fluorescence intensity was calculated using the FluoView software program (Olympus).

Materials, antibodies, and plasmid

Blood cholesterol and lipoprotein analyses

Cholesterol and lipoprotein-deﬁcient serum were from SigmaAldrich (St. Louis, MO), methyl-␤-cyclodextrin (M␤CD) andcompactin were from Tokyo Chemical Industry (Tokyo, Japan),and ezetimibe was from AdooQ Bioscience (Irvine, CA). Rabbit
anti-NPC1L1 antibody was from Novus Biologicals (Littleton,
CO). Alexa 594-conjugated goat anti-rabbit IgG was from Thermo
Fisher Scientiﬁc (Waltham, MA). pCMV6-hNPC1L1-turboGFP
plasmid vector was from OriGene (Rockville, MD).

Plasma total cholesterol was analyzed using the Cholesterol
E-Test kit (Wako Pure Chemical Industries) per the manufacturer’s
instructions. Serum lipoproteins were analyzed by an HPLC system at Skylight Biotech (Akita, Japan) according to the proceduredescribed by Usui et al. (25).

Cell culture
HEK293T cells were cultured in DMEM (Nacalai Tesque,
Kyoto, Japan) supplemented with 10% FBS, 100 U/ml penicillin,and 100 μg/ml streptomycin at 37°C in a 5% CO2 atmosphere.
Cholesterol-depleting medium was DMEM containing 5% lipoprotein-deﬁcient serum, 2 μM compactin, and 25 mM HEPES.
Cholesterol-M␤CD complex was prepared as described previously(22).

Generation of GM3S-deﬁcient HEK293T cells
A single exon of the human ST3GAL5 (GM3S) gene containing
the coding sequence for sialyl motif L was selected for the designof targeting guide RNAs using an online CRISPR design tool (23).
Guide oligos (5′-CACCGCAAGACCTGTCGGCGCTGTG-3′ and5′-AAACCACAGCGCCGACAGGTCTTGC-3′) were annealed andinserted into plasmid pSpCas9(BB) (Addgene, Cambridge, MA).
The plasmid was introduced into HEK293T cells using Lipofectamine 2000 (Thermo Fisher) per the manufacturer’s instructions, clonal cell lines were obtained by single-cell cloning, andexpression levels of gangliosides were evaluated by TLC.

Measurement of cellular cholesterol
Cells were seeded in DMEM in 60-mm dishes at a density of 8 ×
5
10 cells/dish and transfected at 24 h with the indicated plasmidusing Lipofectamine LTX (Thermo Fisher) per the manufacturer’s
instructions. The medium was replaced by cholesterol-depleting
medium at 48 h, cells were cultured overnight, and cholesterolM␤CD complex was added to the medium the next day. For ezetimibe treatment, cells were preincubated with 30 μM ezetimibe for
30 min, incubated with cholesterol-M␤CD complex for 60 min,

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Lipid and LC/MS/MS analyses
Lipids were analyzed as described previously (26). Intestinal
samples were obtained by perfusing mice with saline and scraping
off small-intestinal mucosa with a plastic spatula. LC/MS/MS
analysis was performed as described previously (27).

Intestinal cholesterol absorption rate
The fecal dual-isotope ratio method (28) was used to determine the intestinal cholesterol absorption rate in 6- to 8-week-old
shl
shl
⫺/⫺
mice. Animals were orally gaApoE mice and ApoE /GM3S
14
vaged with 100 μL corn oil containing [ C]cholesterol (1 μCi)
3and [ H]sitostanol (2 μCi), feces were collected once per day
for 3 days, and [14C]cholesterol and [3H]sitostanol levels were
determined.

Cholesterol feeding and immunohistochemical
analysis of NPC1L1
The mice were fasted for 16 h and then gavaged with 200 μL
corn oil containing 40 mg/ml cholesterol, anesthetized after
30 min, and perfused with saline. Intestinal tissues were removed,
ﬁxed in 4% paraformaldehyde in PBS, dehydrated in 30% sucrose
overnight at 4°C, embedded in OCT compound (Sakura Finetek,
Sendai, Japan), and frozen at ⫺80°C. Sections (thickness: 8 μm)
were prepared by cryostat, blocked with 3% BSA for 1 h, incubated with anti-NPC1L1 primary antibody (1:200) for 24 h at 4°C,
washed twice with PBS, incubated with Alexa 594-conjugatedsecondary antibody (1:500) for 30 min at room temperature,and viewed by ﬂuorescence microscopy (Axioskop2; Carl Zeiss,
Göttingen, Germany).

Statistical analysis
Data were expressed as mean ± SD, and means were comparedby Student’s t-test or ANOVA followed by Tukey’s post hoc test.

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Animals

RESULTS
To evaluate the possible involvement of gangliosides(GM3) in NPC1L1-mediated cholesterol absorption, we ﬁrst
examined cholesterol content in control HEK293T and
GM3S-deﬁcient (GM3S KO) cells. The two cell lines wereturbo

, and cellular cholestransfected with NPC1L1-GFP
terol content was measured. GM3S deﬁciency had no effecton endogenous cholesterol levels (Fig. 1). In NPC1L1expressing control cells, the cholesterol level was increased bycholesterol supplementation, and the increase was blockedby pretreatment with ezetimibe. In NPC1L1-expressing

Fig. 2. Cholesterol-dependent internalization of
NPC1L1-GFPturbo is ameliorated by GM3S depletion.
A: Control HEK293T and GM3S KO cells were seeded
in 0.001% poly-L-lysine-coated 35-mm glass-bottom
dishes on day 0 and transfected with NPC1L1-GFPturbo
on day 1. The medium was replaced with medium containing 5% lipoprotein-deﬁcient serum and 2 μM
compactin on day 2 to deplete cellular cholesterol,and cells were supplemented with 60 μg/ml cholesterol with or without ezetimibe treatment on day 3.
Time-lapse images were taken by confocal microscopy.
Representative images are shown. B: Quantiﬁcation ofplasma membrane-localized NPC1L1-GFPturbo in thecells shown in panel A. Intensity at time zero was deﬁned as 100%. *P < 0.05 for comparison of control
versus GM3S KO.

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Fig. 1. GM3S deﬁciency inhibits cholesterol uptake
via an NPC1L1-dependent pathway. NPC1L1-expressing
control HEK293T cells and GM3S KO cells wereincubated in cholesterol-depleting medium to reduce
cellular cholesterol. For cholesterol replenishment,cholesterol-M␤CD complex (30 μg/ml) was added
directly to the medium, and cells were cultured for
60 min with or without ezetimibe pretreatment. Total
lipid extraction was performed, and cholesterol andphospholipid contents were measured. Cholesterol
content was normalized relative to phospholipid content. **P < 0.01.

GM3S KO cells, cholesterol uptake was signiﬁcantly lower
than in control cells, and ezetimibe pretreatment had no notable effect (Fig. 1). Previous studies indicate that the dynamic translocation of NPC1L1 between the cell surface
and intracellular region is essential for NPC1L1-mediatedFig. 4. Lipoprotein proﬁles of ApoEshl and ApoEshl/GM3S⫺/⫺ mice. Lipoprotein-associated cholesterol levels in 16- to 18-week-old male mice
were determined by HPLC. A, B: Representative HPLC patterns of (A) ApoEshl and (B) ApoEshl/GM3S⫺/⫺ serum. A 5 ␮l serum sample wasinjected onto two tandem gel permeation columns and eluted with TSK eluent LP-1 at a ﬂow rate of 0.7 ml/min. Pink lines represent cholesterol, and blue lines represent triglyceride. Serum total cholesterol and total triglyceride levels are 587 ± 65 and 59 ± 33 mg/dl (A) and
259 ± 75 and 35 ± 11 mg/dl (B), respectively. Lipoprotein subclasses determined from observed elution times are presented. C–F: Chylomicron, VLDL, LDL, and HDL, respectively (n = 3 per group). *P < 0.05 and **P < 0.01.

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Fig. 3. Hypercholesterolemia is ameliorated in GM3S KO mice.
A: Plasma total cholesterol was measured in four groups as indicated (n = 18–35 per group). B: Diet-induced hypercholesterolemia
was normalized in GM3S KO mice. Plasma cholesterol levels were
determined after 10 weeks of a normal diet (cholesterol-free; ND)
or high-cholesterol diet (1.25% cholesterol; HCD) (n = 10–30 pergroup). **P < 0.01.

cholesterol uptake and that cholesterol is required for theactive endocytosis of NPC1L1 (6, 10).
Next, experiments were performed to evaluate theeffect of GM3S deﬁciency on NPC1L1 translocation. In
control cells, cholesterol supplementation following
cholesterol depletion resulted in the translocation of
NPC1L1 from the plasma membrane to the intracellularregion. In contrast, NPC1L1 translocation was much
lower in GM3S KO cells and at a level similar to that ofezetimibe-treated cells (Fig. 2). These ﬁndings indicate
the involvement of GM3 in NPC1L1-dependent cholesterol absorption.
We accordingly hypothesized that experimentally induced hypercholesterolemia in GM3S KO (GM3S⫺/⫺)
mice can be ameliorated by inhibiting NPC1L1-mediatedintestinal cholesterol uptake. To test this hypothesis, wecrossed apoE-deﬁcient, spontaneously hyperlipidemic
mice (ApoEshl) with GM3S⫺/⫺ mice and examined plasmacholesterol levels. Plasma cholesterol was not signiﬁcantly
reduced in GM3S⫺/⫺ mice, whereas levels in ApoEshl/
GM3S⫺/⫺ mice were strikingly lower than the high levels in
ApoEshl mice (Fig. 3A). Next, we examined the possible resistance of GM3S⫺/⫺ mice to diet- induced hypercholesterolemia. Plasma cholesterol levels were increased by ahigh-cholesterol diet in WT mice but not in GM3S⫺/⫺ mice
(Fig. 3B). These ﬁndings indicate that GM3S⫺/⫺ mice wereresistant to hypercholesterolemia induced by either apoE
deﬁciency or a high-cholesterol diet.

shlFig. 6. LC/MS/MS analysis of GM3 molecular species in mouseintestinal mucosa. Six species were detected (arranged according toabundance in WT mice).

To test the hypothesis that resistance to hypercholesterolemia in GM3S⫺/⫺ mice is due to impaired NPC1L1 function, we compared the intestinal cholesterol absorption
rates of ApoEshl versus ApoEshl/GM3S⫺/⫺ mice based on theoral administration of radiolabeled cholesterol. Uptake ofcholesterol from the intestine was signiﬁcantly lower in
ApoEshl/GM3S⫺/⫺ than in ApoEshl mice (Fig. 7).
It has been demonstrated that the oral administration ofcholesterol in mice induces the translocation of NPC1L1

Fig. 5. TLC analysis of ganglioside composition in mouse intestinal mucosa. Acidic (A) and neutral (B) GSLs equivalent to 2 or 1 mg
intestinal mucosa wet weight, respectively, were applied to plates.

Fig. 7. GM3 plays an important role in intestinal cholesterol uptake. Mice (n = 5–6 per group) were orally gavaged with [14C]cholesterol and [3H]sitostanol, and the cholesterol absorption rate wasdetermined by the fecal dual-isotope ratio method as described inMaterials and Methods. **P < 0.01.

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Plasma lipoprotein proﬁles were obtained for ApoE
and ApoEshl/GM3S⫺/⫺ mice. In ApoEshl/GM3S⫺/⫺ mice,cholesterol content was signiﬁcantly reduced in chylomicron, VLDL, and LDL fractions but not in the HDL fraction (Fig. 4). The reduction of cholesterol content wasmost striking for the chylomicron fraction (Fig. 4C), indicating defective intestinal cholesterol absorption inthese mice.
We next examined the GSL composition of intestinal
mucosa, where NPC1L1-mediated cholesterol absorption
occurs. It has been reported that intestinal GM3S expression level is high in neonatal mice and declines during thecourse of development (29). We detected GM3 expressionin WT and ApoEshl mice by TLC and LC/MS/MS analyses.
Trace amounts of GM3 molecular species were also detected in GM3S KO mice (Figs. 5A, 6), likely as the result of
a newly identiﬁed transcriptional variant in GM3S⫺/⫺ mice
generated by targeting exon 3 of the GM3S gene (30). Levelsof neutral GSLs in intestinal mucosa were not notably
altered in GM3S⫺/⫺ mice (Fig. 5B).

from the intestinal epithelial surface to the intracellular region (31, 32). We examined the possibility that GM3S
deﬁciency impairs cholesterol-dependent NPC1L1 translocation in vivo by immunostaining of intestinal NPC1L1. In
both ApoEshl and ApoEshl/GM3S⫺/⫺ mice, in the absence ofcholesterol feeding, NPC1L1 localized mainly at the apicalside of enterocytes (Fig. 8). Cholesterol feeding induced
NPC1L1 internalization in ApoEshl but not in ApoEshl/
GM3S⫺/⫺ mice. Taken together, these ﬁndings clearly indicate that GM3 and/or related gangliosides are essential for
NPC1L1-dependent intestinal cholesterol absorption.

DISCUSSION

Fig. 8. Immunostaining of NPC1L1 in the small intestine of
ApoEshl and ApoEshl/GM3S⫺/⫺ mice with or without oral cholesteroladministration. Frozen sections were stained with anti-NPC1L1 antibodies. Arrows: plasma membrane-localized NPC1L1. Arrowheads:
internalized NPC1L1.

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The authors are grateful to all members of the Inokuchi andFukase laboratories for valuable scientiﬁc discussions andtechnical support, to the Tohoku Medical and Pharmaceutical
University Center for Laboratory Animal Science for their services,and to S. Anderson for English editing of the manuscript.

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The protein NPC1L1 is known to be localized in detergent-resistant, ganglioside-enriched microdomains (11, 12,32). The role of gangliosides in NPC1L1-dependent cholesterol absorption is unknown. Our previous studies haveshown that GM3 plays key roles in certain metabolic disorders and that the inhibition of GM3 biosynthesis may help
ameliorate metabolic imbalance (27, 33, 34). Results fromthe present study indicate that GM3S deﬁciency promotes
resistance to hypercholesterolemia by inhibiting NPC1L1mediated cholesterol uptake. NPC1L1-expressing GM3S
KO cells displayed cholesterol uptake signiﬁcantly lower
than that of control cells (Fig. 1) and impairment of thecholesterol-dependent translocation of NPC1L1 from theplasma membrane to the intracellular region (Fig. 2).
Consistent with these ﬁndings, GM3S⫺/⫺ mice showed reductions of intestinal cholesterol uptake and cholesteroldependent translocation of NPC1L1 (Figs. 7, 8). Plasmacholesterol levels in WT, GM3S⫺/⫺, ApoEshl, and ApoEshl/
GM3⫺/⫺ mice are summarized in Fig. 3A. GM3S-deﬁcient
mice were resistant to hypercholesterolemia induced by
the high-cholesterol diet, and the hypercholesterolemia
characteristic of ApoEshl mice was signiﬁcantly ameliorated
in ApoEshl/GM3S⫺/⫺ mice. On the other hand, plasma cholesterol levels were similar for WT and GM3S⫺/⫺ mice fed anormal diet. Taken together, these observations suggest
functional involvement of GM3S in the exogenous pathwaysof cholesterol metabolism, including intestinal NPC1L1
activity.
Developmental changes in intestinal GSL composition
have been found to be synchronized with expression levels
of intestinal nutrient transporters (29). The knockdownof intestinal glucosylceramide synthase resulted in retarded growth and early death in mice because of defects in intestinal intracellular vesicular transport (35).
These studies suggest that GSLs are physiologically important for intestinal nutrient absorption, but they did
not address the role of GSLs in the NPC1L1 pathway. It
has been shown that NPC1L1 requires a cholesterolenriched membrane microdomain to function as a cholesterol transporter (11, 12, 32).
In the present study, cholesterol uptake by NPC1L1 wasreduced in GM3S-deﬁcient cells and mice. We therefore
conclude that NPC1L1 requires not only cholesterol but
also GM3 (or related gangliosides) to form functional
membrane microdomains for cholesterol transport. Two
possibilities can be considered: i) gangliosides interact directly with NPC1L1 via electrostatic interactions with multiple oligosaccharide chains to facilitate conformationalchange leading to translocation from lipid rafts to clathrincoated pits, and ii) gangliosides are required for the association of NPC1L1 with proteins such as ﬂotillins. The
present ﬁndings provide novel insights into the mechanism of NPC1L1-mediated cholesterol absorption, which
can be regulated by membrane lipid composition as well
as by protein- protein interactions. The detailed mechanisms whereby GM3 and related gangliosides function in
NPC1L1-mediated cholesterol absorption remain to beelucidated.
Gangliosides, particularly GM3, and its synthesizing enzyme GM3S appear to be potential targets for hypercholesterolemia therapy. Genetic variation in NPC1L1 is closely
associated with interindividual variation in response toezetimibe (36). Moreover, the loss of ezetimibe-binding
mutations in the extracellular loop of NPC1L1 has beenreported (9). Taken together, one can speculate that mutations in the region lead to unresponsiveness to the ezetimibe treatment. Membrane lipid modiﬁcation such as
GM3S inhibition can be used regardless of the binding afﬁnity of compounds to the NPC1L1 and provide an alternative therapy for nonresponsive individuals.

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International Immunology
doi:10.1093/intimm/dxy082
Advance Access publication 16 December 2018

© The Japanese Society for Immunology. 2018. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com

Kaoru Toshima1,*, Masakazu Nagafuku1*, Toshiro Okazaki2, Toshihide Kobayashi3and Jin-ichi Inokuchi1
1

Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical
University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan2
Department of Hematology and Immunology, Kanazawa Medical University, 1ಣ1 Daigaku, Uchinada, Ishikawa 920-0293,
Japan
3
Faculté de Pharmacie, Université de Strasbourg, 74 route du Rhin, F-67401 Illkirch, France
Correspondence to: J.-i. Inokuchi; E-mail: jin@tohoku-mpu.ac.jp*These authors equally contributed to this study.
Received 28 September 2018, editorial decision 4 December 2018, accepted 13 December 2018

Abstract
Sphingomyelin (SM) in combination with cholesterol forms specialized membrane lipid
microdomains in which specific receptors and signaling molecules are localized or recruited tomediate intracellular signaling. SM-microdomain levels in mouse thymus were low in the early
CD4+CD8+ double-positive (DP) stage prior to thymic selection and increased >10-fold during lateselection. T-cell receptor (TCR) signal strength is a key factor determining whether DP thymocytes
undergo positive or negative selection. We examined the role of SM-microdomains in thymocyte
development and related TCR signaling, using SM synthase 1 (SMS1)-deficient (SMS1−/−) mice
which display low SM expression in all thymocyte populations. SMS1 deficiency caused reduced
cell numbers after late DP stages in TCR transgenic models. TCR-dependent apoptosis induced byanti-CD3 treatment was enhanced in SMS1−/− DP thymocytes both in vivo and in vitro. SMS1−/− DPthymocytes, relative to controls, showed increased phosphorylation of TCR-proximal kinase ZAP70 and increased expression of Bim and Nur77 proteins involved in negative selection following
TCR stimulation. Addition of SM to cultured normal DP thymocytes led to greatly increased surface
expression of SM-microdomains, with associated reduction of TCR signaling and TCR-induced
apoptosis. Our findings indicate that SM-microdomains are increased in late DP stages, function
as negative regulators of TCR signaling and modulate the efficiency of TCR-proximal signaling topromote thymic selection events leading to subsequent developmental stages.
Keywords: lipid raft, SM-microdomain, TCR signaling, thymic selection

Introduction
T-cell development in the thymus requires multiple stages
to generate a T-cell population having a huge repertoire of
antigen speciﬁcities but lacking autoreactivity determinedby the T-cell receptor (TCR) (1–3). The early T-cell progenitors, CD4−CD8− double-negative (DN) thymocytes, produce
a functional pre-TCR which promotes further development
into CD4+CD8+ double-positive (DP) thymocytes. During
the DP thymocyte stage, developing cells rearrange their
TCRα genes and express a mature TCR on their surface.
Only cells that bear a TCR subsequently undergo both positive and negative selection to become mature CD4 or CD8

single-positive (SP) thymocytes, which then exit the thymus
to enter the peripheral circulation.
DP thymocytes that recognize major histocompatibility
complex (MHC) molecules are either positively or negatively
selected, depending on the degrees of afﬁnity and avidityof the TCR for MHC–peptide complexes, and the strengthof intracellular signaling following TCR engagement (4, 5).
Negative selecting ligands with high avidity induce stronger
TCR signaling, leading to a variety of intracellular signalingcascades such as ERK5 and other MAPK activation pathways (6). These signaling pathways cooperatively trigger

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Plasma membrane sphingomyelin modulates
thymocyte development by inhibiting TCR-induced
apoptosis

Page 2 of 13

Role of sphingomyelin in thymocyte development
thymocytes, resulting in increased apoptosis induced by TCR
stimulation. Normal DP thymocytes (which normally have no
SM-microdomains), when treated with SM, acquired surface
expression of SM-microdomains and became resistant to
TCR-induced apoptosis. Our ﬁndings, taken together, indicatethat (i) membrane SM lipids suppress apoptosis of DP thymocytes by reducing TCR signaling and (ii) SM-microdomains
modulate the TCR signal intensity and the outcomes of thymic
selection.
Methods
Mice
SMS1−/− mice, from the C57BL/6 background, were established as described previously (25). OT-I and HY TCR transgenic mice were obtained from Dr K. Ikuta (Kyoto University,
Japan) (26, 27). All mice were maintained under deﬁned
pathogen-free conditions in the animal facility of Tohoku
Medical and Pharmaceutical University, in compliance withinstitutional guidelines. Animals used in experiments wereusually aged 5–7 weeks. Gender- and age-matched littermates of wild-type and heterozygotes were used as controls.
Antibodies and FACS analysis
Thymus and spleen cells were stained with appropriate ﬂuorochrome-conjugated antibodies. The following mAbs werefrom BioLegend (San Diego, CA, USA): CCR7 (4B12), CD4(GK1.5), CD5 (53-7.3), CD8α (53-6.7), CD11b (M1/70), CD24
(M1/69), CD25 (PC61), CD28 (E18), CD44 (IM7), CD62L(MEL-14), CD69 (H1.2F3), cKit (2B8), CD16/32 (93), Gr-1
(RB6-8C5), NK1.1 (PK136), TCRβ (H57-597), TCRγδ (GL3),
TCRVα2 (B20.1), TCRVβ5.1 5.2 (MR9-4), HY (MR14-1), Qa2(695H1-9-9), TER119 (TER-119). For regulatory T-cell analysis, CD4SP thymocytes were identiﬁed by anti-CD4/CD8 surface staining, then ﬁxed and permeabilized for intracellular
staining with PE-conjugated anti-Foxp3 (MF-14, BioLegend)
mAb. PE-conjugated, isotype-matched mAbs were used ascontrols. Stained cells were analyzed using a FACSAria II cell
sorter (BD Biosciences, San Jose, CA, USA) and the FlowJo
software program (Tree Star Inc., Ashland, OR, USA). Datashown are mean ± SD.
Detection of SM using the speciﬁc probes
The physical status of SM present in biological membranes
was assessed using two SM-binding proteins: lysenin (Lys)from Eisenia fetida (earthworm) and equinatoxin II (EqtII) fromActinia equina (sea anemone) (28). These are pore-forming
protein toxins that bind speciﬁcally to SM on target cell membranes, but their manners of binding are different: Lys binds
to clusters of 5–6 SM molecules, whereas EqtII binds to single
SM molecules (Supplementary Figure S1A) (29, 30). A construct of non-toxic Lys fused with enhanced green ﬂuorescentprotein (EGFP-NT-Lys), pQE30/His6-EGFP-NT-Lys containing
EGFP at the N-terminus of the DNA fragment of Lys aminoacids 161–297 was prepared as described previously (31).
A construct of EqtII fused with EGFP (EqtII-EGFP), pET28/
EqtII (8-69)-EGFP-His6 containing EGFP at the C-terminus of
EqtII sequences was provided by the RIKEN BioResource
Research Center (BRC) through the National Bio-Resource

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overt expression of Nur77 family and proapoptotic Bim proteins, resulting ultimately in mitochondrial dysfunction andapoptosis (7, 8). Both loss- and gain-of-function studies of
TCR signaling regulators have demonstrated that the threshold between positive and negative selection is deﬁned by thebalance between positive and negative signaling (9).
Studies during the past two decades have shown that theinitial events of T-cell activation involve movement of the TCR
into specialized membrane lipid microdomains, often termed‘lipid rafts’, on the cell surface (10). The structure of suchmicrodomains plays a crucial role in efﬁciency of TCR signaling; in both thymocytes and peripheral mature T cells, thesemicrodomains are clearly involved in TCR signaling and in
the localization and function of proximally located proteins(11, 12).
The term ‘lipid raft’ was introduced by K. Simons andE. Ikonen because of the close association of cholesteroland various sphingolipids, including glycosphingolipids(GSLs) and phosphocholine-containing sphingomyelin (SM),as components of the detergent-resistant complex of signaling molecules present in membrane lipid microdomains (13,14). Ganglioside-microdomains on the cell surface are easily
detected using cholera toxin B subunit (CTx-B), which binds
with high sensitivity and speciﬁcity to gangliosides GM1 andextended-GM1b (15, 16), and the role of these microdomains
in thymocyte development has been extensively studied(17). However, the role of lipid microdomains in thymocytes
remains controversial because (i) thymocytes express far
fewer GSLs including gangliosides than do peripheral mature
CD4 and CD8 T cells and (ii) GM1 and extended-GM1b are
extremely minor ganglioside species in thymus (18). Popovic
et al. showed recently that conditional deletion very earlyin T-cell development of glucosylceramide synthase, a key
enzyme in GSL biosynthesis, resulted in near-total absence of
GSLs in DP thymocytes but had no effect on the developmentof conventional T-cell populations in the thymus (19).
Individual lipid species were recently shown to be localized
in distinct, non-overlapping microdomains in plasma membrane; e.g. GM1-microdomains, GM3-microdomains andSM-microdomains (20). Lactosylceramide-microdomains are
directly involved in phagocytosis mediated by the integrin
CD11b/CD18 and chemotaxis induced by a kind of beta-glucan from Candida albicans (21, 22). SM is the most abundant
sphingolipid in mammalian plasma membranes and is accumulated in TCR activation domains immunoisolated by antiCD3 monoclonal antibody (mAb) from TCR-stimulated Jurkat
cells (23); however, little is known regarding the involvementof SM-microdomains (in comparison with microdomains easily detected by CTx-B) in thymocyte TCR signaling eventsand thymic selection.
We investigated the role of SM-microdomains on TCR
signaling and selection events in DP thymocytes. SM synthase 1 (SMS1)-deﬁcient (SMS1−/−) mice were used for thispurpose because SMS1 is the enzyme responsible for denovo SM synthesis (24). In normal mouse thymocytes, levelsof SM-microdomains and SMS1 gene were low at the early
DP stage but much higher in the late DP stages. Thymocytesof SMS1−/− mice showed reduced DP populations andalmost no SM expression. TCR-mediated signals involvedin negative selection were strongly enhanced in SMS1−/− DPRole of sphingomyelin in thymocyte development

Page 3 of 13

stimulated cells were stained with CD4 and CD8, ﬁxed andpermeabilized with a True-Nuclear Transcription Factor Buffer
Set (BioLegend), and incubated with PE-conjugated antiNur77 (eBioscience) and mouse IgG1 control (BioLegend)
mAbs. Total thymocytes were stimulated with soluble antiCD3 plus anti-CD4 mAbs for 5 min at 37°C. DP thymocytes
were identiﬁed by anti-CD4/CD8 surface staining, then ﬁxed
and permeabilized for intracellular staining with allophycocyanin (APC)-conjugated anti-phospho ZAP-70 (Y319) and
FITC-conjugated ZAP-70 mAbs (BioLegend). Fluorochromeconjugated, isotype-matched antibodies were used as controls. Labeled cells were analyzed using a FACSAria II.

Immunohistochemistry

Immunoblotting analysis

Whole thymic lobes were ﬁxed in 4% paraformaldehyde–
PBS, dehydrated in 20% sucrose, embedded in OCT compound (Sakura Finetek, Tokyo, Japan) and cut into 8-µm
sections by cryostat (Microm HM560 MV, Thermo Scientiﬁc).
For Lys staining, sections were blocked with 3% bovine
serum albumin–PBS, and then serially incubated with Lys(Peptides International, Louisville, KY, USA) for 45 min, Lysanti-serum (1:500 dilution, Peptides International) or control rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) for 1 h, and Alexa594-conjugated anti-rabbit
IgG antibody (1:200 dilution, Invitrogen) for 30 min at 20°C.
Images were acquired with an Axioskop 2 Plus (Carl Zeiss,
Inc., Thornwood, NY, USA).

Thymocytes were stimulated with anti-CD3 plus anti-CD28
mAbs for 2 or 6 h at 37°C and lysed with ice-cold lysis buffer
containing 25 mM Tris–HCl (pH 7.6), 120 mM NaCl, 5 mM ethylenediaminetetraacetic acid (pH 8.0), 5 mM Na3VO4, 10 mM
NaF, 1% TX-100, 1% deoxycholate-Na, 1 mM phenylmethylsulfonyl ﬂuoride (PMSF) and the Complete Protease Inhibitor
Cocktail (Roche) for 30 min. Cell lysates were resolved bysodium dodecyl sulfate–polyacrylamide gel electrophoresis,
transferred onto PVDF membranes (Immobilon-P, Millipore,Bedford, MA, USA), and probed with the following mAbs:
anti-phospho-ERK5 (T218/T220), anti-ERK5, anti-Bim (Cell
Signaling Technology, Beverly, MA, USA) and anti-β-actin(Sigma-Aldrich).

Lipid analysis by thin-layer chromatography

Statistical analysis

Lipid analysis of plasma and thymocytes was performed as
described previously (34, 35). Total lipids were extractedwith chloroform/methanol and separated into neutral andacidic lipid fractions using DEAE-Sephadex A-25 columns(GE Healthcare, Chalfont St. Giles, UK). Fractions weredried, treated with 0.1 M NaOH in methanol for cleavageof phospholipids and neutralized. Solutions were desalted
with Sep-Pak C18 reversed-phase cartridges (Waters Corp.,
Milford, MA, USA). Neutral lipid samples with equal protein
content were separated on silica gel thin-layer chromatography (TLC) plates, half-developed with chloroform/methanol/
water (60:25:4), dried and redeveloped with 1-butanol/acetic acid/water (3:1:1, for SM analysis) or with hexane/diethyl
ether/acetic acid (50:50:1, for neutral sphingolipid analysis).
Phosphorus-containing lipids such as SM were visualizedby spraying plates with Dittmer reagent (36), and total lipids
were visualized by spraying with 3% cupric acetate/8% phosphoric acid reagent.

Values presented are mean ± SD. Homoscedasticity was veriﬁed by F-test. Means were compared by unpaired Student’s
t-test or Welch’s t-test for two-group comparison. For datawith non-Gaussian distribution, comparisons were made by
Mann–Whitney U-test. For multigroup analysis, homoscedasticity was veriﬁed by Bartlett’s test, and data were comparedby the Tukey–Kramer test or Scheffe’s F-test. Notations used
to indicate signiﬁcant differences were: *P < 0.05, **P < 0.01,***P < 0.001.

In vitro thymocyte stimulation
Total thymocytes were incubated with various concentrations of immobilized anti-CD3 (2C11, BioLegend) plus antiCD28 (PV-1, Southern Biotechnology, Birmingham, AL, USA)mAbs, with phorbol 12-myristate 13-acetate (PMA)/ionomycin(50 ng ml−1/1 µM, BioLegend) or with dexamethasone (3 nM,
Wako Pure Chemical, Osaka, Japan) at 37°C for various
time periods. Apoptotic cells were determined by annexinV and 7-AAD staining with a Vybrant Apoptosis Assay Kit 2(Molecular Probes, Invitrogen). For Nur77 expression assay,

Results
Expression of SM-microdomains in thymocytes increases
greatly in late stages of thymic selection
SM is expressed ubiquitously in mammalian cell membranes
and, in combination with cholesterol, forms specialized membrane lipid microdomains that regulate membrane ﬂuidity, protein trafﬁcking and signal transduction. To investigate the roleof SM-microdomains in thymocyte development in our mousemodel, we ﬁrst examined SM expression proﬁles in thymocyte
sub-populations. TLC analysis revealed that the SM content
decreased sharply when DN thymocytes developed into DP
thymocytes, and then increased when these developed into
SP thymocytes (Fig. 1A). SM expression was higher in CD4SP
than in CD8SP thymocytes.
Lys binds to clustered SM molecules, while EqtII binds tosingle SM molecules (see Methods “Detection of SM using
the speciﬁc probes”). We therefore presumed that Lys reactivity characterized SM-enriched membrane microdomains

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Project of MEXT (Ministry of Education, Culture, Sports,
Science and Technology), Japan. These lipid probes wereprepared as described previously (32). To block hemolytic
activity of EqtII without disrupting its SM-binding activity, adisulﬁde bridge was introduced between Cys8 and Cys69
of EqtII-EGFP by incubation with 0.5 mM 1,10-phenanthroline and 0.1 mM CuSO4 for 1 h at 30°C (33). For FACS
analysis, thymocytes were washed with phosphate-bufferedsaline (PBS) and labeled with EGFP-NT-Lys or EqtII-EGFP
in PBS, together with cell surface markers described in
Supplementary Figure S2. Stained cells were analyzed using
a FACSAria II.

Page 4 of 13

Role of sphingomyelin in thymocyte development

B

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CD8
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12
10
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15
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Fig. 1. SM-microdomain levels are greatly increased in late stages of thymic selection. (A) Total SM expression in mouse thymocyte sub-populations deﬁned by CD4/CD8 expression. Neutral lipids puriﬁed from DN, DP, CD4SP and CD8SP sub-populations were separated on TLC plates,
and SM was detected by Dittmer reagent. Std: standard lipid. (B) Cell surface staining of gated DN, DP and SP sub-populations with EGFP-NTLys (left; n = 4) and EqtII-EGFP (right; n = 3). (C, D) Cell surface staining with EGFP-NT-Lys (C) and EqtII-EGFP (D), together with thymocyte
developmental markers as described in Supplementary Figure S2. n = 3. (E) Relative mean ﬂuorescence intensity (MFI) for EGFP-NT-Lys and
EqtII-EGFP from small DP to CD4+CD8int populations. Data are relative to MFI values from the small DP population. (F) Immunohistochemistry(using Lys) of thymus. For negative control staining, Lys anti-serum (b) was replaced by non-immune rabbit polyclonal IgG (a). Scale bar:
100 µm. (G) Five stages of thymocyte development (designated I through V) were deﬁned according to CD69 and CCR7 expression as indicated. Dot plots of CD4 versus CD8α and histograms of Lys, EqtII and TCRβ staining are shown for each stage. Solid lines: with staining.
Dashed lines: without staining. Data shown are representative of three independent experiments. * P < 0.05, ** P < 0.01.

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**

*
7

Role of sphingomyelin in thymocyte development

selected thymocytes up-regulate surface TCR expression to
become SP thymocytes (43). The time course of appearanceof the TCRhi population during stages I to V was similar toor slightly earlier than that of the Lyshi population (Fig. 1G),
even though thymocytes in stage II had already received TCR
signals. These ﬁndings indicate that SM-microdomains areformed in the late stages of positive selection, up to or justbefore the DP-to-SP transition, probably as a consequenceof TCR signals.
GSLs and SM form a variety of microdomains in plasma
membranes, some of which are based on a physical association of cholesterol with SM (44–46). We used CTx-B (binds
to GM1 and extended-GM1b) and ﬁlipin III (binds to cholesterol) to determine whether cell surface levels of GSLs andcholesterol ﬂuctuate similarly to that of SM during thymocyte
development. Levels of CTx-B-binding gangliosides did
ﬂuctuate, but (in contrast to SM-microdomain levels) did notincrease from small DP to CD4+CD8int stages (Supplementary
Figure S3A). Cholesterol level increased in parallel with
the SM-microdomain level from small DP to CD4+CD8int
stages (Supplementary Figure S3B). These ﬁndings suggestthat the SM-microdomain level speciﬁcally increases after
versus before thymic selection, because SM is able to form
membrane microdomains in combination with cholesterol.
SM expression in mammalian cells is controlled by two SM
synthases, SMS1 and SMS2 (Supplementary Figure S4) (24,47). SMS1 is primarily responsible for de novo SM synthesisin the Golgi, while SMS2 is involved in regenerating SM fromceramide hydrolyzed by sphingomyelinases on the cell surface (24, 47, 48). We detected SMS1 mRNA, but not SMS2
mRNA, in thymocytes (Supplementary Figure S5A). In contrast, expression of SMS2 mRNA was much higher than thatof SMS1 mRNA in liver, consistent with ﬁndings by Jiang's
group (49, 50). Changes in SMS1 expression patterns among
DN, DP and SP thymocytes as revealed by quantitative PCR
analysis (Supplementary Figure S5B) were comparable tothose of SM-microdomains (Fig. 1B). Thus, SMS1 but not
SMS2 is evidently the enzyme primarily responsible for SM
biosynthesis in mouse thymocytes.
To further evaluate the similarity of expression patterns of
the SMS1 gene and SM-microdomains during DN-to-SP thymocyte development, we examined global microarray datasets from the Immunological Genome Project (www.immgen.
org), which detail gene expression in >200 mouse immune
cell populations (51). The pattern of SMS1 expression during DN-to-SP thymocyte development (Supplementary
Figure S5C) was similar to that of Lys ﬂuorescence intensity(Fig. 1C). Thus, cell surface SM-microdomain levels during
thymocyte development appear to be strictly regulated by
SMS1 gene expression levels.
SM-microdomains are essentially absent in
SMS1-deﬁcient thymocytes
The role of SM in thymocyte development was further evaluated using SMS1−/− mice. Jiang et al. reported that serum
SM levels in SMS1−/− were ~50% of levels in controls (49).
Similarly, our TLC analyses showed that in SMS1−/−, 46%
of SM remained in plasma. In SMS1−/− thymocytes, cellular SM was present in <10% of thymocytes in control mice

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(termed ‘SM-microdomains’), whereas EqtII reactivity characterized single SM molecules dispersed in membrane(termed ‘dispersed-SM’) (Supplementary Figure S1A) (19,20). Consistent with this presumption, treatment of thymocytes with 4 mM methyl-β-cyclodextrin (MβCD), a cholesterol-depletion reagent commonly used for disruption of lipid
rafts, completely eliminated Lys staining but not EqtII staining (Supplementary Figure S1B). We therefore used thesetwo probes to evaluate the physical status of SM present inplasma membranes at each stage of thymocyte development.
Expression patterns of SM-microdomains (detected by
EGFP-NT-Lys) and of dispersed-SM (detected by EqtIIEGFP) on the cell surface were similar to those of total SM
analyzed by TLC (Fig. 1A and B). To assess SM-microdomain
levels during thymocyte development, we labeled whole
thymocytes with Lys and with several surface markers that
identify various sub-populations during progression from DN
to SP thymocytes and performed FACS analysis (Fig. 1C).
Surface markers, and the hierarchical gating strategy
used (37), are summarized in Supplementary Figure S2.
SM-microdomain levels were lower in DP stages than in DN
and SP stages in general, and particularly low at the small
DP stage prior to thymic selection. After the small DP stage,
the SM-microdomain level increased during progression from
CD69+ DP to CD4+CD8int thymocytes following thymic selection (Fig. 1C). There was a consequent >10-fold increase ofcell surface SM-microdomains after versus before thymic
selection. The dispersed-SM level was lowest at the small
DP stage and nearly twice as high in the CD4+CD8int stage(Fig. 1D). There was a striking difference in up-regulation of
the SM-microdomain level (>10-fold) versus the dispersedSM level (<2-fold) during DP thymocyte development(Fig. 1E). These ﬁndings suggest that de novo synthesized
SM molecules are used almost entirely for microdomain formation rather than simply ﬂoating on the plasma membrane.
T-progenitor cells, after entry into the thymus, differentiate
into DP thymocytes in the cortex (38, 39). DP thymocytes
receive survival signals and undergo further differentiation
into SP thymocytes. These signals also induce relocalizationof thymocytes from the cortex to the medulla; therefore, mostthymocytes in the medulla are SP thymocytes (40). Staining
intensity by Lys for SM-microdomains was much higher inthe medulla than in the cortex, indicating high formation of
SM-microdomains during DP-to-SP differentiation (Fig. 1F). To
examine this process in more detail, we performed multicolor
staining of thymocytes with Lys or EqtII, in combination withthe early activation marker CD69 and the chemokine receptor CCR7. CD69 and CCR7 are used to classify developing
thymocytes into ﬁve stages in terms of selection, migrationand maturation (41, 42). CD69−CCR7− thymocytes are localized in the cortex and have not yet received TCR selection
signals (stage I). CD69+CCR7− thymocytes have received
TCR signals and show increased CD69 expression (stage
II). CD69+ thymocytes that receive continuous TCR signals
begin to express CCR7 (stage III) and migrate to the medulla.
CD69+CCR7hi medullary thymocytes (stage IV) show reduced
CD69 expression and then become competent to exit the thymus (stage V) (42). Lyshi thymocytes, which show increased
SM-microdomain expression, begin appearing in stage
III, and are predominant in stage IV (Fig. 1G). Positively

Page 5 of 13

Page 6 of 13

Role of sphingomyelin in thymocyte development

ASM

originNo staining (control)
EGFP-NT-Lys (control)
EGFP-NT-Lys (SMS1-/-)B

No staining (control)
EqtII-EGFP (control)
EqtII-EGFP (SMS1-/-)

CD4SP

Count normalized to Mode

DP

Count normalized to Mode

DN

Aberrant thymic selection in SMS1−/− mice

CD8SP

EGFP-NT-Lys
Thymocytes

25

25
20
15
10
5

D

% of total DN cells

0

80
60

15
10
5

E

Control
SMS1-/-

40
20
0

0.8

DN2

DN3

% of
CD4SP cells% oftotal thymocytes

20

0

DN1

F

Splenocytes

0.6
0.4
0.2
0
ɤδ T

% of
total thymocytes

Cell number (107)30

Cell number (107)C

EqtII-EGFP**

80

Control
SMS1-/-

60
40
20
0

**

**

*

DN4

6
5
4
3
2
1
0

Control
SMS1-/-

Treg

Fig. 2. SMS1−/− thymocytes lack surface SM-microdomains. (A) Total
cellular SM in SMS1−/− thymocytes. Neutral lipids were puriﬁed fromcontrol and SMS1−/− thymocytes and separated on TLC plates. SM
was detected by Dittmer reagent. Std: standard lipid. Data shown
are representative of three independent experiments. (B) Cell surface staining of gated DN, DP and SP sub-populations of control
and SMS1−/− thymocytes labeled with EGFP-NT-Lys (left) and EqtIIEGFP (right). Data shown are representative of three independent
experiments. Bold lines: littermate control thymocytes. Thin solidlines: SMS1−/− thymocytes. Dashed lines: non-labeled control thymocytes. (C) Absolute numbers of total thymocytes and splenocytes.
Dots: individual mice; n = 19 (left), n = 7 (right). Bars: means. (D)CD4−CD8− DN thymocytes were subdivided into DN1 (CD25−cKit+),
DN2 (CD25+cKit+), DN3 (CD25+cKit−) and DN4 (CD25−cKit−) subsets
by multicolor FACS analysis. Data shown are ratios of cell numbers of
sub-populations to total DN thymocytes. n = 3. (E) Ratios of numbersof DN, DP, CD4SP and CD8SP thymocytes to total thymocytes. n = 3.
(F) Ratios of numbers Ǫδ T cells (TCRγδ+) to total thymocytes (left)
and Treg cells (CD25+Foxp3+) to CD4SP thymocytes (right). n = 3.
*P < 0.05, **P < 0.01.

Thymocyte cellularity was evaluated in SMS1−/− mice. Total
numbers of thymocytes and splenocytes were similar in
SMS1−/− versus control mice (Fig. 2C). The DP sub-population
was smaller in SMS1−/− versus control thymocytes (Fig. 2E),
but DN sub-populations were similar (Fig. 2D). There are no
signiﬁcant differences in Ǫδ T and Treg populations between
control and SMS1−/− mice (Fig. 2F).
Developmental defects associated with thymic selection
can be masked by compensatory changes in the TCR repertoire. We therefore introduced single transgenes encoding αβTCRs into SMS1−/− mice to limit compensation, andcrossed HY and OT-I TCR transgenic mice with SMS1−/− mice.
Numbers of TCR transgenic positive thymocytes were much
lower in OT-I SMS1−/− and female HY SMS1−/− mice than in
TCR transgenic controls (Fig. 3A and B). Among both OT-I
SMS1−/− and female HY SMS1−/− thymocytes, numbers of DP
and CD8SP thymocytes were greatly reduced (Fig. 3C and
D). In OT-I SMS1−/− mice, numbers of stage II CD69hiCCR7− tostage IV CCR7hiCD69hi thymocytes were reduced (Fig. 3E).
These ﬁndings demonstrate that SMS1 deﬁciency inhibits
positive selection.
Finally, we examined the effect of SMS1 deﬁciency in male
HY TCR transgenic mice. Total HY TCR+ cell number did not
differ notably in SMS1−/− versus control thymocytes (Fig. 4A).
One possible explanation is that HY TCR recognizes a malespeciﬁc antigen, such that male HY mice undergo massive
deletion of thymocytes by negative selection upon reaching
the DP stage (52). On the other hand, the CD8SP population
was reduced in SMS1−/− thymocytes (Fig. 4B), suggestingthat SMS1 deﬁciency promotes negative selection.
TCR stimulation in SMS1−/− DP thymocytes induces
up-regulation of apoptosis-related molecules involved innegative selection and apoptotic cell death
Thymic selection depends on TCR-mediated intracellularsignaling. We examined the effect of SMS1 deﬁciency on thymocyte apoptosis induced by anti-CD3 mAb administration.
Following in vivo anti-CD3 mAb treatment, annexin-V+ thymocytes were more numerous in SMS1−/− versus control thymus(Fig. 5A). In in vitro culture, DP thymocyte apoptosis induced
by anti-CD3 + anti-CD28 mAb treatment was also higher in
SMS1−/− versus control mice (Fig. 5B). SMS1 deﬁciency had
no effect on apoptosis induced by PMA/ionomycin (whichbypasses TCR-proximal signaling) or by dexamethasone(a synthetic glucocorticoid) (Fig. 5B). These ﬁndings, taken

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(Fig. 2A and Supplementary Figure S6A). SM-microdomains
were essentially absent in SMS1−/− thymocytes, and a small
amount of dispersed-SM was present (Fig. 2B), suggestingthat the amount of cell surface SM was insufﬁcient to cluster
and form SM-microdomains. We next examined the effect of
SMS1 deﬁciency on the content of ceramide, the precursorof numerous sphingolipids, including gangliosides and SM.
Levels of ceramide on the cell surface and inside cells were
not notably altered in SMS1−/− thymocytes (Supplementary
Figure S6A and B), indicating that SMS1 deﬁciency did notcause a compensatory increase of ceramide. In SMS1−/−
mice, levels of CTx-B-binding gangliosides were slightly
increased in CD4SP thymocytes, and unchanged in othertypes of thymocytes (Supplementary Figure S6C).

Role of sphingomyelin in thymocyte development

B

6
5
4
3
2

*

1

7
6

Cell number (107)

Cell number (107)

7

5
4
3
1

0

0

C

D

OT-I
OT-I SMS1-/2.5*

2.0

Cell number (107)

Cell number (107)

**

2

***

1.5
1.0
0.5
0
0.0
DN

DP

E

CD69

III

I

female HY
female HY SMS1-/-

*

**

DN

CD8SP

DP

CD8SP

OT-I SMS1-/-

OT-I

II

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
0.0

IV

V

Cell number (106)

CCR7
20
18
16
14
12
10
8
6
4
2
0

OT-I
OT-I SMS1-/-

*

*

I

II

III

IV

V

Fig. 3. Reduced positive selection in SMS1−/− mice. (A, B) Absolute numbers of transgenic TCR-positive thymocytes gated on Vα2+Vβ5+ cells
for OT-I mice (A), and on HY TCR+ cells for female HY mice (B). Dots: individual mice. Bars: means. Each group, n = 6 (A). Female HY mice,n = 5; female HY SMS1−/− mice, n = 4 (B). (C, D) Absolute numbers of DN, DP and CD8SP thymocytes deﬁned by CD4/CD8 expression in transgenic TCR-positive thymocytes as in A and B. Each group, n = 6. (C). Female HY mice, n = 5; female HY SMS1−/− mice, n = 4 (D). (E) Absolute
numbers of OT-I thymocytes, subdivided into ﬁve stages of thymocyte development (I through V) as in Fig. 1(G). Each group, n = 3. *P < 0.05,**P < 0.01, ***P < 0.001.

together, suggest that aberrant thymic selection in SMS1−/−
mice is a consequence of aberrant TCR signal intensity.
We next examined the role of TCR-mediated signaling in
negative selection. ZAP-70, a Syk-family tyrosine kinase,plays a key role in both positive and negative selection (53).
The amount of phosphorylated ZAP-70 recruited by a phosphorylated TCR complex affects the fate of DP thymocytes;

i.e. differentiation into SP thymocytes or elimination by apoptosis (54). ZAP-70 phosphorylation induced by anti-CD3 +anti-CD4 mAb treatment was higher in SMS1−/− than in control
DP thymocytes (Fig. 5C). TCR-proximal signaling, including
ZAP-70, is divided into two cascades (ERK5 pathway and Bim
pathway) in negative selection signals (55). ERK5 activity regulates the expression of proapoptotic Nur77 family members.

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A

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Page 8 of 13

Role of sphingomyelin in thymocyte development

B

Cell number (106)20

male HY
11.7±1.1

15

2.5±1.5

male HYSMS1-/-***
5.9±1.2

1.0±0.9105CD878.8±5.2087.7±7.0

CD4

Fig. 4. Enhanced negative selection in SMS1−/− mice. (A) Absolute numbers of transgenic TCR-positive thymocytes gated on HY TCR+ thymocytes for male HY and male HY SMS1−/− mice. Dots: individual mice. Bars: means. Male HY mice, n = 6; male HY SMS1−/− mice, n = 4. (B) Dot
plots of CD4 versus CD8α for HY TCR+ thymocytes. Numbers in quadrants: mean ± SD of percentage of each thymocyte population deﬁned
by CD4 and CD8. Male HY mice, n = 5; male HY SMS1−/− mice, n = 3. ***P < 0.001.

Proapoptotic Bim plays a key role in negative selection independently of the ERK5-Nur77 pathway (56, 57). TCR stimulation of SMS1−/− thymocytes resulted in increases of ERK5
phosphorylation, Bim expression (Fig. 5D) and Nur77 induction (Fig. 5E). The CD5 expression level is developmentally
regulated by TCR signaling and TCR avidity, and is therefore
used as a marker of TCR signal intensity in thymocytes (58).
CD5 expression in SMS1−/− DP thymocytes was up-regulated
by TCR stimulation (Fig. 5F). The enhancement of TCR signal
intensity in SMS1−/− DP thymocytes increased the sensitivity
to TCR-mediated apoptosis.
SM-microdomains function as negative regulators of TCR
signal intensity in normal DP thymocytes
The above-described ﬁndings suggest that SM-microdomains
function as negative regulators of TCR signal intensity andthymocyte apoptosis during thymic selection. Expression of
SM-microdomains in normal DP thymocytes was low prior tothymic selection, and increased greatly in the latter stage of
positive selection (Fig. 1C and G). We examined the possibility that addition of SM to normal DP thymocytes leads toa reduction of TCR signaling and TCR-induced apoptosis.
TCR stimulation of whole DP thymocytes resulted in a strong,dose-dependent increase of SM-microdomain formation in
the latter stage of positive selection (Fig. 6A). By SM addition,
TCR stimulation also signiﬁcantly reduced CD5 expression
and enhanced cell viability (Fig. 6B and C). These ﬁndings
indicate that the SM content of normal DP thymocytes controls the formation of SM-microdomains, and consequent
reduction of TCR signal intensity.
Discussion
We performed a comprehensive analysis of the expression
and function of plasma membrane SM-microdomains as
related to thymocyte development. Use of EGFP-NT-Lys, aprobe that recognizes SM molecules clustered in microdomains as distinct from single SM molecules dispersed in theouter leaﬂet of the membrane lipid bilayer (Supplementary

Figure S1) (29, 30), allowed us to demonstrate for the ﬁrst
time a great increase in SM-microdomain levels during DP
stages, particularly at the end of thymic selection events(Fig. 1). The process involved three stages: (i) the pre-selection stage, TCRβlowCD69− DP, characterized by the absenceof SM-microdomains because of low SM expression levels; (ii)the CD69+ DP stage, with initiation of TCR signaling required
for thymic selection, and an increase of SM-microdomain levels; and (iii) the post-selection stage, TCRβhiCD69+CD4+CD8
low, in which SM-microdomain levels are >10-fold higher thanin the pre-selection stage. Levels of dispersed, single SM
molecules increased only slightly during this process (Fig. 1).
Up-regulation of the SM-microdomain level and TCR expression paralleled the course of thymic selection. TCR expression levels generally remain very low during thymic selection
and are up-regulated only in successfully selected thymocytes (59, 60).
Cholesterol is physically associated with SM in membrane
microdomains (46). The plasma membrane cholesterol content during pre- to post-selection stages increased in parallel with levels of SM but not of gangliosides (Supplementary
Figure S3B). Expression levels of CTx-B-binding gangliosides
were very low in the DP stage, increased upon differentiation
into the CD8 SP stage, and then declined upon differentiation
into the CD4SP stage, consistent with a report by de Mello
Coelho et al. (61). Ganglioside-microdomains are polarized to
TCR activation sites in response to TCR stimulation in mature
peripheral T cells, but not in DP thymocytes (62). Theseﬁndings, taken together, suggest that SM-microdomains(not ganglioside-microdomains) are formed preferentially inthe plasma membrane during the course of selection eventsin DP thymocytes.
Analysis of SM-microdomains is necessary to elucidate
the role of membrane lipid microdomains in thymocyte TCR
signaling and its outcomes. We used SMS1−/− mice for thispurpose because they express only a trace amount of SM inthymocytes and have no Lys-positive DP thymocytes; i.e. DP
thymocytes in these mice have no surface SM-microdomains(Fig. 2). SMS1−/− mice show a slight (although signiﬁcant)Downloaded from https://academic.oup.com/intimm/advance-article-abstract/doi/10.1093/intimm/dxy082/5250428 by Tohoku Yakka Daigaku Toshokan user on 28 February 2019A

Role of sphingomyelin in thymocyte development

8
6
4
2
0
Saline2
1.5

CD3/CD28
TCR stim PMA/Iono

1
0.5

--

Dex

1.4

-

Nur77+
CD3/
CD28

*

1
0.8
0.6
0.4
0.2

SMS1-/Counts

Counts0/0Countsβ-actin18.8±0.4

䕕Control 䕔 SMS1-/-

1.2

Bim

Controlanti-CD3+ antiCD3/CD4
CD4

Nur77+ERK5

CD3/CD28(μg/mL)*E

Control
SMS1-/hr
6
0 2
0 2
6
- + - + CD3/CD28- + - +

p-ERK5F䕕 Control䕔 SMS1-/-0(-)-

anti-CD3

D***

Nur77+ (fold change)10

䕕 Control䕔 SMS1-/-

pZAP-70/ZAP-70(fold change)*䕕 Control䕔 SMS1-/-

80
70
60
50
40
30
20
10
0

Counts12

Annexin-V+ cells (%)

Annexin-V+ cells (%)

C

B
14

0

15.2±0.2 **

Nur77

TCR
stim(-)TCR
CD3/
stim(+)
CD28

62.5±3.5

Counts32.5±5.3

Counts

10/50

Counts

1/5CountsControl
SMS1-/47.2±9.5

77.2±5.0 *CD5Fig. 5. Enhanced TCR signaling in SMS1−/− DP thymocytes, accompanied by up-regulation of apoptosis-related molecules and of TCR-induced
apoptotic cell death. (A) Thymocyte apoptosis induced by anti-CD3 mAb administration. Littermate control and SMS1−/− mice were injected i.p.
with 3 µg anti-CD3 mAb or saline twice, at 24-h intervals. Mice were killed 24 h after the last injection, thymocytes were isolated and stainedwith CD4, CD8 and annexin-V, and stained cells were analyzed by FACS. Data shown are percentages of annexin-V+ DP thymocytes. (B–F) In
vitro assays with TCR stimulation. (B) Thymocytes were cultured in the absence (−) or presence of immobilized anti-CD3 (1 µg ml−1) plus antiCD28 (5 µg ml−1) mAbs (CD3/CD28), 50 ng ml−1 PMA plus 1 µM ionomycin (PMA/Iono) or 3 nM dexamethasone (Dex). Cells were cultured for
18 h, harvested and analyzed as in (A). Data shown are mean ± SD from three independent experiments. (C) Intracellular staining of phosphoZAP-70. Thymocytes were unstimulated (−) or stimulated with anti-CD3 plus anti-CD4 mAbs (CD3/CD4) for 5 min, and analyzed as describedin Methods. MFI values for phospho-ZAP-70 and total ZAP-70 were calculated in the DP-gated population. Relative phosphorylation of ZAP-70
in stimulated cells was normalized relative to total ZAP-70 protein for each group. Data shown are fold change values relative to unstimulated
thymocytes of control mice, normalized to one. n = 4. (D) Thymocytes were unstimulated (−) or stimulated (+) with anti-CD3 (10 µg ml−1) plus
anti-CD28 (20 µg ml−1) for the indicated periods. ERK5, phospho-ERK5, Bim and β-actin were detected by immunoblotting analysis. Data shown
are from ﬁve independent experiments. (E) Thymocytes were cultured in the absence (−) or presence of immobilized anti-CD3 (1 µg ml−1) plus
anti-CD28 (5 µg ml−1) mAbs (CD3/CD28) for 2 h, and analyzed as described in Methods. Histograms at left: intracellular Nur 77 expression on
the gated DP population. Nur 77 MFI values in the Nur77+ cells were calculated for each group. Bar graphs at right: ratios of values to those ofunstimulated cells of control mice, normalized to one. n = 3. (F) Thymocytes were cultured with immobilized anti-CD3 plus anti-CD28 mAbs for
16 h at the indicated concentrations, stained with anti-CD5 mAb and analyzed by FACS. Histograms: CD5 expression gated on the DP population. Horizontal lines: percent CD5+ cells. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001.

reduction of DP thymocytes. Numbers of SMS1−/− thymocytes bearing positively selected transgenic TCRs are
relatively quite low (Fig. 3A and B). CD69+CCR7+ late developmental stages of thymic selection are particularly affected(Fig. 3E). Male SMS1−/− mice bearing HY TCR transgenes
show a signiﬁcant decrease in the CD8SP compartment(Fig. 4C). Positive selection generally precedes negative

selection. In SMS1−/− mice, positive selection was abolishedat the end of selection stages, and negative selection was
substantially enhanced.
A difference in ligand afﬁnity for the TCR is essential for
shifting the threshold from positive to negative selection signals via the TCR complex (63). Outcomes of positive andnegative selection are determined by not only the afﬁnity and

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A

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Role of sphingomyelin in thymocyte development

A
EGFP-NT-Lys binding(102 MFI)

䕕䕔 CD3/CD28

40
35
30

**

25
20
15
10
5
0
0 μg/ml

10 μg/ml

B

25 μg/ml*

8䕕䕔 CD3/CD28

CD5 MFI (103)

7
6
5
4
3
2
1
0
0 μg/ml
0

C

10 10
μg/ml

25 μg/ml
25*
Annexin-/7-AAD- cells (%)

80

䕕䕔 CD3/CD28

70
60
50
40
30
20
10
0
0 μg/ml

10 μg/ml

25 μg/ml

Fig. 6. Addition of SM to normal DP thymocytes lacking
SM-microdomains reduces their TCR signal intensity. Thymocytes
were cultured with or without immobilized anti-CD3 (1 μg ml−1) plus
anti-CD28 (5 μg ml−1) mAbs (CD3/CD28) in the presence of indicated concentrations of SM. Cells were cultured for 16 h, harvested,
stained with Lys (A), CD5 (B) or annexin-V plus 7-AAD (C), and analyzed by FACS. (A, B) Mean ± SD of MFI of Lys and CD5 gated on
DP thymocytes. (C) Percentage of annexin-V−/7-AAD− cells. n = 3.
*P < 0.05, **P < 0.01.

avidity of TCR-antigen engagement, but also the strength andduration of TCR signals (64). Induction of thymocyte apoptosis by in vivo administration of anti-CD3 mAb was enhanced
in SMS1−/− mice (Fig. 5A). In mature T cells, membrane lipid
microdomains serve as a platform for TCR-proximal proteins, e.g. coreceptors CD4 and CD8, Src family kinases
Lck and Fyn, ZAP-70, and the transmembrane adaptor LAT(65, 66). We demonstrated previously that splenic CD4+ T
cells from SMS1−/− mice showed impairment of clonal expansion, IL-2 and IFNγ production and phosphorylation of ZAP70 and LAT upon CD3- and CD4-mediated activation (25).
Protection of SMS1−/− mice was observed in a ConA-induced
hepatitis model (25). We therefore expected to ﬁnd that

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SM-microdomains are involved in TCR signaling associatedwith thymic selection in central tolerance.
Contrary to expectation, SMS1−/− DP thymocytes showedenhanced ZAP-70 phosphorylation and increased expressionof proapoptotic proteins and hyper-apoptotic phenotypes
upon TCR ligation (Fig. 5). This ﬁnding may be attributable
to two qualitative differences between immature thymocytes
and mature T cells: (i) similar but not identical molecules
involved in TCR-proximal signaling (67, 68) and (ii) formation patterns of the immunological synapse structure at thethymocyte/antigen-presenting cell interface are different fromthose observed during mature CD4+ T-cell activation (69–71).
SM expression patterns vary greatly among T-cell developmental stages, e.g. DP, CD4SP and CD8SP thymocytes, andCD4+ and CD8+ T cells (Fig. 1; our unpublished data). The difference in need for SM-microdomains in thymocytes versus
mature CD4+ T cells may be due to these factors.
Positive and negative selection, two contrasting developmental outcomes, depend completely on signals initiatedfrom the TCR, even though the TCR signaling machinery is
qualitatively similar in composition (67). Some key players
in the TCR signaling network responsible for the inductionof negative rather than positive selection have been identiﬁed (63, 72). Negative selecting ligands for the TCR induce
stronger activation of TCR-proximal signaling in thymocytes,
leading to ERK5 phosphorylation, induction of Nur77 family
members, and up-regulation of proapoptotic Bim protein.
ERK5-Nur77 and Bim cascades cooperatively develop mitochondrial dysfunction, resulting in apoptosis. Among theTCR-proximal signaling molecules, ZAP-70 is a well-known
kinase essential for both positive and negative selection(53). Phosphorylation of ZAP-70 at Y319 after 5 min of TCR
stimulation was signiﬁcantly increased by SMS1 deﬁciency(Fig. 5B). In a study by Daniels et al., nearly all thymocytes
stimulated by negative selecting ovalbumin peptides showed
recruitment of phosphorylated ZAP-70 at Y319 to the plasma
membrane within 2 min, while such recruitment was much
lower for positive selecting ovalbumin peptides (9). Thus,
SM-microdomains function as negative regulators of TCRproximal signaling during thymic selection.
Expression of the proapoptotic protein Nur77 was correlated with the strength of TCR signaling, and particularly with
ERK5 activation (55, 73, 74). Bim is an antagonist of Bcl-2
that promotes cellular survival by inhibiting the activities ofthe proapoptotic proteins Bax and Bak, thereby inducing
mitochondrial dysfunction. These two major effector pathways of thymic deletion were promoted by SMS1 deﬁciency(Fig. 5C and D). Enhanced TCR signaling in SMS1−/− mice
resulted in increased CD5 expression on DP thymocytes(Fig. 5F). The cell surface glycoprotein CD5 is used as a surrogate marker for TCR signal strength during thymic development; the degree of surface CD5 expression is correlatedwith the strength of TCR signaling generated by cells (58,75). These ﬁndings suggest that SM-microdomains act as aplatform to modulate TCR signaling during thymic selection
events, and are involved in ﬁne tuning of thresholds for positive and negative selection.
SMS1 deﬁciency inhibited the DP-to-SP differentiation intransgenic models of positive selection (Fig. 3), and greatly
reduced (by 33–50%) the overall thymocyte cellularity,

Role of sphingomyelin in thymocyte development

the TCR, SM-microdomains and cholesterol (Fig. 1C andSupplementary Figure S3B) (77, 80). Positively selected
thymocytes multiply surface TCR levels, which potentiallycould lead to triggering stronger TCR signaling, and at thistime they also up-regulate SM-microdomains and cholesterolon their surface (Fig.1G and Supplementary Figure S3B). It
was recently reported that cholesterol binding to the TCRβ
chain maintains the TCR in an inactive status and cholesterol removal causes it to switch to the active status (81). In
contrast, other recent reports claim that cholesterol serves
as a positive regulator of TCR activation because cholesterol
extraction results in the disruption of TCR nanoclustering (82,83). Other membrane lipids interacting with cholesterol may
determine which of these opposing functions dominates;
TCRs and associated signal molecules can perceive theirmicroenvironment, such as the concentration of a speciﬁc
lipid species and the presence of a speciﬁc lipid microdomain, and respond accordingly. In view of these ﬁndings, wehypothesize that initially low amounts of SM-microdomains are
greatly increased to prevent the persistence of TCR signals
in positively selected thymocytes, despite the fact that TCR
expression is strongly up-regulated at the same time in thesecells, which potentially could trigger a stronger TCR signaling
to cause a switch from positive to negative selection.
SM-microdomains are shown in this study to negatively
regulate TCR signal transduction in thymocyte development.
Expression levels of CTx-B-binding gangliosides, in contrast
to those of SM, are higher in CD8SP than CD4SP thymocytes(Supplementary Figure S6A). We demonstrated previouslythat these functions depend on distinct expression patternsof ganglioside species in CD4+ and CD8+ T cells (15). Optimal
expression levels of speciﬁc sphingolipid species in speciﬁctypes of immune cells are presumably necessary for proper
functioning of the cells. We therefore need to elucidate theroles of speciﬁc lipid species in membrane microdomains.
To understand how SM-microdomains reduce TCR signaling
in the plasma membrane of positively selecting thymocytes,
we require microscopic visualization of lipid microdomains
and immunological synapse structures to clarify interactions
between SM-microdomains and TCR-proximal signaling molecules during thymic selection. Conﬁrmation that TCR signaltransduction in thymic selection is controlled by ‘ﬁne tuning’of SM-microdomain levels will provide a basis for future therapy of autoimmune diseases by speciﬁc elimination of autoreactive T cells to establish appropriate negative selection(central tolerance).
Funding
This work was supported by Grants-in-Aid for Scientiﬁc Research(16H04767 to J.-i. I.) from MEXT, Mizutani Foundation for
Glycoscience (to J.-i. I.), Takeda Science Foundation (to J.-i. I.),
Nagai Memorial Research Scholarship from the PharmaceuticalSociety of Japan (to K.T.) and the MEXT-Supported Program for theStrategic Research Foundation at Private Universities.

Acknowledgements
The authors are grateful to Ms F. Shishido (Tohoku Medical andPharmaceutical University) for experimental support, Ms M. Toyosawa(Tohoku Medical and Pharmaceutical University) for assistance with

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particularly CD69+CCR7hi medullary thymocytes (stage
IV). CD69+ thymocytes up-regulate TCR surface expression during stages III and IV, when positively selected
thymocytes migrate to the medulla to undergo negative
selection, and develop very high surface expression of Lyspositive SM-microdomains (Fig. 1G). Evidently, expressionof SM-microdomains is greatly increased when positively
selected thymocytes are no longer able to generate intense
intracellular signaling from mass-produced TCR molecules,
perhaps causing a switch from a positive to a negative selection signal. Thus, SM-microdomains may reduce the TCR signal strength such that positive selection signals are shiftedfrom a positive to a negative pathway in selecting thymocytes, whereas negative selection signals are enhanced.
The roles of lipid rafts in thymocyte development havebeen investigated by many groups. Positive selection signals
occur during the recruitment of TCR signaling molecules tothe raft fraction (11, 12). CD4 lineage commitment from DP
is directed by raft-associated presentation of MHC class II
molecules (76). Fluorescence-labeled CTx-B is often used
for microscopic visualization of lipid rafts and immunological synapse structures; however, CTx-B reacts only with a
few ganglioside species which comprise a minor proportion of raft-forming sphingolipids in DP thymocytes (18).
Speciﬁc lipid species are presumed to be localized in distinct, non-overlapping microdomains in the plasma membrane. TCR signals did not cause polarization of lipid rafts
visualized with CTx-B in DP thymocytes (62). Thus, the validity of the lipid microdomain concept in thymocytes is questionable. We demonstrate here that a reduction or increaseof SM-microdomains in thymocytes causes, respectively,
an increase or reduction of TCR signaling. Zech et al. foundthat SM lipids with cholesterol and saturated phosphatidylcholine were accumulated in TCR activation domains immunoisolated by anti-CD3 mAb from Jurkat cells following TCR
stimulation (23). These ﬁndings, taken together, suggestthat SM-microdomains serve as a platform for TCR-proximal
signaling, and that SM-microdomains (not ganglioside-microdomains) negatively modulate TCR responsiveness during
thymic selection.
Several points remain unresolved. Do SM-microdomains
contain TCR-associated signaling components and TCR per
se following TCR ligation in selecting thymocytes, similarly tomature T cells? If so, is SM-microdomain clustering accompanied by immunological synapse formation on the surfaceof selecting thymocytes? The latter question is of great interest in view of the diversity of immunological synapse structures (77, 78). DP thymocytes interacting with thymic stromalcells did not form a central supramolecular activation cluster; i.e. a central area of immunological synapses observed
between mature CD4+ T cells and B cells, where most of
the TCR and CD28 are accumulated (70). Instead, the above
cells, during positive selection, formed ‘multifocal’ immunological synapses in which small accumulated TCR clusteredat multiple sites in the cell–cell interface (69). The formationof these multifocal synapses was the result of much lower
TCR expression by DP thymocytes relative to peripheral T
cells (79). In this way, pre-selected DP thymocytes inherently
possess the ability to transduce the TCR signaling required
for thymic selection, despite the low surface expression ofPage 11 of 13

Page 12 of 13

Role of sphingomyelin in thymocyte development

mouse breeding and genotyping and Dr S. Anderson for English editing of the manuscript.

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Page 13 of 13

REVIEW ARTICLE

The regulatory roles of glycosphingolipid-enriched lipid
rafts in immune systems
Hitoshi Nakayama1,2, Masakazu Nagafuku3, Akemi Suzuki3, Kazuhisa Iwabuchi1,2,4 andJin-Ichi Inokuchi3
1 Laboratory of Biochemistry, Juntendo University Faculty of Health Care and Nursing, Urayasu, Japan
2 Institute for Environmental and Gender-speciﬁc Medicine, Juntendo University Graduate School of Medicine, Urayasu, Japan
3 Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai,Japan
4 Infection Control Nursing, Juntendo University Graduate School of Health Care and Nursing, Urayasu, Japan

Correspondence
K. Iwabuchi, Laboratory of Biochemistry,
Juntendo University Faculty of Health Careand Nursing, Urayasu, Chiba 279-0023,
Japan
Fax: +81 47 353 3178
Tel: +81 47 353 3171
E-mail: iwabuchi@juntendo.ac.jp
andJ. I. Inokuchi, Division of Glycopathology,
Institute of Molecular Biomembrane and
Glycobiology, Tohoku Medical andPharmaceutical University, 4-4-1,
Komatsushima, Aoba-ku, Sendai, Miyagi
981-8558, Japan
Fax: +81 22 727 0076
Tel: +81 22 727 0117
E-mail: jin@tohoku-mpu.ac.jp(Received 12 June 2018, revised 9 October
2018, accepted 10 October 2018, available
online 27 October 2018)

Lipid rafts formed by glycosphingolipids (GSLs) on cellular membranes play
important roles in innate and adaptive immunity. Lactosylceramide (LacCer)
forms lipid rafts on plasma and granular membranes of human neutrophils.
These LacCer-enriched lipid rafts bind directly to pathogenic components,
such as pathogenic fungi-derived b-glucan and Mycobacteria-derived lipoarabinomannan via carbohydrate-carbohydrate interactions, and mediate innate
immune responses to these pathogens. In contrast, a-series and o-series gangliosides form distinct rafts on CD4+ and CD8+ T cell subsets, respectively,
contributing to the respective functions of these cells and stimulating adaptive
immune responses through T cell receptors. These ﬁndings suggest that gangliosides play indispensable roles in T cell selection and activation. This
Review introduces the involvement of GSL-enriched lipid rafts in innate andadaptive immunity.
Keywords: acquired immunity; glycosphingolipid; innate immunity;
intracellular parasitism; lipid raft; neutrophil; phagocytosis; T cell
activation; T cell selection

doi:10.1002/1873-3468.13275
Edited by Sandro Sonnino

Glycosphingolipids (GSLs) are composed of hydrophilic
sugar structures and hydrophobic ceramide [1]. The ceramide moiety of GSLs interacts hydrophobically withcholesterol sterol-ring systems via van der Waal’s forces
and hydrogen bonds [2]. Moreover, cis interactions
between sugar moiety of GSLs facilitate lateral

Abbreviations
BCR, B cell antigen receptor; CSBG, Candida solubilized cell wall b-glucan; CTB, cholera toxin B subunit; EMRN, encephalomyeloradiculoneuropathy; Gb3, globotriaosyl ceramide; GM3S, GM3 synthase; GSLs, glycosphingolipids; LacCer, lactosylceramide; LAM, lipoarabinomannan; LPS, lipopolysaccharide; MAC, Mycobacterium avium-intracellulare complex; MAM, mitochondria-associated membranes;
ManLAM, mannose capped LAM; MHC, major histocompatibility complex; MbCD, methyl-b-cyclodextrin; NKT, natural killer T; OVA, ovalbumin; PAMP, pathogen-associated molecular pattern; PILAM, phospho-myoinositol capped LAM; PRRs, pattern-recognition receptors; PtdGlc,
phosphatidylglucoside; RA, rheumatoid arthritis; ROS, reactive oxygen species; SLE, systemic lupus erythematosus; SPR, surface plasmon
resonance; Stx, Shiga toxin; SV40, simian virus 40; TCRs, T cell receptors; WT, wild-type.

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H. Nakayama et al.

interactions of these molecules with other membranecomponents. These interactions lead to the formation ofcellular membrane microdomains, so called lipid rafts,
which are rich domains in cholesterol, GSLs, sphingomyelin, glycosylphosphatidylinositol, and membraneanchored molecules [3,4]. GSL-enriched lipid rafts
contribute innate and adaptive immune systems [5–12].
The innate and adaptive immune systems are involved
in resistance to invading pathogens, but the two systems
are functionally distinct.
Innate immune responses are the initial step in resistance to invading pathogens, including viruses, bacteria,
and fungi. Many viruses, bacteria, and bacterial toxins
have been shown to bind to the carbohydrate components of GSLs on host cell surfaces [13]. Binding studies
have revealed that several glycolipids are components offunctional receptors for microorganisms and bacterial
toxins [14]. For example, the neutral GSL, Lactosylceramide (LacCer, CDw17), forms LacCer-enriched lipid
rafts on the plasma and granular membranes of humanneutrophils. LacCer-enriched lipid rafts play essential
roles in innate immune responses to bacterial pathogens,including Candida and Mycobacteria. Indeed, LacCerenriched lipid rafts are capable of binding directly tobacterial components, such as C. albicans-derived b-glucan and Mycobacteria-derived lipoarabinomannan(LAM) through carbohydrate–carbohydrate interactions [15,16]. The binding of bacterial pathogens to LacCer-enriched lipid rafts activates signaling cascades
involving Src family kinases, inducing chemotaxis,
phagocytosis, and phagolysosome formation (phagosome maturation). Interestingly, pathogenic mycobacteria, such as M. tuberculosis, target LacCer-enriched
lipid raft-dependent activation of the Src family kinase
Hck on phagosomal membranes of neutrophils, thereby
inhibiting phagolysosome formation [15]. These ﬁndings
show that GSLs can act as direct and functional receptors for pathogen-derived ligands.
In addition to their roles as functional receptors,
GSLs can act together with other receptors via cis
interactions. Lipid rafts formed by the ganglioside (sialylated GSL) GM3 are associated with insulin receptor
in adipocytes [17]. GM1 binds to TrkA protein andregulates the receptor function [18], and GM1-enriched
lipid rafts work as signaling platforms for laminin-1induced TrkA/b1 integrin-associated neurite outgrowth[19]. GM1 was also found to interact with the G protein-coupled receptor serotonin-1A receptor via thesphingolipid-binding domain of the latter [20], suggesting the involvement of GM1 in neurodegenerative diseases. In innate immune responses, the phagocytosis of
non-opsonized microorganisms, such as Mycobacteria,
in human neutrophils is regulated jointly by CD11b/
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CD18 (aMb2)-integrin and LacCer-enriched lipid rafts
coupled with Src family kinase Lyn [15,21]. Theseobservations suggest that GSL-enriched lipid rafts regulate receptor functions, resulting in various physiological activities.
In addition to modulating innate immune systems,
GSLs have been found to play critical roles in adaptive immune systems. In adaptive immunity, a-series
gangliosides (a-GMs) and o-series gangliosides are
involved in the function and stimulation of T cell
receptors (TCRs) on CD4-positive (CD4+) and CD8positive (CD8+) T cells, respectively [22]. GQ1b anda-GalCer were shown to function as immunomodulators [23–25]. Moreover, a-GalCer, derived from a marine sponge, was found to act as a lipid antigen that
activates natural killer T (NKT) cells [23], which control innate and adaptive immune responses [26].
a-Linked glycosylceramides were shown to be themajor endogenous ligands of NKT cells [27]. Theseﬁndings suggest that GSL-mediated NKT activation
may be a potential immunotherapeutic target.
Although GSLs can mediate both innate andadaptive immune functions through a variety of pathways, the pathogenesis of autoimmune diseases likely
involves immune system responses to GSL antigens.
Interestingly, the anti-LacCer monoclonal antibodies(mAbs) T5A7 and Huly-m13 can recognize LacCer onhuman neutrophils, whereas T5A7 alone recognizes
LacCer on mouse neutrophils [28]. Moreover, Hulym13 can be used for immunoprecipitation, whereas
T5A7 cannot [21]. These observations suggest that the
speciﬁcities of antibodies against the same GSL antigens contribute to differences in the structural featuresof GSL-enriched lipid rafts.
This review describes the mechanisms by which
GSLs and GSL-enriched lipid rafts recognize theirligands and receptors, as well as describing the regulatory roles of GSL-enriched lipid rafts in innate andadaptive immunity.

GSLs as immune modulators
Glycosphingolipids are abundantly expressed on theouter leaﬂets of cell membranes. In addition, some gangliosides are broadly distributed in body ﬂuids of mostvertebrates. Some GSLs have been demonstrated tofunction as unintentional binders of bacteria in speciﬁc
tissues.
Binding of GSLs to microorganisms
Microorganisms and toxins target GSLs for the adhesion of microorganisms to speciﬁc tissues (Table 1).

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The role of glycosphingolipids in immunity

Table 1. Binding of GSLs to pathogens and toxins.
GSLs

Pathogens or toxins

Ref. No.

GD1a,
GT1b
GM1

Polyomavirus[29]

Simian virus 40
Brucella suis
Cholera toxin B subunit
Escherichia coli enterotoxin
Vibrio cholerae enterotoxin
Shiga toxin, Verotoxin B subunit
Pseudomonas aeruginosa,
Biﬁdobacterium biﬁdum, Lactobacillus
Mycobacterium tuberculosis
Mycobacterium avium-intracellulare complex
Mycobacterium gordonae
Mycobacterium smegmatis
Mycobacterium tuberculosis ManLAM
Mycobacterium avium-intracellulare
complex ManLAM
Mycobacterium smegmatis PILAM
Candida albicans b-glucan
Helicobacter pylori
Pneumocystis carinii b-glucan
Bacillus dysenteriae
Bordetella pertussis
Saccharomyces cerevisiae PGG-glucan
Escherichia coli
Propionibacterium granulosum
Propionibacterium freudenreichii
Yersinia pestis
Escherichia coli CFA/I protein
Neisseria gonorrhoeae
Borrelia burgdorferi
Shigella LPS

[29]
[36]
[37,38][39][42][30–33][34,35]

Gb3
AsialoGM1
LacCer

GM1,
GM3

[15]
[15]
[15]
[15]
[15]
[15]
[15]
[16]
[44]
[45]
[46]
[47]
[48]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[58]

For instance, polyomavirus invades human erythrocyte
through the binding to GD1a and GT1b [29]. Globotriaosyl ceramide (Gb3) binds to Shiga toxin (Stx) andverotoxin B subunit [30–33]. Human epithelial andendothelial cell surfaces contain binding sites for Stx[30–33], with cell surface Gb3 binding to the B subunitof Stx [30–32]. Epithelial cells-expressing asialo GM1(GA1) binds to Pseudomonas aeruginosa, Biﬁdobacterium biﬁdum, and Lactobacillus [34,35]. Simian virus
40 (SV40) [29] and Brucella suis [36] were found tobind to GM1. Cholera toxin B subunit (CTB) also
binds to GM1 [37,38]. GM1 expressing on epithelial
cells also binds to CTB and Escherichia coli enterotoxin [39]. In addition to GM1, CTB binds to othergangliosides, especially fucosylated GM1 [40,41]. Gangliosides in human milk are thought to protect intestinal tissues from Vibrio cholerae and E. coli
enterotoxins [42]. Ganglioside content and distributionof human milk individually alter by lactation period.

While GM3 is the main ganglioside in mature milk,
GD3 is the most abundant in colostrum. Orally
administered gangliosides are likely to catch microorganisms, resulting in the intestinal protection. Indeed,
the dietary effects of ganglioside on the production ofcytokines and IgA, which are involved in the development of the intestinal immunity, have been examinedin animal models [7]. GD3 but not GM3 was shownto inhibit lipopolysaccharide (LPS)-induced murine
dendritic cell maturation and to modulate LPSinduced inﬂammatory cytokine release [43]. However,the detailed mechanisms underlying the different activities of GM3 and GD3 remain to be elucidated.
Lactosylceramide is expressed not only on neutrophils but also on intestinal epithelial cells, and binds
to various microorganisms, such as Mycobacterium
avium-intracellulare complex (MAC), M. tuberculosis,
C. albicans, Bacillus dysenteriae, Bordetella pertussis,
E. coli, and Propionibacterium freudenreichii [15,16,44–
56]. Human and bovine milk contain LacCer as amajor GSL component [57], indicating that milkderived LacCer is a putative trap and blocking pathogen binding in the intestines, and that milk-derived
LacCer protects the host from invading pathogens.
Interactions between the sugar moieties of gangliosides
and the polysaccharide moieties of Shigella LPS were
found to facilitate binding of bacteria to humanCD4+ T cells [58]. Taken together, these observations
suggest that speciﬁc carbohydrate-carbohydrate interactions between sugar moieties of GSL and of pathogen-associated molecular pattern (PAMP) may berelated to various immune disorders.
Effects of GSL regulation on innate immunity
Innate-like T lymphocytes, such as NKT cells, recognize GSLs presented by CD1d and control innate andadaptive immune responses against not only someinﬂammatory diseases but also against cancers andinfectious diseases [26]. It has been found that amarine sponge-derived a-GalCer is a lipid antigen
molecule, which activates NKT cells [23]. Moreover,
a-GalCer was found to enhance immune reactions tovarious infectious microorganisms and several cancers,
as well as inhibiting autoimmune disorders, allograft
rejection, and graft-versus-host disorders [59]. aLinked glycosylceramides were determined to be themain endogenous ligands on NKT cells [27]. Macrophage inducible C-type lectin (Mincle) responds tob-GlcCer, which is an endogenous ligand and possess
immunostimulatory activity [60]. These ﬁndings suggest that GSL-mediated NKT activation is a potential
target for immunotherapy of several disorders.

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Glycosphingolipids form lipid rafts on the plasma andgranular membranes of host immune cells and act asfunctional pattern-recognition receptors (PRRs) forinvading pathogens. LacCer and gangliosides recognize
pathogen-speciﬁc carbohydrate structures. These GSLs
mediate a variety of physiological activities, including
chemotaxis, phagocytosis, cytokine production, apoptosis, and autophagy. In some cases, intracellularpathogens, such as Mycobacteria, alter GSL-enriched
lipid raft-mediated innate immune functions, allowing
these pathogens to escape killing by host phagocytes.

solubilized cell wall b-glucan (CSBG) and mycobacterial LAM. In addition, LacCer-enriched lipid rafts
serve as signaling platforms for both inﬂammatory
responses and phagocytosis through aMb2-integrin thatis devoid of signaling motif in the cytoplasm region.
b-Glucan from Pneumocystis carinii has been shown tostimulate the production of large amounts of MIP-2
via LacCer on rat alveolar epithelial cells [45]. LacCer
is expressed on the surfaces of human dendritic cells[71]. LacCer contained GSL-enriched lipid rafts havebeen reported to participate in the activation of humandendritic cells by Pneumocystis b-glucans [72]. Theseﬁndings suggest that GSL-enriched rafts containing
LacCer are involved in the initiation of b-glucan cellular responses and IL-23 release from dendritic cells.
CSBG induces the migration of human neutrophils
and this migration is completely inhibited by Src family kinase inhibitor PP1 or LacCer liposomes [70]. In
contrast, capsular polysaccharide from Streptococcus suis inhibits LacCer-mediated macrophage phagocytosis [73]. We recently reported that both mannose
capped LAM (ManLAM) from M. tuberculosis andphospho-myoinositol capped LAM (PILAM) from
Mycobacterium smegmatis induce Lyn phosphorylationand subsequent aMb2-integrin-mediated phagocytosis
by human neutrophils [15]. These observations suggestthat LacCer-enriched lipid rafts act as not only a signaling PRR inducing inﬂammatory responses but alsoa phagocytic PRR inducing and supporting phagocytosis of microorganisms.

GSL-enriched lipid rafts as pattern-recognition
receptors

Organization and signal transduction by
GSL-enriched lipid rafts

Mammalian innate immune responses are initializedby binding of PAMPs, which are essential components
required for the microbial survival and are not present
on mammalian cells [61], to PRRs expressed on innate
immune cells. PRRs are categorized according to theirfunctions. Toll-like receptors [62,63], LPS-binding
receptor CD14 [64], and C-type lectin receptor dectin-1[65] are PRRs that sense a variety of PAMPs, andmainly mediate PAMPs-induced inﬂammatory signaling pathways. In contrast, macrophage mannose
receptor CD206 [66], scavenger receptors [67,68], andaMb2-integrin [69] are phagocytic PRRs that directly
recognize PAMPs covering microorganisms and induce
phagocytosis of microorganisms. Not only glycoproteins but also GSLs such as LacCer [16,21,70] andGM1 [29,36], which form lipid rafts, are capable ofbinding and sensing PAMPs. As described above, LacCer has binding capacities for microbial PAMPs,
including pathogenic fungi b-glucan Candida

Biological membranes consist of various molecules,
which form several kinds of clusters based on their
physicochemical properties and cis interactions. These
properties provide physical boundaries between outside
and inside of the cells. Therefore, cell membrane components can undergo nonhomogeneous lateral distribution, leading to the formation of membrane domains
with a highly distinguished molecular composition andsupramolecular structures, which are stabilized by lateral interactions among membrane components. The
physicochemical properties of GSLs suggest that they
form deﬁned clusters and that certain proteins cannot
undergo free and continuous lateral diffusion, butrather are transiently conﬁned to microdomains (lipid
rafts) [74–76]. Several molecules can enter into these
domains to form functional domains. In addition,
palmitoylated proteins, such as Src family kinases andsmall G proteins, tend to enter GSL-enriched lipid
rafts [77–80], suggesting that GSLs are involved in

However, the in vivo mechanism by which GSLs recognize their receptors is still unclear.
GSL regulation of adaptive immunity
The ganglioside GQ1b was shown to promote the production of cytokines from T cells, without alteringcytokine productions in B cells and monocytes [24].
When B cells were cocultured with GQ1b pretreated T
cells, B cells produced Ig. Addition of IL-6 or IL-10 to
B cells increased Ig production, with the two together
displaying additive effects. These ﬁndings indicate that
GQ1b induces T cell production of IL-6 and IL-10,
resulting in B cell production of Ig cells [24] and thatextracellular or exogenously added gangliosides may
modulate adaptive immune responses.

GSLs in innate immunity

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H. Nakayama et al.

signal transduction from outside into the cells. Many
studies of the organization and immunological functions of GSL-enriched lipid rafts involve LacCer[3,15,16,21,28,70,71,81,82], a molecule that is selectively expressed on the plasma and granular membranes of human neutrophils, and that forms lipid
rafts with other molecules [15,21,70]. The C24 fatty
acid chain of LacCer on plasma membranes directly
binds to Lyn and Gai [81], with ligand binding tothese LacCer clusters mediating outside-in signaling(Fig. 1) [16,21,82]. Interestingly, immunoelectron
microscopy found that only one-quarter of LacCerenriched lipid rafts is coupled with Lyn on cellular
membranes [82], indicating that different types of LacCer-enriched lipid rafts are present on individual membranes. Although two anti-LacCer monoclonal
antibodies T5A7 and Huly-m13 bind to the lactose
moiety of LacCer, the two were shown to recognize
different areas of LacCer-enriched domains on artiﬁcial lipid layers [28]. Binding experiments suggested
that Huly-m13 binds to lactose clusters in LacCerenriched domains, while T5A7 binds to dispersed
LacCer clusters in the phase boundary regions of LacCer-enriched domains. Two different LacCer-enriched
lipid rafts, e.g., Lyn- and Hck-coupled LacCerenriched lipid rafts are present on phagosomal
membranes of neutrophils [15]. Among them, only
Hck-associated LacCer-enriched lipid rafts can mediate
phagolysosome formation. Interestingly, Hck is absent
in LacCer-enriched lipid rafts of resting neutrophils.
Thus, LacCer-mediated functions during phagocytosis
require the reorganization of LacCer-enriched lipid
rafts and the association with appropriate molecules
during phagosome maturation. LacCer should gather
with appropriate molecules to form several kinds oflipid rafts, each of which may express different molecular patterns and mediate different functions on thesame membranes.
It is unclear, however, whether LacCer in LacCerenriched lipid rafts can mediate outside-in signal transduction. The human acute myeloid leukemia cell line
HL-60 can be induced to differentiate into neutrophilic
lineage cells by treatment with dimethyl sulfoxide. The
resulting D-HL-60 cells acquire superoxide generating
activity, but cannot phagocytose microorganisms under
non-opsonized conditions [21,70]. LacCer on plasma
membranes of D-HL-60 cells consists primarily of
C16:0-LacCer, with little C24-LacCer present [82]. In
contrast, the main molecular species of LacCer on theplasma membranes of human neutrophils are C24:0-,
C24:1-, and C16:0-LacCer. Importantly, Lyn is able toenter into LacCer-enriched lipid rafts of D-HL-60 cells
that are loaded with C24:0- and C24:1-LacCer

The role of glycosphingolipids in immunity

molecules. On the other hand, the loading of C16:0- andC22:0-LacCer molecules hardly reconstruct Lyn-associated LacCer-enriched lipid rafts. LacCer-mediatedimmunological functions can be reconstructed in the
C24:0- or C24:1-LacCer molecule-loaded D-HL-60
cells. Moreover, C24- but not C16-LacCer molecules
have been demonstrated to be directly associated with
Lyn and Gai by the crosslinking experiments with photoactivatable tritium-labeled LacCer analogues [81].
The interactions between C24 fatty acid chains of LacCer and palmitic chains of Lyn and Gai are thought tobe indispensable for LacCer-enriched lipid raftsmediated outside-in signaling (Fig. 1).
aMb2-Integrin mediates immunological functions ofneutrophils and macrophages, such as adhesion,
migration, chemotaxis, and phagocytosis of opsonized
and non-opsonized microorganisms [83]. aMb2-Integrin
does not possess a catalytic motif responsible for signaling inside of the cells [84,85]. Rather, Src family
kinases are the responsible signal transduction molecules for aMb2-integrin-induced functions [86,87].
Thus, the aMb2-integrin requires adaptor molecules forconnecting to Src kinases. aMb2-Integrin initiates
innate immune responses through binding to several
ligands. The aM subunit can bind not only to C3bi but
also to some kinds of PAMPs, including b-glucan and
LPS [88,89]. In plasma membranes of resting neutrophils, aMb2-integrin and LacCer-enriched lipid rafts
are close to but not colocalized with each other. Upon
ligand binding to aM integrin subunit, aMb2-integrins
move to Lyn-associated LacCer-enriched lipid rafts(Fig. 1C). Anti-LacCer antibody Huly-m13 was shown
to pull down the b2 subunit from the detergentresistant membrane fraction (DRM) of zymosancontaining phagosomes, whereas the b2 subunit was
not isolated from the plasma membrane DRM of resting neutrophils [21]. Gene-silencing experiments
showed that aMb2-integrin-mediated activation by
non-opsonized microorganisms depended on Lyn-associated LacCer-enriched lipid rafts in human neutrophils [15,21] (Table 2). These ﬁndings suggest that
Lyn-associated LacCer-enriched lipid rafts act as signaltransduction platforms for signaling motif-deﬁcientmolecules, such as aMb2-integrin.
Mechanism of the binding of LacCer-enriched
lipid rafts to pathogens
As described in section I, several kinds of pathogenic
microorganisms bind to LacCer, suggesting existenceof the common pattern(s) on the membranes of thesemicroorganisms, which can be recognized by LacCer
clusters. Fungi express b-glucans, which are

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The role of glycosphingolipids in immunity

Fig. 1. LacCer-enriched lipid rafts. (A) Schematic image showing the direct association of C24-LacCer with Lyn and Gai. LacCer forms Lynand Gai-coupled lipid rafts on the plasma membrane of human neutrophils. The C24 fatty acid chains of LacCer interdigitate into the inner
leaﬂet of plasma membranes and interact directly with the palmitic chains of Lyn and Gai. These interactions allow association of thesesignal transduction molecules with LacCer to mediate signaling from outside to inside, resulting in neutrophil functions, including
chemotaxis, phagocytosis, and superoxide generation. In addition, LacCer-enriched lipid rafts also act as a signal transduction platform foraMb2-integrin (CD11b/CD18), which is devoid of a signaling motif in its cytoplasmic region. (B) Effect of C24-LacCer on the functions of DHL-60 cells. Loading of C24-LacCer but not C16-LacCer into D-HL-60 cells reconstituted cellular functions, such as chemotaxis andphagocytosis, to almost the same levels as human neutrophils. (C) Schematic image showing the interactions of Lyn-associated LacCerenriched lipid rafts with aMb2-integrin. Upon the binding of bacterial PAMPs, such as CSBG or LAM, to aMb2-integrin and LacCer-enriched
lipid rafts on host cells, the aM subunit is activated and undergoes a conformational change, inducing the accumulation of cytoskeletal
proteins, including talin and a-actinin (actin rearrangement). aMb2-Integrin subsequently translocates into LacCer-enriched lipid rafts,
stimulating Lyn by the direct or indirect interaction between LacCer and residues 514–553 in the C-terminal portion of the conserved
domain of the b2 subunit. These signaling cascades induce the formation of phagocytic cups, resulting in phagosome formation.

Table 2. Associations of GSLs with partner molecules to mediate immune responses.
GSLs

Partner molecules

Cell type

Phenotype

Ref. No.

LacCerNeutrophils (Human)

GM1

PreTCRa

[114]

a-series
gangliosides
o-series
gangliosides
GM1
GM3

CD4, TCR

Monocytes (Human)
Lung epithelial cells NCIH292 (Human)
Lung adenocarcinoma epithelial
cell line A549 (Human)
SCID thymocyte-derived cellline SCB.29 (Mouse)
T cells (Mouse)b-glucan- and LAM-induced phagocytosis of
zymosan and mycobacterial species, respectively
LT-IIb-B5-induced NFjB activation
Flagellin-induced autocrine release of ATP
Clostridium perfringens alpha-toxin-induced
IL-8 release
PreTCR signaling[15,21]GD1aAsialo GM1
GM1a

CD11b/CD18(aMb2)-integrin
TLR2/TLR1
TLR5
TrkA

Helper T cell activation[22]

CD8, TCR

T cells (Mouse)

Killer T cell activation[22]

IgM-BCR
Caspase-8(upon CD95/Fas
stimulation)

Immature B cells (Mouse)
Lymphoblastoid T cells (Human)

Removal of autoreactive immature B cells (apoptosis)
Apoptosis

[153]
[157]

heterogeneous glucose polymers, consisting of b-1,3linked b-D-glucopyranosyl units with or without b-1,6linked side chains of various distributions and lengths[90,91]. CSBG consists of a b-1,3 glucopyranose glucan
backbone, with b-1,6 long glucopyranose side chainsand b-1,3 monoglucopyranose branches [91]. CSBG
binds to GSLs having galactose as the terminal residue, such as GalCer, LacCer, and Gb3 [16]. The structure of yeast-derived PGG-glucan resembles that of
CSBG [92]. PGG-glucan also binds to GSLs having
galactose as the terminal residue [48].
Mycobacteria abundantly express glycolipid LAM
on their cell wall [93]. Pathogenic mycobacteria, including M. tuberculosis and MAC, express a terminal ManLAM, while nonpathogenic mycobacteria, including
M. smegmatis, express a terminal PILAM or no capped
LAM [94]. LAMs have the same mannan core structure, consisting of a linear chain of a-1,6

[169]
[170]
[171]

mannopyranose backbone with a-1,2 mannopyranoseside branches [95]. Both M. tuberculosis-derived ManLAM and M. smegmatis-derived PILAM bind to LacCer [15]. Surface plasmon resonance (SPR) experiments
demonstrated that M. tuberculosis-derived ManLAM
binds to LacCer but not to GM3 (Fig. 2B). M. smegmatis a-1,2 mannosyltransferase-deﬁcient mutant(DMSMEG_4247) expresses mutated PILAM, whichlacks a-1,2 monomannose side branches of the mannancore. Human neutrophils cannot phagocytose
DMSMEG_4247 bacteria. DMSMEG_4247-derived
LAM does not bind to LAM (Fig. 2C). Therefore, thea-1,2 monomannose side branches of mannan core in
LAM of both nonpathogenic and pathogenic mycobacteria are important for phagocytosis of mycobacteria
by neutrophils through the binding of LacCer to LAM
and lipomannan. LacCer-enriched lipid rafts bind to
b-1,6 long glucopyranose side chains with b-1,3

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H. Nakayama et al.

Fig. 2. Binding speciﬁcity of LacCer to the structures of b-glucans and LAM. (A) Schematic images showing binding speciﬁcities of bglucans and LAMs to LacCer. b-Glucans are a heterogeneous group of glucose polymers, consisting of b-1,3-linked b-D-glucopyranosyl units
with b-1,6-linked side chains of various distributions and lengths. LacCer does not bind to b-glucan, which lacks b-1,6-linked long b-Dglucopyranose side branches, such as curdlan. LAMs are major glycolipid components of all mycobacterial species. All mycobacterial LAMs
have a mannan core structure, consisting of a 21–34 residue a-1,6-mannopyranose backbone and 5–10 a-1,2 mannopyranose side chains.
LacCer recognizes the a-1,2-monomannose side branches of LAM, which are expressed by both pathogenic (M. tuberculosis) andnonpathogenic (M. smegmatis) mycobacteria. (B) SPR analysis of the binding speciﬁcity of LAMs to LacCer. SPR analysis indicated that
M. tuberculosis-derived ManLAM binds speciﬁcally to LacCer, but not to GM3. RU, resonance unit. (C) Binding speciﬁcity of LAMs to
LacCer. ManLAM and M. smegmatis LacCer bound to PILAM derived from WT, but not from the M. smegmatis a-1,2-mannosyltransferase
deletion mutant (DMSMEG_4247), as the latter lacks the a-1,2-monomannose side branches of the LAM mannan core (reproduced from
Science signaling 9, ra101 (2016)) with slight modiﬁcations. **P < 0.01. Thus, LacCer binds not only to b-1,6 long glucopyranose side chainswith a b-1,3 glucopyranose branches of CSBG but also to a-1,2-monomannose side chain-containing mannan cores of LAM, suggesting that
LacCer recognizes the common structural patterns of fungal b-glucans and mycobacterial LAMs.

glucopyranose branches of CSBG [16]. Therefore,
these observations provide a novel mechanism that
LacCer clusters bind to the comb-shaped common
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three-dimensional structures between fungal b-glucans
and mycobacterial LAMs through carbohydrate–carbohydrate interactions.

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H. Nakayama et al.

GSL-enriched lipid raft-mediated immune
evasion by intracellular pathogens
The engulfment and killing of microorganisms byphagocytes are well-characterized functions of innate
immunity. PRRs on phagocytes recognize microorganisms, either directly or indirectly, acting as direct sensors of non-opsonic infectious microorganisms in hostphagocytes. Phagocytes efﬁciently engulf opsonized
microorganisms through complement component C3bi
and IgG. Intracellular pathogens, including Listeria,
Salmonella, and Mycobacteria, have been shown totarget lipid rafts [96] as well as proteinaceous PRRs on
host plasma membranes. Above all, pathogenic
mycobacteria are known to exploit lipid rafts and
PRRs to gain entry into phagocytes under non-opsonized conditions [97,98]. These intracellular pathogens
are able to survive inside cells by manipulating a variety of signaling pathways. Once intracellular pathogens
are engulfed by phagocytes, they inhibit the fusion oflysosomes to nascent microorganism-containing
phagosomes or escape from phagocytic vesicles toreach the cytosol [98–105]. Lysosomes, which containseveral kinds of digestive enzymes, effectively fuse toserum-opsonized M. tuberculosis-containing phagosomes, but do not fuse to non-opsonized bacteria-containing phagosomes [106,107], suggesting that signalingcascades differ under opsonized and non-opsonizedconditions.
Although, regardless of pathogenicity, human neutrophils phagocytose mycobacteria through Lyn-associated LacCer-enriched lipid rafts and aMb2-integrin,
the fates of these phagocytosed bacteria are distinct.
Neutrophil phagocytosis of nonpathogenic mycobacteria results in the fusion of lysosomes to bacteria-containing phagosomes [108]. By contrast, phagocytosed
pathogenic mycobacteria prevent lysosomal fusion tobacteria-containing phagosomes, resulting in the survival of mycobacteria inside phagocytes. Neutrophil
granules highly express LacCer-enriched lipid rafts on
their membrane [70,109]. LacCer-enriched lipid rafts
are also expressed on bacteria-containing phagosome
membranes [15,21]. The Src family kinase Hck, which
localizes to the granular membranes [110], is essential
for lysosomal fusion to phagosomes [111]. Alongwith phagosome formation, Hck associates with
LacCer-enriched lipid rafts on membrane of phagosomes containing the nonpathogenic mycobacterium
M. gordonae but not the pathogenic mycobacterium
MAC [15]. Furthermore, these molecular associations
can be observed on phagosomes containing nonpathogenic M. smegmatis-derived PILAM- but notpathogenic M. tuberculosis-derived ManLAM-coated

The role of glycosphingolipids in immunity

beads. Therefore, the mannose cap motif of ManLAM is thought to abrogate the formation of Hckassociated LacCer-enriched lipid rafts on phagosomal
membranes. ManLAMs derived from pathogenic
mycobacteria may therefore affect the reorganizationof phagosomal-membrane LacCer-enriched lipid rafts,
disturbing Hck association with the LacCer domains,resulted in enhancing subsequent mycobacterial escapefrom neutrophil killing (Fig. 3).

Gangliosides in adaptive immunity
T cells are indispensable players in the cell-mediatedimmunity and differentiate into subsets with distinctivefunctions in the thymus which is a central organ for
the education and maturation of T cells. T cell activation through T cell antigen receptors (TCRs) requires
the recruitment of both membrane molecules including
costimulatory receptors and intracellular molecules
participating in signal transduction, into the lipid rafts[12]. T cells differentiate into helper T (CD4+ T) cells,
which are characterized with the expression of CD4 on
their cell surface, and cytotoxic T (CD8+ T) cells
which are distinguished with the expression of CD8 on
their cell membrane. CD4+ and CD8+ T cells achieve
distinctive immunological functions, and have similar
mechanisms of TCR-mediated signaling via particular
intracellular signaling events. Th and Tc cells also
express different types of gangliosides at different
levels [22]. This section covers glycan structure-speciﬁcfunctions of gangliosides involved in the activation of
CD4+ and CD8+ T cells and the roles of gangliosides
in immune diseases.
Gangliosides in T cell development
Precursor cells migrating from bone marrow develop
into conventional T cells in the thymus. Developing T
cells in the thymus, usually called thymocytes, can bebroadly divided into four subsets, based on theirexpression of the speciﬁc surface markers CD4 andCD8: DN (CD4CD8), DP (CD4+CD8+), CD4SP(CD4+CD8), and CD8SP (CD4CD8+). Cells in the
DN subset, which are regarded as the least mature
stage, can be further subdivided into four substages,
DN1 to DN4. DN3 is a prominent stage in the courseof thymocyte development. During this stage, TCRb
chain genes undergo productive rearrangement, leading
to the expression of a preTCR, which induces cell survival, proliferation and expression of both CD4 andCD8 coreceptors [112].
Unlike TCR signaling in mature T cells, preTCR
signaling in DN3 cells does not require the recognition

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H. Nakayama et al.

Fig. 3. The capping motif of LAM affects LacCer-enriched lipid raft-mediated phagosome maturation. Schematic images showing
mycobacterial phagocytosis in human neutrophils. Human neutrophils engulf mycobacterial species through the binding of LacCer-enriched
lipid rafts to the LAM mannan core region. Human neutrophils can therefore engulf both pathogenic and nonpathogenic mycobacteria using
the same mechanism. In contrast, pathogenic mycobacteria, such as M. tuberculosis and MAC, disrupt the reorganization of Hck-coupled
LacCer-enriched lipid rafts on phagosomal membranes through the mannose-capping motif of ManLAM, arresting phagosome maturation.

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H. Nakayama et al.

of major histocompatibility complex (MHC) plus antigen on antigen presenting cells. Rather, preTCR canmediate autonomous signaling, based on the ability ofthe preTCR component pTa to spontaneously form
oligomers [113]. Patch formation by preTCR molecules
can be assessed by ﬂuorescence microscopy. PreTCR
can be palmitoylated and is partially enriched in lipid
rafts [114]. CTx staining, followed by ﬂuorescent-activated cell sorting, showed that ganglioside expression
is higher in the DN than in the DP and SP populations, with ganglioside expression being much higher
at stages DN3 to DN4 than in other thymocyte populations. CTx-reacting ganglioside species (GM1 andextended-GM1b) were shown to colocalize with
preTCR patches on DN cell surfaces [114] (Table 2).
These ﬁndings suggest that CTx-reacting gangliosides
can assist in preTCR clustering in lipid rafts enrichedwith gangliosides. Phosphorylation of ZAP-70 kinase
following preTCR activation can be abolished bytreatment with methyl-b-cyclodextrin (MbCD), which
is widely used for lipid raft disruption. Forcibly targeting CD3Σ, a signaling component of the preTCR complex, in lipid rafts by palmitoylation of the CD3Σassociated molecule calnexin in Rag recombinase null
DN cells, which are defective in preTCR expression,
allows the further development of DN cells [78]. However, mutated forms of preTCR, which lack putative
palmitoylated sites or all intracellular domains, havenormal preTCR signaling capacity [115,116]. Further
research is necessary to better clarify the physiological
functions of gangliosides in preTCR signaling and DN
cell development.
PreTCR signaling has been associated with cell survival and proliferation and with expression of both
CD4 and CD8 coreceptors. The resulting cells are termed DP thymocytes. After TCR rearrangement, DP
cells express mature TCR and undergo positive andnegative selection to become mature CD4 and CD8 SP
cells, respectively. Because DP cells have fewer raftforming lipids, GSLs, and sphingomyelin, than thymocytes of any other developmental stage, the proﬁlesand functions of lipid rafts in DP cells remain unclear.
TCR-mediated activation, such as the recruitment ofsignaling molecules into immunological synapses during positive selection of DP cells, does not involve theaccumulation of CTx-reacting gangliosides [117]. DN4cells in TCR-null mice exhibited greater Ca2+
responses than DP cells upon stimulation with an antiTCR antibody. The forced expression on thymic
epithelium of CD80, a costimulatory molecule thatcooperates with CD28 on thymocytes, induces GM1
accumulation at the site of contact between thymocytes and thymic epithelium, as well as reducing

The role of glycosphingolipids in immunity

positive selection and increasing apoptosis like negative selection [118]. These results suggest that DP cells
have a higher signal threshold for TCR activation andtherefore require stronger stimulation than DN cells,
as the structure and dynamics of lipid rafts on DP
cells are limited by the reduced availability of gangliosides and sphingomyelin.
Although conditional targeted disruption of the
GlcCer synthase (GlcCerS) gene in early DN stage thymocytes resulted in almost complete reduction of GSLs
derived from GlcCer, conventional T cell development
was not affected [119]. This was likely caused by theability of trace amounts of GSLs to form speciﬁc microdomains and induce TCR-mediated signal transduction.
Alternatively, it may have been due to the sphingomyelin microdomain essential for TCR-mediated DN
thymocyte development, in that activation with antiCD3 antibody induced the accumulation of sphingomyelin in a microdomain containing TCR [120].
Roles of gangliosides in TCR-dependent
activation of T cells
Antigen-presenting cells carrying MHC-peptide complexes present peptide antigens to TCRs on the T cells,
triggering T cell activation. CD4 and CD8 as costimulatory molecules on T cells bind to nonpolymorphic
regions of the MHC and facilitate the signaling. During the activation, TCR and CD4 or CD8 on the cell
membrane and intracellular signaling molecules need
to be recruited into speciﬁc cell membrane region
called lipid rafts.
The lipid rafts are composed of sphingomyelin andcholesterol as the major raft lipid components andGSLs or gangliosides as the minor but signiﬁcant components. Cholesterol interacts with hydrocarbon chainsof sphingolipids, holds the raft assembly together likea “glue”, and plays a critical role in maintaining membrane ﬂuidity. The functions of cholesterol in the lipid
rafts are very well documented on the basis of results
obtained by MbCD treatment which depletes cholesterol from cell membranes and disrupts cellular functions [121]. Despite concerns about other effects
produced by the treatment [122], experimental
approaches using MbCD have been widely used. In
contrast, fewer studies have addressed the role of gangliosides in raft functions due to the lack of convenient
procedures manipulating the presence of GSLs andgangliosides in the cell membrane.
Important aspects of ganglioside expression of T cells
can be seen in GM3 synthase knockout (GM3S/) and
GM2/GD2 synthase knockout (GM2/GD2S/) mice[22]. GM3S transfers sialic acid to galactose residue of

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LacCer for making GM3, which the simplest moleculeof the “a-series” gangliosides. GM2/GD2S transfers
GalNAc to LacCer, GM3 or GD3, and expressed GA2,
GM3, and GD2. In wild-type (WT) mice, a-series
GM1a expression of CD4+ T cells is higher than CD8+
T cells. In contrast, expressions of o-series GM1b, GalNAc-GM1b, and extended GM1b are higher in CD8+
T than in CD4+ T cells. TCR-induced proliferation andcytokine production were severely disordered in CD4+
T cells of GM3S/ mice, and these defects can be canceled by pretreatment of the cells with GM3 and GM1a,but not b-series, gangliosides. The severe disorder ofTCR-dependent proliferation and cytokine production
also can be seen in T cells of GM2/GD2S/ mice, andthese defects were also canceled by pretreatment of thecells with o-series gangliosides or their precursor GSLs,
GM1b and GA1, but not with a- or b-series gangliosides. These observations indicate that CD4+ andCD8+ T cell subsets have speciﬁc raft domains composed of distinct kinds of gangliosides, and this distinction is the basis of different function of TCR
stimulation of these two subsets (Fig. 4; Table 2). An
Helper T eel I(CD4+ T cell)a-series
o-series

Thymocytes

Activation

Involvement of gangliosides in T cell-mediatedautoimmune and allergic diseases&
Killer T cell
(CD8 + T cell)
TCR CD8

Activation
Fig. 4. Distinct expression signatures of ganglioside molecules during
T cell development. The maturation of immature thymocytes (doubly
positive for CD4+and CD8+) to mature singly positive T cell
subpopulations was accompanied by selective ganglioside expression.
Gene expression patterns, together with ganglioside analysis,
conﬁrmed that CD8+ T cells exclusively express o-series gangliosides
due to downregulation of GM3S and upregulation of GM2/GD2S
expression. In contrast, GM1a expression was maintained in CD4+ T
cells due to the upregulation of GM3S, suggesting that each T cellsubset has unique rafts composed of different ganglioside molecules
and that these rafts provide distinct functions during different
intracellular events following receptor-mediated stimulation. This
ganglioside selection process may be indispensable in the formation of
distinct and functional lipid rafts in mature T cells.

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interesting possibility is proposed as that CD4 may
interact with the glycan structure common to GM3 andGM1a, Siab2-3Galb1-4Glc, and CD8 with that ofGM1b and GA1, Galb1-3GalNAcb1-4Galb1-4Glc.
Another possibility is the involvement of ceramide
structures of these gangliosides. LC-MS analysis indicated GM1a and b, GD1, GalNAc-GM1b, andextended GM1b carry ceramides composed of d18:116:0, -18:0, -20:0, -22:0, -24:1, and -24:0. Ceramide
structures of these gangliosides were not so much different between CD4+ and CD8+ cells except for higher
content of d18:1-22:0 carrying gangliosides in CD8+
cells. Is this difference critical for the ganglioside preference by CD4 and CD8? These possibilities need to beaddressed and, for doing this, further development ofimaging mass spectrometry which is able to cover them/z values of these gangliosides with much higher sensitivity would become an indispensable approach. CD4
and CD8 localize to lipid rafts by palmitoylation, a process during which acyl chains are attached to core proteins, but raft localization is not determined solely by
this process [123,124]. To ensure the movement of CD4
and CD8 to speciﬁc and correct locations on the membrane, these molecules must likely interact with rafts
carrying speciﬁc gangliosides. Taking these issues into
consideration, the speciﬁc roles of individual gangliosides in regulating membrane microenvironments
remain to be determined as critically important molecular mechanism for maintaining membrane functions.

Negative selection of T cells is indispensable for theprevention of autoimmune diseases during T cell development in the thymus. Survival of self-reactive T cells
could lead to autoimmune disorders and allergic diseases. There are several subsets of CD4+ T cells, e.g.,
Th1, Th2, Th17, and Treg cells. These CD4+ T cellsubsets produce different kinds of cytokines andchemokines, and showing their speciﬁc functions. The
functional speciﬁcity of CD4+ T cell subsets may be
due to differences in the organization of TCR signaling complexes in lipid rafts [125].
Allergic asthma is deﬁned as a type 1 hypersensitivity reaction, which is a manifestation of immediateand late-phase reactions in the lungs. Innate immune
cells, including mast cells, basophils, and eosinophils,
are effector cells during airway inﬂammation [126,127].
CD4+ T cells contribute to the initiation of allergic
reactions by producing Th2 cytokines, such as IL-4
and IL-13, which initiate production of IgE antibodies
against harmless antigen from B cells [128,129].

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H. Nakayama et al.

Administration of ovalbumin (OVA) induces allergic
airway inﬂammation and airway hyperresponsivenessin OVA-sensitized mice [22]. The OVA-induced allergic
airway inﬂammation includes OVA-speciﬁc IgE production, airway inﬁltration by inﬂammatory cells,
mucus hypersecretion, and increased Th2 cytokine
levels in serum. Importantly, these allergic airway
responses markedly reduced in the OVA-sensitized
GM3S/ mice (Fig. 5). The reduction of allergic
responses in GM3S/ mice is thought to be due to alack of GM1a in CD4+ T cells [22]. The expression ofGM1a is enhanced in self-reactive CD4+ T cells, andthis enhancement persists abnormal cell activation[130,131]. Administration of antisense oligonucleotides
against the GM3S gene suppressed airway inﬂammation [132]. These ﬁndings indicate that the a-GMs such
as GM1a are essential for T helper cell function

The role of glycosphingolipids in immunity(Fig. 4). The balance between effector Th2 cells andsuppressive Treg cells during allergic airway inﬂammation was skewed toward Th2 predominance [133]. The
particular role of Th17 cells in asthma is still
unknown. But, Th17 cells could be involved in neutrophilic, steroid-resistant severe asthma and enhance
the airway inﬂammation caused by Th2 cells [134].
Th17 cells from GM3S/ mice reduced the number ofTh17 cells in vitro [135].
One of the most common autoimmune diseases issystemic lupus erythematosus (SLE), which presentswith multisystem clinical manifestations, including
rash, arthritis, glomerulonephritis, hemolytic anemia,
thrombocytopenia, and central nervous system involvement [136,137]. The pathogenesis of SLE involves theappearance of many types of autoantibodies andautoreactive T and B cells. T cells from patients with

–/–

GM3S

Fig. 5. The role of gangliosides in the pathogenesis of allergic asthma. a-GMs in CD4+ T cells are critical for signal transduction throughimmunological synapses, resulting in the synthesis and release of Th2 cytokines, such as IL-4, IL-5 and IL-13. These cytokines play a central
role in the development of airway narrowing by activating eosinophils, B cells and mast cells, which cause mucus secretion and airway
hyperresponsiveness.

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SLE were reported to show intrinsic alterations in lipid
components of lipid rafts [130,138,139]. The levels of
expression of GM1a in CD4+, but not CD8+, T cells
were signiﬁcantly higher in SLE patients than in
healthy controls. LacCer, GA2, Gb3, GM1a, andGD1a were also upregulated in SLE CD4+ T cells[140]. The increase in GM1a was greater in CD45ROpositive CD4+ memory T cells from patients withactive than inactive SLE [130]. In addition, the expression of GM1a is enhanced in activated human T cells[130,139,141,142] and in self-reactive CD4+ T cells,
resulting in the persistence of abnormal cell activation[130,131]. Taken together, these ﬁndings indicate thatgangliosides are key factors in the pathogenesis andpathology of SLE. GlcCerS inhibitor normalized theincreases in GM1a and LacCer levels in SLE CD4+ T
cells after stimulation with anti-CD3 and anti-CD28
mAbs, as well as partially reversing the defects in TCR
signaling [140]. The levels of expression of cellular
GSLs are systematically controlled by de novo synthesis, turnover, and recycling [143]. The biosynthesis of
GSLs and their trafﬁcking to and from plasma membranes are increased in SLE T cells, leading to theaberrant accumulation of gangliosides in lipid rafts[144].
Rheumatoid arthritis (RA) is a typical autoimmune
disease and typically results in warm, swollen, and painful joints, and adaptive immune systems are involved in
RA development [145]. In RA patients, macrophages,
Th1 cells, Th17 cells, and activated B cells migrates into
inﬂamed tissues, and inﬂammatory cytokines, including
IL-1, IL-6, IL-8, IL-17, TNFa, and interferon-gamma,
can be detected in the ﬂuids surrounding inﬂamed synovia and joints [146,147]. Moreover, the gene expressionof GM3S and the GM3 expression levels were higher inthe synovia of patients with RA than with osteoarthritis[148]. In GM3S/ mice, the progression of collageninduced inﬂammatory arthritis, a mouse model of RA,
was accelerated, and the induction of IL-17-producing
cells in regional lymph nodes was enhanced by collagen
immunization [148]. In contrast, GM3S/ mice exhibited a smaller number of Th17 cells after culture in vitro[135]. Further studies are needed to clarify the pathological signiﬁcance of GM3 molecular species in RA development.
CD4+ T cells can be subclassiﬁed as GD1c-positiveIL-2-producing Th1-like cells and GD1c-negative IL-4producing Th2-like cells [149]. The lipid rafts of eachof these subpopulations have a unique, characteristic
ganglioside expression pattern, with these patterns
being responsible for the speciﬁc functions of Th effector cells.

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Role of GM1 in B cell antigen receptor (BCR)
signaling
B cell antigen receptors (BCRs) are critical for the clonal selection of B cells and the differentiation of B
cells into plasma cells. Mature B cells express not only
IgM-type but also IgD-type BCRs. These IgM andIgD type BCRs possess identical antigen-binding sites,
but differ in their membrane-bound heavy chain isoforms. GM1-enriched membrane lipid rafts involved in
BCR signaling may be responsible for the gangliosiderelated immune functions of B lymphocytes [150–153].
IgM-BCR is not closely localized to GM1-enriched
lipid rafts in resting B cells, whereas IgD-BCR is
located adjacent to these lipid domains [151]. BCR
stimulation induces the translocation of IgM-BCRs
into GM1-enriched lipid rafts in a caveolin-1-dependent manner, whereas IgD-BCRs are excluded fromthese lipid domains [153], resulting in the clustering of
IgM- and IgD-BCR nanoclusters. The mechanisms bywhich IgM and IgD nanoclusters are organized in
GM1-enriched lipid rafts and modulate BCR signaling
remain unknown.

GSL-enriched lipid raft-mediatedapoptosis and autophagy in immunity
Apoptosis is generally recognized as a principal mechanism that modulates cell death, occurring not only incellular damages or stress but also in the normal development and morphogenesis [154]. In innate immunity,
apoptosis is a signiﬁcant mechanism for the elimination of neutrophils from inﬂamed tissues by tissue-resident macrophages, which are crucial for conﬁning
inﬂammatory tissue injury and subsequent resolutionof inﬂammation [71]. Phosphatidylglucoside (PtdGlc)
is a unique glycophospholipid that forms PtdGlcenriched raft-like domains on human neutrophil
plasma membranes [71]. Furthermore, PtdGlc-enrichedraft-like domains mediate the death receptor CD95/
Fas-mediated apoptosis pathway. These ﬁndings suggest that lipid raft-mediated apoptosis regulation isessential in innate immune systems.
GM3 is the major component of lipid rafts inhuman lymphocytic cells [155]. Furthermore, thedisialoganglioside GD3 is also abundant, and concentrated in lipid rafts [156]. Following treatment of
human lymphoblastoid T cells with anti-CD95/Fas,
GM3 was reported to associate with caspase-8, a component of the death-inducing signaling complex [157],
suggesting that gangliosides are structural componentsof the membrane multi-molecular signaling complex
involved in the apoptosis pathway mediated by CD95/

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H. Nakayama et al.

Fas receptor. In general, two different cascades are
known to occur during apoptosis, the plasma membrane-associated death receptor-mediated cascade andthe mitochondria-mediated cascade. GD3 permeabilizes mitochondria in MH1C1 rat hepatoma cells in amanner dependent on reactive oxygen species (ROS)
and calcium [158]. TNF-a stimulation induces physicalassociation and GD3 accumulation in mitochondria ofrat hepatocytes [159]. Moreover, GD3-enriched raftlike domains have been reported present on the mitochondrial membranes that recruit Bcl-family proteinsin human lymphoblastoid T cells [160]. Taken
together, these ﬁndings suggest that GSLs play pivotal
roles in apoptotic pathways of both innate and adaptive immune cells. In addition, GSLs form lipid rafts
not only on plasma membranes but also on subcellularcompartments, with these subcellular GSL-enriched
lipid rafts mediating important signaling pathways
involved in various physiological functions. Indeed,
LacCer forms lipid rafts on both granular compartments and nascent microorganism-containing phagosomes [15], with the association of phagosomal
LacCer-enriched lipid rafts with Hck, which facilitateslysosomal fusion, being indispensable for the maturation of microorganism-containing phagosomes.
Apoptosis and autophagy are well regulated andintertwined biological processes [154,161]. Under stressconditions, autophagy is unable to assist cell survival,
and apoptosis pathways are activated in cells to ensure
their effective elimination without triggering local
inﬂammation [162]. Thus, efﬁcient regulation of crosstalk between apoptosis and autophagy is likely to beadvantageous to cells [154]. Interestingly, GD3 wasshown to associate with phosphatidylinositol(3)phosphate (PtdIns3P) and microtubule-associated protein
light chain 3 in ﬁbroblasts, indicating the importantroles of GD3 in the timing of initiation of autophagy
and autophagosome biogenesis [163]. Formation of
autophagosomes at the endoplasmic reticulum–mitochondria contact site [mitochondria-associated membranes (MAM)] in mammalian cells has been suggested[164]. Recently, GD3 was shown to form lipid raft-likedomains in MAM [165] and to interact with coreinitiator proteins of autophagy. These observations
suggest that lipid raft-like domains in MAM are important in the organelle scrambling activity that leads
to autophagosome biogenesis. Detailed elucidation of
the mechanisms by which GSLs interact with autophagy-related proteins and contribute to autophagosome
biogenesis may suggest new treatments for many humandiseases, because autophagy has been shown to be an
important regulator not only of innate and adaptive
immunity but also of other biological conditions,

The role of glycosphingolipids in immunity

including cellular responses to starvation, cell death,cancer, and neurodegenerative diseases [166].
Perspectives
Glycosphingolipids expressed on cells play essential
roles in a variety of immunological functions. Neutral
GSLs, such as LacCer and Gb3, are important in initializing host defense responses through binding totheir ligands [15,30]. Exogenously added C24-LacCer
can regulate cellular functions, such as chemotaxis andphagocytosis [21,81]. a-Linked glycosylceramides wereidentiﬁed as the major endogenous ligands of NKT
cells [27], which control innate and adaptive immune
responses. In addition, b-GlcCer was identiﬁed as an
endogenous ligand for Mincle [60], suggesting that
GSL-receptor trans-interactions may regulate immune
responses. GSLs may play important roles in tuning
the cross talk of signaling pathways in immune systems, by regulating their expression and release.
Immune reactions to GSL antigens may be involved in
the pathogenesis of autoimmune diseases, such as
encephalomyeloradiculoneuropathy(EMRN),
asautoantibodies against LacCer are present in patientswith EMRN [167]. The binding sites of anti-LacCer
antibody clones differ, even in LacCer-enriched rafts[28], suggesting that the antigenic speciﬁcity of GSLs iscomplicated on plasma membranes.
Recent studies have indicated that individual CD4+
T cell subsets express different ganglioside-enriched
lipid rafts on plasma membranes, which affect thepathogenesis of allergic and autoimmune diseases.
Thymus T cell differentiation is associated with differential expression patterns of gangliosides in individual
T cell subsets. In peripheral lymphoid organs, the subpopulations of effector CD4+ T cells possess distinct
lipid rafts with unique ganglioside patterns, which areresponsible for speciﬁc Th functions. Therefore, theselective ganglioside expression is indispensable for theformation of distinct, functional lipid rafts of T cells
during their maturation. Allergic reactions have somecommon features. However, these reactions are somehow reﬂected by greatly different molecular patterns
responsible for antigens speciﬁcities. Reduction of aGMs by suppression of GM3S causes changing of different ganglioside-enriched lipid rafts. Such kind oftechnique is a potentially powerful tool for the treatment of immune system disorders.
Glycosphingolipids having different types of sugar
motifs and/or acyl chains are likely able to form distinct lipid domains [71,168]. Elucidating the functionsof GSLs requires further technical advances to identify
raft-associatedmolecules
that
maintain
their

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H. Nakayama et al.

physiological status. These technical advances maylead to the development of new pharmacologicalagents for several types of disease, including infectious,
inﬂammatory and autoimmune disorders.

Acknowledgements
The authors’ studies described in this Review were
supported in part by grants from Foundation ofStrategic Research Projects in Private Universities (toHN and KI, S1201013 and S1311011; to JI,
S1201031); MEXT KAKENHI (to KI, 16017293 and24659293; to HN, 25860831 and 15K19592; to JI,
16H04767); AMED under Grant Number JP17gm0910006 (to KI) and the Naito Foundation (to NH
and JI), Mizutani Foundation for Glycoscience (JI andKI), ONO Medical Research Foundation (JI), Uehara
Memorial Foundation (JI).

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Glycobiology, 2019, vol. 29, no. 3, 260–268
doi: 10.1093/glycob/cwy105
Advance Access Publication Date: 20 December 2018
Original Article

Globo-series glycosphingolipids enhanceToll-like receptor 4-mediated inﬂammationand play a pathophysiological role in diabetic
nephropathy
Takahiro Nitta2,3, Hirotaka Kanoh2, Kei-ichiro Inamori2, Akemi Suzuki2,
Tomoko Takahashi3, and Jin-ichi Inokuchi2,12Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology and 3Division of
Pathophysiology, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Tohoku Medical
and Pharmaceutical University, Sendai 981-8558, Japan

To whom correspondence should be addressed: Tel: +81-22-727-0117; Fax: +81-22-727-0076; e-mail: jin@tohoku-mpu.
ac.jp
1

Received 4 October 2018; Revised 14 November 2018; Editorial decision 20 November 2018; Accepted 21 November 2018

Abstract
Alteration of glycosphingolipid (GSL) expression plays key roles in the pathogenesis and pathophysiology of many important human diseases, including cancer, diabetes and glycosphingolipidosis. Inﬂammatory processes are involved in development and progression of diabetic
nephropathy, a major complication of type 2 diabetes mellitus. GSLs are known to play roles ininﬂammatory responses in various diseases, and levels of renal GSLs are elevated in mouse models of diabetic nephropathy; however, little is known regarding the pathophysiological role ofthese GSLs in this disease process. We studied proinﬂammatory activity of GSLs in diabetic
nephropathy using spontaneously diabetic mouse strain KK. Mice were fed a high-fat diet (HFD)(60% kcal from fat) or normal diet (ND) (4.6% kcal from fat) for a period of 8 wk. HFD-feeding
resulted in quantitative and qualitative changes of renal globo-series GSLs (particularly Gb3Cer),
upregulation of TNF-α, and induction of renal inﬂammation. Gb3Cer/Gb4Cer treatment enhanced
inﬂammatory responses via TLR4 in TLR4/MD-2 complex expressing cells, including HEK293T,
mouse bone marrow-derived macrophages (BMDMs) and human monocytes. Our ﬁndings suggest that HFD-induced increase of Gb3Cer/Gb4Cer positively modulate TLR4-mediated inﬂammatory response, and that such GSLs play an important pathophysiological role in diabetic
nephropathy.
Key words: diabetic nephropathy, Globo-series GSLs, renal inﬂammation, TLR4 ligands, Toll-like receptor 4

Introduction
Diabetic nephropathy, a major complication of type 2 diabetes mellitus (T2DM), is deﬁned histologically by renal changes such as diffuse mesangial cell proliferation, mesangial matrix expansion,diffuse or nodular glomerulosclerosis, podocyte loss, glomerularbasement membrane thickening, tubulointerstitial ﬁbrosis andatrophy, hyaline arteriolosclerosis, and arterial sclerosis, and is associated with (micro)albuminuria and reduced glomerular ﬁltration
rate (Alsaad and Herzenberg 2007; Fioretto and Mauer 2007).
Morphological changes as above involve genetic factors, and are
based on biochemical and hemodynamic changes (Vardarli et al.
2002; Lee et al. 2005; Freedman et al. 2007; Kanwar et al. 2011).

© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

260

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Immunology

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Globo-series glycosphingolipids and diabetic nephropathy

diabetic nephropathy, and observed for the ﬁrst time increased levelsof renal globo-series GSLs under such condition. Globo-series
GSLs enhanced TLR4-mediated inﬂammation in both mouse bonemarrow-derived macrophages (BMDMs) and human monocytes.
Our ﬁndings indicate that globo-series GSLs act as a positive modulator on TLR4-mediated inﬂammatory response, and play a role inpathophysiology of diabetic nephropathy.

Results
HFD feeding induces obesity, hyperglycemia and renal
inﬂammation in KK mice
Inbred mouse strain KK was established in 1957 from an inbred strainof native Japanese mice that spontaneously developed diabetes of polygenic origin. Many diabetic characteristics (glucose intolerance, insulin
resistance, hyperinsulinemia, moderate hyperglycemia, hyperlipidemia,
hypercholesterolemia, hyperleptinemia, histological changes of renal
glomerulus) are associated with obesity in KK mice (Makino et al.
1985; Igel et al. 1998; Suto et al. 1998; Okazaki et al. 2002), and thesemice are often used in studies of diabetes and diabetic complications.
To elucidate the pathophysiological role of GSLs in diet-induced diabetic nephropathy, we introduced HFD at age 8 wk in one group of
KK mice, while another group was maintained on normal diet (ND) asa control. Initial body weight (at age 8 wk) and ﬁnal weight (at age 16wk) for the two groups are shown in Figure 2A. Comparative values ofthree parameters at 16 wk in the two groups (hereafter referred to simply as “HFD” and “ND” for convenience) were: ﬁnal body weight(ND, 35.4 ± 2.5 g; HFD, 45.9 ± 2.4 g; P < 0.001) (Figure 2A), weightgain (ND, 127 ± 8.5%; HFD, 163 ± 8.5%; P < 0.001) (Figure 2B),
non-fasting blood glucose (ND, 214 ± 51 mg/dL; HFD, 328 ± 126 mg/
dL; P < 0.01) (Figure 2C). Each of these parameters was signiﬁcantly
increased in HFD relative to ND. Blood glucose level in HFD was indicative of hyperglycemia. Renal inﬂammation was assessed by measuring TNF-α gene expression level by quantitative PCR. TNF-α level was
signiﬁcantly higher in HFD than in ND (Figure 2D). These ﬁndings
indicate that HFD feeding induced nephritis in KK mice.

Increased levels of renal GSLs in HFD-fed KK mice
GSL levels are elevated in kidneys of mice with various renal diseases (diabetic nephropathy, lupus nephritis, polycystic kidney disease) (Deshmukh et al. 1994; Nowling et al. 2015; Subathra et al.
2015). We examined quantitative and qualitative changes of GSLs
in kidneys of ND and HFD. HPTLC analysis revealed that levels ofA

B

Neutral GSLs
Ugcg
Cer

B4galt5B4galt6
Cer
GlcCerAcidic GSLs

A4galtCer
LacCer
St3gal5

Gb3Cer

R
N

Cer

H

Gb4CerO

αAcyl chain (n = 1–9)

(CH2)n – CH3

Ceramide Portion

Cer
GM3
St8sia1

a-series

Cer
GD3b-series

Galactose

Sphingosine (d18:1)

Sugar(s)B3galnt1
Cer

OH Phytosphingosine

N-Acetylgalactosamine(t18:0)R

Sugar(s)
N OH

H

O

α

Acyl chain (n = 1–9)

(CH2)n – CH3

Phytoceramide Portion

Gangliosides
GlucoseOH

Globo-series

Sialic acid

R = OH : α-hydroxy fatty acidR = H : Non -hydroxy fatty acid

Fig. 1. Biosynthetic pathways and structural diversity of glycosphingolipids. (A) GSL biosynthetic pathways. (B) Structures of GSL molecular species.

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Biochemical changes arise from immune cell-mediated inﬂammation,a key causative factor in pathogenesis of diabetic nephropathy.
Inﬂammatory cytokines (e.g., TNF-α, IL-6, IL-1β) are secreted frominﬁltrated and activated macrophages, and are enhanced by oxidative stress through increased levels of reactive oxygen species (ROS)(Pai et al. 1996; Das and Elbein 2006; Yoshida and Tsunawaki
2008; Elmarakby and Sullivan 2012; Badal and Danesh 2014).
Glycosphingolipids (GSLs) consist of hydrophilic oligosaccharides and hydrophobic ceramide (Figure 1A). Ceramide species arecomposed of sphingosine linked to a particular fatty acyl chain byamide bond (Figure 1B). Ceramide acyl chains vary in chain length,
degree of saturation and presence/absence of α-hydroxylation(Figure 1B). GSLs containing a variety of ceramide structures are
crucial components of plasma membranes. GSLs play key roles infundamental biological processes such as development, cell adhesion, and signaling (Hakomori and Igarashi 1995; Hakomori et al.
1998; Tagami et al. 2002; Yu et al. 2009). Numerous previous
reports have demonstrated pathophysiological involvement of GSLs
in kidney diseases, including lupus nephritis, polycystic kidney disease, Fabry disease, renal cancer, hemolytic uremic syndrome anddiabetic nephropathy (Obrig et al. 1993; Zador et al. 1993;
Deshmukh et al. 1994; Biswas et al. 2006; Biancini et al. 2012;
Grove et al. 2014; Nowling et al. 2015; Subathra et al. 2015). A
2015 study showed involvement of glucosylceramide (GlcCer) andlactosylceramide (LacCer) in ﬁbrosis, extracellular matrix production, and hypertrophy of kidney cells (Subathra et al. 2015). Asidefrom this, little is known regarding roles of GSLs in pathophysiology
of diabetic nephropathy. GSLs evidently play important roles ininﬂammatory responses contributing to various diseases (Kim et al.
2002; Ohmi et al. 2011; De Francesco et al. 2013), but no study todate has focused on such role of GSLs in diabetic nephropathy.
To test the hypothesis that GSLs play a pathophysiological rolein inﬂammatory responses contributing to diabetic nephropathy, weused a mouse model of T2DM with high-fat diet (HFD). T2DM anddiabetic nephropathy in humans are complex diseases that involve
polygenic factors and environmental factors, such as obesity andsedentary lifestyle (Vardarli et al. 2002; Lee et al. 2005; Freedman
et al. 2007; Kanwar et al. 2011; Wu et al. 2014). We analyzed associations between renal GSLs and renal inﬂammation using KK mice.
This strain is a useful model for studies of T2DM and diabetic
nephropathy because it displays correlations between polygenic factors and susceptibility to these disease processes (Shike et al. 2001;
Fan et al. 2003). We fed KK mice with HFD to more closely simulate pathological conditions associated with human T2DM and262

T Nitta et al.

**

40
30
20
10

**
150
100
50

0

0
Initial(8wks)

500

D 20

*

Relative expression

ND
HFD

C

200

Blood glucose (mg/dL)

50
Body weight (g)B

60

Weight gain (%)

A

24:0, in Gb3Cer/Gb4Cer species (Figure 4A, B). Total Gb3Cer level(calculated as sum of values from the 26 molecular species) was
6.2-fold higher for HFD than for ND (Figure 4C), consistently withresults from HPTLC/densitometry (Figure 3C). Total Gb4Cer level(calculated as sum of values from the 18 molecular species) was
2.8-fold higher for HFD than for ND (Figure 4D).
For our analyses, we deﬁne fatty acids (FAs) with chain length≤20 carbons as long chain fatty acids (LCFAs) and those with chain
length >20 carbons as very long chain fatty acids (VLCFAs) according to previous report (Kihara 2012). In KK mouse kidney, the threemost abundant species, d18:1-16:0, d18:1-22:0 and d18:1-24:0,
accounted for more than half of the total in Gb3Cer (Figure S1E) or
Gb4Cer (Figure S1F) species. Among these species, the proportionof d18:1-22:0 species, VLCFA, was increased markedly in HFD(Figure S1E, F). The elevated levels of renal Gb3Cer/Gb4Cer carrying VLCFAs (saturated FAs: 22:0, 23:0, 24:0; monounsaturated
FAs: 24:1; α-hydroxy FAs: h22:0, h23:0, h24:0; monounsaturated
and α-hydroxy FAs: h24:1) were higher than the elevated levels of
Gb3Cer/Gb4Cer carrying LCFAs (saturated FAs: 16:0, 18:0, 20:0;

400
300
200
100

HFD

*

15
10
5
0

0
ND

Final
(16wks)

Tnf

ND

ND

HFD

HFD

Fig. 2. Obesity, hyperglycemia, and renal inﬂammation in HFD-fed KK mice (hereafter referred to as “HFD”). (A) initial (age 8 wk) and ﬁnal (age 16 wk) bodyweight (n = 8 for each group). (B) Weight gain (n = 8 for each group). (C) Non-fasting blood glucose of ND and HFD fed for 8 wk (n = 13 for each group). Datashown are mean ± SD. *P < 0.01, **P < 0.001 for comparison with ND (by Student’s t-test in A, B; Welch’s t-test in C). (D) mRNA expression level of TNF-α
shown as box plot (n = 4 for each group). *P < 0.05 vs. ND, by Mann–Whitney unpaired test.

A

B

Neutral GSLs

SM4

HexCerLacCer

GM3
GM2
GM1

Gb3Cer
Gb4CerND

Std

D

Gb3Cer
22
20
18
16
14
12
10
8
6
4
2
0

**

ND

HFD

Relative expression

μg/mg Protein

C

Acidic GSLs

Std

HFD

A4galt
2.8

*

2.4
2
1.6
1.2
0.8
0.4
0

ND

HFD

ND

HFD

Fig. 3. Strong increase in renal globo-series GSL levels in HFD. (A, B) Neutral (A) and acidic (B) GSLs extracted from ND and HFD kidneys were spotted on
HPTLC plates and developed as described in Materials and methods. (C) Comparison of kidney Gb3Cer levels under ND and HFD. (D) Quantitative RT-PCR analysis of Gb3Cer synthase gene A4galt in kidney. n = 4 for each group. *P < 0.01, **P < 0.001 vs. ND, by Student’s t-test. Data shown are mean ± SD.

Downloaded from https://academic.oup.com/glycob/article-abstract/29/3/260/5199375 by Tohoku Yakka Daigaku Toshokan user on 28 February 2019neutral GSLs were signiﬁcantly higher in HFD, whereas acidic GSL
levels did not differ notably between the two groups (Figure 3A, B).
Increase in HFD was particularly notable for globo-series GSLs
(Gb3Cer and Gb4Cer) (Figure 3A). We further analyzed Gb3Cerlevel, which showed the greatest increase. Densitometric analysis of
HPTLC data showed a 4.7-fold increase of Gb3Cer level in HFD(Figure 3C). Gb3Cer is synthesized from LacCer by A4GALT, andis then converted to Gb4Cer by B3GALNT1 (Figure 1A). In a searchfor possible causes of Gb3Cer metabolism abnormalities in HFD,
we assessed renal expression levels of A4galt (Gb3Cer synthasegene) in HFD vs. ND. A4galt expression level was signiﬁcantly higher in HFD (Figure 3D). The higher contents of Gb3Cer/Gb4Cer in
HFD can therefore be explained, at least in part, by increased
Gb3Cer synthase expression level. LC-ESI-MS/MS analysis detected
26 molecular species of Gb3Cer and 18 molecular species of
Gb4Cer carrying ceramide (Figure 4A, B) or phytoceramide (phytoGb3Cer, phyto-Gb4Cer) (Figure S1). These species were increased
signiﬁcantly in HFD compared to ND. In particular, there were
elevated levels of three species, d18:1-16:0, d18:1-22:0 and d18:1-

263

Globo-series glycosphingolipids and diabetic nephropathy

ND
HFD

30
25

Ratio to internal standard

**
**

**

20
15

**

5

**

**

**

**
t.a. **

0

Total Gb3Cer
160
140
120
100
80
60
40
20
0

*

D

**
**

*

15
10
5

HFD

HFD

6

*

**

5

***

4
3
2

*
***

***

**

E

ND

*

n.d. n.d. ***F

Gb3Cer
120

ND

100

HFD

**
†

80
60
40
20
0

0
ND

7

**

0

Total Gb4Cer

20

ND

1

**

25
Relative abundanceC**

Relative abundance

10

Gb4Cer
8

HFD

LCFA
VLCFA
(C16, C18, C20) (C22, C23, C24)

Gb4Cer
18
16
14
12
10
8
6
4
2
0

ND
HFD

*
†

LCFA
VLCFA
(C16, C18, C20) (C22, C23, C24)

Fig. 4. LC-ESI-MS/MS analysis of renal Gb3Cer and Gb4Cer molecular species showing increased levels in HFD relative to ND. (A, B) 14 ceramide-carrying
Gb3Cer species (A) and 12 ceramide- carrying Gb4Cer species (B) were detected, and are shown as relative abundance in bar graphs. n = 4 for each group. *P <0.05, **P < 0.01, ***P < 0.001 vs. ND, by Welch’s t-test and Student’s t-test. t.a., trace amount; n.d., not detected. (C, D) Total levels of Gb3Cer (C) and Gb4Cer(D), calculated as described in Materials and Methods, were compared in HFD vs. ND (n = 4 for each group). *P < 0.01 vs. ND, by Welch’s t-test (C) andStudent’s t-test (D). (E, F) The levels of globo-series GSL molecular species (E, Gb3Cer; F, Gb4Cer) carrying VLCFAs (C22-C24) and LCFAs (C16-C20) and comparison between HFD and ND. n = 4 for each group. *P < 0.05, **P < 0.01 vs. LCFAs, by Student’s t-test; †P < 0.01 vs. ND (VLCFAs), by Welch’s t-test (E) andStudent’s t-test (F). Data shown are mean ± SD.

α-hydroxy FAs: h16:0, h18:0, h20:0) in comparison between ND
and HFD (Figure 4E, F). These ﬁndings indicate that HFD feedingproduces quantitative and qualitative changes of renal Gb3Cer and
Gb4Cer, and suggest that these alterations of Gb3Cer and Gb4Cer
molecular species plays a pathophysiological role in diet-induced
diabetic nephropathy.

Involvement of globo-series GSLs in renal
inﬂammatory responses
In view of the greatly altered renal Gb3Cer/Gb4Cer levels in HFDinduced diabetic nephropathy, we examined the roles of globo-series
GSLs in inﬂammatory responses associated with diabetic nephropathy. Enzyme-linked immunosorbent assay (ELISA) was used tomeasure production of TNF-α by mouse BMDMs or human monocytes stimulated with LPS (0.25 ng/mL and 0.1 ng/mL, respectively)
plus Gb3Cer or Gb4Cer (each 0, 2.5, 5, 10 μM) (Figure 5A, B). We
used commercially available Gb3Cer/Gb4Cer carrying VLCFAs(C22, C24) as major molecular species (Figure S2), and thesemolecular species were selectively increased in kidney by HFD feeding (Figure 4A, B). A higher TNF-α level was produced by costimulation with LPS and Gb3Cer/Gb4Cer than by stimulation withLPS alone (Figure 5A, B), but the degree of this effect declined as
Gb3Cer/Gb4Cer concentration increased (Figure 5A). Human IL-6
and IL-12/23 levels also higher by co-stimulation with LPS and these
GSLs than by stimulation with LPS alone (Fig. S4).

To determine the selectivity of Gb3Cer/Gb4Cer for TLR4, we
assessed speciﬁcity of these GSLs for TLRs using TLR4/MD-2-overexpressing HEK293T cells or TLR ligands for TLR4, 1/2, 5, 7/8 and2/6. We observed that a higher NF-κB activity was induced in
response to co-stimulation with LPS (20 ng/mL) and Gb3Cer/
Gb4Cer (each 3, 10, 30 ng/mL) than by stimulation with LPS alone
in TLR4/MD-2 overexpressing HEK293T (Figure 5C). The degreeof NF-κB activity declined as these GSLs increased, consistent withthe result of co-stimulation of mouse BMDMs as shown in ﬁgure
5A. Gb3Cer/Gb4Cer also selectively enhanced human IL-6 (hIL-6)
production induced by TLR4 ligands, LPS and HMGB1, but not byligands for TLR1/2, 5, 7/8 and 2/6 (Figure 5D). However, these
GSLs alone did not have the ability to produce hIL-6 via TLR4.
These results indicated that Gb3Cer/Gb4Cer selectively and positively modulates TLR4-mediated inﬂammatory response in humanand mouse monocytes/macrophages.

Discussion
Diabetic nephropathy is a major complication of T2DM arisingfrom genetic factors and environmental factors. Associations
between renal GSLs and diabetic nephropathy have been reported inseveral animal models (Masson et al. 2005; Grove et al. 2014;
Subathra et al. 2015). The db/db mouse model used in the above
studies has a diabetic phenotype resulting from a single geneDownloaded from https://academic.oup.com/glycob/article-abstract/29/3/260/5199375 by Tohoku Yakka Daigaku Toshokan user on 28 February 2019

Ratio to internal standard

35

Relative abundanceB

Gb3Cer40

Relative abundance

A

264

T Nitta et al.

100

**

80

††

††

†

**

60
40
20

0
GSL(μM)

-

-

2.5

5

10

2.5

5

10

35
30

Human monocytes
Gb3
Gb4

**

20
15
10
5

0
GSL(μM)

-

-

16
14

††

6
4
2

0
GSL(μM)

-

-

3

10

30

3

10

30

LPS (20 ng/ml)**

12
Concentration(ng/ml)

5

**

8

hIL-6

Human monocytes
Gb3 (5 μM)
Gb4 (5 μM)

10

5

10

LPS (0.1 ng/ml)

LPS (0.25 ng/ml)

D

12

**

25

NF-κB
HEK293T (TLR4/MD-2)
Gb3
Gb4

**

8

**

6

**

4
2
0
-

LPS
(0.2 ng/ml)

HMGB1(10 μg/ml)
TLR4

Pam3CSK4(0.4 μg/ml)

Flagellin(50 ng/ml)

R848(0.5 μg/ml)

MALP-2(1.0 ng/ml)

TLR1/2

TLR5

TLR7/8

TLR2/6

Fig. 5. Globo-series GSLs selectively affect inﬂammatory responses via TLR4. A: ELISAs for murine TNF-α in BMDM culture supernatant following treatment
with LPS (0, 0.25 ng/mL) plus Gb3Cer or Gb4Cer (0, 2.5, 5, 10 μM). TNF-α amount was normalized to nontreated control and expressed as ratio. n = 4 for eachgroup. **p < 0.01 vs. group treated with LPS only; †p < 0.05, ††p < 0.01 vs. group treated with LPS only, by Dunnett’s test. B: ELISAs for human TNF-α in humanmonocyte culture supernatant following treatment with LPS (0, 0.1 ng/mL) plus 5 μM Gb3Cer or Gb4Cer. TNF-α amount was normalized to nontreated control
and expressed as ratio. n = 3 for each group. **p < 0.01, by Dunnett’s test. C: TLR4/MD-2-overexpressing HEK293T cells were transfected with NF-κB luciferase
construct and stimulated with LPS (0, 20 ng/mL) plus Gb3Cer or Gb4Cer (0, 3, 10, 30 μM). Luciferase activity was normalized to nontreated control and expressed
as relative luciferase activity. n = 4 for each group. **p < 0.01 vs. group treated with LPS only; ††p < 0.01 vs. group treated with LPS only, by one-way ANOVA.
D: ELISAs for human IL-6 in human monocyte culture supernatant following treatment with TLR ligands (TLR4: LPS, human recombinant HMGB1; TLR1/2:
Pam3CSK4; TLR5: Flagellin; TLR7/8: R848; TLR2/6: MALP-2) plus 5 μM Gb3Cer or Gb4Cer. n = 4 for each group. **p < 0.01, by Dunnett’s test. Data are shown asmean ± SD.

mutation for leptin receptor. However, obesity, T2DM, and diabetic
nephropathy in humans are polygenic diseases. We therefore usedmouse strain KK, a polygenic disease model, for analysis of diabetic
nephropathy. Levels of renal globo-series GSLs (Gb3Cer, Gb4Cer)
were strikingly increased in HFD (Figure 3A). These GSLs were also
increased by HFD feeding in nondiabetic C3H/HeN mice
(Figure S3). L.J. Siskind’s group (Subathra et al. 2015) and J.A.
Shayman’s group (Zador et al. 1993) reported increased levels of
renal GSLs (HexCer, LacCer, GM3) in db/db mice with diabetic
nephropathy and in Sprague-Dawley rats with streptozotocininduced diabetes. Our ﬁndings and previous reports implicated the
GSL species in pathophysiology of diabetic nephropathy.
In the present study, enhanced levels of globo-series GSLs
resulted, at least in part, from increased gene expression of Gb3Cer
synthase (Figure 3D). Increased Gb3Cer synthase expression has
been observed in response to LPS and inﬂammatory cytokines suchas IL-1 and TNF-α (van Setten et al. 1997; Hughes et al. 2000;
Okuda et al. 2010; Kondo et al. 2013). Indeed, renal expression of

TNF-α gene was higher in HFD than in ND in the present study(Figure 2D).
We used LC-ESI-MS/MS to evaluate differential levels of renal
Gb3Cer/Gb4Cer molecular species in HFD vs. ND. Contents of
d18:1-16:0, 22:0, 24:0 species were signiﬁcantly higher in HFD thanin ND (Figure 4A, B). The proportion of d18:1-22:0, VLCFA, to thetotal Gb3Cer/Gb4Cer species was increased markedly in HFD compared to ND (Figure S1E, F). These results suggest that these species,
speciﬁcally VLCFA species, are involved in pathophysiology ofdiabetic nephropathy. Hyperglycemia typically results in renal
macrophage inﬁltration and renal inﬂammation, both of which contribute to pathogenesis and pathophysiology of diabetic nephropathy(Chow et al. 2004; Navarro-Gonzalez et al. 2011). Therefore, we
assessed proinﬂammatory activity of Gb3Cer/Gb4Cer in TLR4/MD2-overexpressing HEK293T cells, mouse BMDMs and humanmonocytes (Figure 5A–D). Gb3Cer/Gb4Cer enhanced inﬂammatory
response in the presence of TLR4 ligands, LPS and HMGB1, but neither in the absence of the ligands nor in the presence of the otherligands (Figure 5D). The effects of Gb3Cer/Gb4Cer on TLR4-

Downloaded from https://academic.oup.com/glycob/article-abstract/29/3/260/5199375 by Tohoku Yakka Daigaku Toshokan user on 28 February 2019**C

hTNF-α40Nontreated control ratio

120
Nontreated control ratio

B

mTNF-α
Mouse BMDMs
Gb3
Gb4

Relative luciferase activity

A

265

Globo-series glycosphingolipids and diabetic nephropathy

A

B

Homeostasis

binds to the other side of TLR4/MD-2 units (Figure 6B). However,
Gb3Cer/Gb4Cer alone would not be able to induce activation of
TLR4/MD-2 complex (Figure 6C).
Previous reports (van Setten et al. 1997; Legros et al. 2017)
showed that Gb3Cer and Gb4Cer are abundant in human renal
glomerular cells, and that these cells and KK mouse kidney cells
have similar expression patterns of certain molecular species.
Gb3Cer in human renal glomerular cells is increased in response toproinﬂammatory cytokines (van Setten et al. 1997). Circulating
endotoxin and HMGB1 levels, TLR4 ligands, are elevated in bothhumans and mice with T2DM (Brun et al. 2007; Creely et al. 2007;
Al-Attas et al. 2009; Wang et al. 2015). HMGB1 is also upregulated in mouse mesangial cells under high glucose levels (Chenet al. 2015). Given that Gb3Cer and Gb4Cer are up-regulated byproinﬂammatory cytokines and affect TLR4-expressing cells, suchas macrophage and mesangial cells (Kaur et al. 2012), these GSLs
might exacerbate and prolong renal inﬂammation in diabetic
nephropathy.
Taken together, our ﬁndings suggest that the effect of Gb3Cer/
Gb4Cer via TLR4 play an important role in the pathophysiology ofdiet-induced diabetic nephropathy.

Materials and methods
Animals and feeding conditions
Eight-week-old male KK/Ta mice (CLEA Japan; Tokyo, Japan) weredivided randomly into two groups. The control group was fed normal diet (ND) (CE-2; CLEA Japan), while the HFD group was fed
high-fat diet (HFD) (D12492; Research Diet; New Brunswick, NJ)
ad lib for 8 wk. Kidneys and blood (from right ventricle) were harvested after sacriﬁcing animals. Non-fasting blood glucose was measured using Accu-Chek Aviva strips (Roche DC; Japan).

C

Pathological state(Obesity, Diabetes, etc)

Gb3CerLPS

LPS

Gb4Cer

Gb3Cer (VLCFA)

Gb3Cer

Gb4Cer (VLCFA)

Gb4CerActivation
MD-2

Activation
MD-2

MD-2

MD-2

MD-2

Activation
MD-2

MD-2

MD-2

Monomer

Monomer
Signaling

MD-2

MD-2

Monomer
Signaling

NF-κB

Host Defence

MD-2

TLR4

TLR4

TLR4

MD-2

NF-κB

Chronic Inflammation

Fig. 6. Working hypothesis for the action of Gb3Cer/Gb4Cer on TLR4 signaling. (A) TLR4 signaling by LPS. Dimerization of TLR4/MD-2 units requires, at least,
one molecule of LPS (Saitoh et al. 2004). (B) Synergistic activation of TLR4/MD-2 by LPS and Gb3Cer/Gb4Cer. Gb3Cer/Gb4Cer associates with TLR4/MD-2 complex as a modulator in presence of LPS, and enhances inﬂammatory response through trans-interaction. (C) Gb3Cer/Gb4Cer alone would not induce activationof TLR4/MD-2 complex.

Downloaded from https://academic.oup.com/glycob/article-abstract/29/3/260/5199375 by Tohoku Yakka Daigaku Toshokan user on 28 February 2019

mediated inﬂammatory response declined as concentration increased(Figure 5A, C). Furukawa’s group has reported that Gb4Cer is an
endogenous ligand of TLR4/MD-2, and that Gb4Cer negatively
regulated TLR4/MD-2 complex at a dosage higher than the one weused for immune cells (Kondo et al. 2013). Therefore, Gb4Cer
appears to be capable of enhancing inﬂammatory response at lower
concentrations, and suppressing inﬂammatory response at higher
concentrations (Figure 5A, C). These results indicated that Gb3Cer/
Gb4Cer alone would not be able to induce inﬂammatory response
via TLRs but selectively and positively modulate TLR4-mediatedinﬂammatory response in presence of TLR4 ligands. However, themechanism how Gb3Cer/Gb4Cer enhances TLR4-mediated inﬂammatory response remains unclear. Previous studies have been demonstrated that GSLs implicated in TLR4 signaling on the microdomain(Cai et al. 2013; Nikolaeva et al. 2015; Mobarak et al. 2018).
However, our results support that Gb3Cer/Gb4Cer associates with
TLR4 as a modulator, and induces TLR4 signaling through transinteraction rather than cis-interaction between these GSLs and TLR4
on the microdomain because Gb3Cer/Gb4Cer did not enhanceinﬂammatory response via TLR1/2, TLR5, and TLR2/6 that locatedon the plasma membranes. In addition, Furukawa’s group hasreported recently that Gb4Cer bind to MD-2 by binding assay using
radiolabeled Gb4Cer (Kondo et al. 2013). Therefore, we propose thecurrent model (Figure 6) whereby Gb3Cer/Gb4Cer modulates
TLR4-mediated inﬂammatory response in the presence of TLR4
ligands by binding to TLR4/MD-2 complex. It is known that TLR4
signaling is required for the homo-dimerization composed of twounits of TLR4/MD-2/LPS complex after LPS binding to TLR4/MD-2
units (Park et al. 2009). Interestingly, Saitoh et al. reported that, atleast, a single TLR4 ligand could induce TLR4 dimerization(Figure 6A) (Saitoh et al. 2004), suggesting that there is still roomfor one more ligand to interact with dimeric TLR4/MD-2 complex.
Therefore, Gb3Cer/Gb4Cer might bind to the empty side of TLR4/
MD-2 units, and enhances TLR4 signaling mediated by LPS which

266

Analysis of GSLs

RNA isolation, reverse transcription and quantitative
PCR
Total RNA was extracted using Sepasol-RNA I super G (Nacalai
Tesque; Kyoto, Japan) and Fast Gene RNA Premium Kit (Nippon
Genetics; Tokyo). cDNA was generated from 500 ng total RNA
using PrimeScript RT Reagent kit (Takara Bio). Primer-probe sets
for mouse A4galt (Mm01307145_m1) and Tnf (Mm00443258_m1)
were from Applied Biosystems (Foster City, CA) and mouse Gapdh(Mm.PT.39a.1) was from Integrated DNA Technologies (Coralville,
IA). Quantitative PCR was performed on StepOnePlus Real-Time
PCR System (Applied Biosystems) using these primer-probe sets and
THUNDERBIRD Probe qPCR Mix (Toyobo Co.; Osaka, Japan).
Relative mRNA levels were calculated from a standard curve andnormalized to GAPDH expression.

Mass spectrometric (MS) analysis
Gb3Cer and Gb4Cer molecular species were quantiﬁed by HPLC
coupled with electrospray ionization tandem MS (LC-ESI-MS/MS)
in multiple reaction monitoring (MRM) positive ionization mode
(Supplementary Table 1). A triple stage quadrupole (TSQ) Vantage
AM instrument (Thermo Fisher; Waltham, MA) was calibrated bydirectly infusing a neutral GSL mixture containing Gb3Cer and
Gb4Cer species. All ion source parameters and ionization conditions
were optimized to improve sensitivity. Neutral GSLs from mouse
kidney extract were dissolved in 100 μL methanol, and 5 μL of thesolution was injected into an HPLC pump (model Accela 1250,
Thermo Fisher) and separated on a Develosil C30 column (C30-UG3-1 × 50 mm; Nomura Chemical Co.; Japan).
Gradient program and parameters were as follow: start with
100% solvent A (20% H2O/50% 2-propanol/30% methanol containing 0.1% acetic acid and 0.1% ammonia) for 5 min; ramp up to
100% solvent B (2% H2O/50% 2-propanol/48% methanol containing 0.1% acetic acid and 0.1% ammonia) over 30 min; maintain
100% solvent B for 5 min; return to 100% solvent A over 1 min,hold there for 9 min; ﬂow rate throughout chromatographic run:
60 μL/min; +3000 V potential applied between ion source, electrospray needle, and nitrogen gas; collision energy 60 eV for Gb3Cer
molecular species, 52 eV for Gb4Cer molecular species. Gb3Cer(d18:1-17:0) (Matreya, LLC; State College, PA) was added to neutral GSL samples from mouse kidney extract as internal standard
and relative abundance of each Gb3Cer/Gb4Cer molecular species
was determined based on ratio to internal standard. Total Gb3Cer/
Gb4Cer value was calculated as sums of relative abundance for the
26 and 18 (respectively) molecular species detected. A caveat regarding interpretation of these MS data is that ionization efﬁciencies are
not the same for all molecular species. In view of the limited availability of pure molecular species standards, we assumed that all

species have ionization efﬁciencies comparable to those of theinternal standards used.

Puriﬁcation of human monocytes
Heparinized fresh human peripheral blood was diluted to double
the volume with cold PBS (4°C; endotoxin-free; Nacalai Tesque)containing 1 μg/mL polymyxin B (Sigma-Aldrich; St. Louis, MO).
Diluted blood was overlaid on cold lymphocyte separation solution(4°C, Nacalai Tesque) containing 1 μg/mL polymyxin B, and centrifuged for 25 min at 4°C, 800 × g. Peripheral blood mononuclear cell
(PBMC) fraction was collected and diluted to double the volume ofwash solution (PBS, 1% heat-inactivated FCS (Biosera), 5 mM
EDTA, pH 7.5 (Nacalai Tesque), 1 μg/mL polymyxin B). PBMCs
were separated by centrifugation for 10 min at 4°C, 600 × g, andwashed twice. PBMCs were resuspended in 750 μL wash solution
and incubated with 120 μL anti-human CD14 magnetic particles(BD Biosciences) for 30 min at room temperature. CD14-positive
cells (monocytes) were separated by magnetic ﬁeld and washed threetimes with wash solution. Puriﬁed cells were resuspended in cold
low-glucose DMEM (Nacalai Tesque) with 0.75% FCS, left on ice
for 45 min, counted, diluted to 2.0 × 105/mL with culture medium(low-glucose DMEM, 0.75% FCS, 40 ng/mL recombinant humangranulocyte-macrophage colony-stimulating factor (GM-CSF)(BioLegend; San Diego, CA), and cultured in 96-well plates (100 μL/well) overnight at 37°C in 5% CO2 atmosphere.

Differentiation of mouse bone marrow-derived
macrophages (BMDMs)
Femoral and tibial bone marrows of 12- to 16-wk-old nondiabetic
C3H/HeN mice (Japan SLC, Inc.) were collected in 1% FCSsupplemented low-glucose DMEM, and erythrocytes were lysed in
RBC lysis buffer. Bone marrow cells were washed in 1% FCS DMEM,
and then cultured for 5–7 days in 10% FCS DMEM supplemented
with 40 ng/mL recombinant human macrophage colony- stimulating
factor (M-CSF) (BioLegend). Non-adhesive cells were washed out
with PBS. Differentiated macrophages were collected in ice-cold PBS(with 1% FCS, 5 mM EDTA) by scraping, washed, counted, diluted
to 2.0 × 105/mL in 1% FCS DMEM, and cultured in 96-well plates(100 μL/well) overnight at 37°C in 5% CO2 atmosphere.

Vector construction
Vector carrying murine MD-2 and TLR4 cDNA (pDUO-mMD2/
TLR4) was from InvivoGen (San Diego, CA). cDNA fragments,
fused with a KpnI site and one Kozak sequence (ACC) at 5’-end anda SalI site at 3’-end, were ampliﬁed by PCR (KOD-Plus-Neo;
Toyobo), and inserted separately into pCDNA3 at KpnI and XhoI
site (Invitrogen). A set of vectors for dual luciferase assay, NF-κB
reporter gene (pGL3-ELAM; a Fireﬂy luciferase gene controlled by
NF-κB-dependent promoter of ELAM-1), and control reporter gene(pRL-TK; a Renilla luciferase gene controlled by constitutive active
promoter of thymidine kinase) were gifts from Dr. Takayuki
Kuraishi (Kanazawa University, Japan).

Cell culture and transfection
HEK293T cells were obtained from RIKEN Bioresource Center(Japan) and maintained in 10% FCS low-glucose DMEM at 37°C in
5% CO2 atmosphere. Prior to transfection, cells were diluted to 2.0
× 105/mL in 1% FCS DMEM and cultured in 96-well plates (100 μL/well) overnight. Cells in each well were transfected with vectors

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Lyophilized kidney tissue was extracted with chloroform/methanol(2:1 and 1:1, v/v). Total lipids were separated into acidic and neutral
fractions on DEAE-Sephadex A-25 anion-exchange columns (GEHealthcare Life Sciences). Acidic and neutral lipids were deesteriﬁed by mild alkaline hydrolysis and desalted using a Sep-Pak
C18 cartridge (Waters; Milford, MA). Acidic and neutral GSLs
(respective protein equivalent 1000 μg and 200 μg) were spotted on
HPTLC plates, developed respectively with chloroform/methanol/
0.2% CaCl2 (55:45:10, v/v/v) and chloroform/methanol/water(60:25:4, v/v/v), and visualized by orcinol/sulfuric acid staining.

T Nitta et al.

267

Globo-series glycosphingolipids and diabetic nephropathy(20 ng pCDNA3-mMD-2, 0.1 ng pCDNA3-mTLR4, 40 ng pGL3ELAM, 20 ng pRL-TK), complexed with 0.5 μL Lipofectamine LTX
and 0.25 μL Plus reagent in 20 μL Opti-MEM (Invitrogen), and subjected to stimulation 24 h after transfection.

Gb3Cer (from pig RBCs; Nacalai Tesque) and Gb4Cer (from pig
RBCs; Fuji-ﬁlm Wako, Japan) were dissolved (1 mM) in DMSO,
and diluted with low-glucose DMEM to 100 μM just before experiments. Cells were incubated with Gb3Cer/Gb4Cer, and stimulated
after 30 min with TLR ligands: LPS from E. coli O111:B4 (SigmaAldrich); human recombinant HMGB1 (Biolegend, San Diego, CA);
Pam3CSK4 (Novus Biologicals, Littleton, CO); Flagellin from S.
Typhmurium (ENZO Life science, Farmingdale, NY); R848 (Fujiﬁlm wako, Japan); MALP-2 (Novus Biologicals, Littleton, CO).
After 18 h culture, media were collected and subjected to ELISA.
ELISA kits for human IL-6, human TNF-alpha, human IL-12/23(p40), and murine TNF-alpha were from BioLegend. Fireﬂy and
Renilla luciferase activities were measured using Dual-Glo
Luciferase Assay System (Promega; Australia) on a microplate
reader (model Inﬁnite 200 PRO; Tecan; Switzerland).

Statistical analysis
Data were analyzed by Student’s t-test, Welch’s t-test, Dunnett’s
multiple comparison test, one-way ANOVA, or Mann Whitney
unpaired test as appropriate, using the Microsoft Excel.

Supplementary data
Supplementary data is available at Glycobiology online.

Funding
This study was supported by JSPS KAKENHI Grants-in-Aid for ScientiﬁcResearch (B) (JP16H04767 to J.I.), Grants-in-Aid for Exploratory Research(JP17K19569 to J.I.), and Grants-in-Aid for Young Scientist (B) (JP17K15450
to H.K.), Takeda Science Foundation (J.I.), Fugaku Trust for Medicinal
Research (J.I.), and Mizutani Foundation for Glycoscience (J.I.).

Acknowledgements
The authors are grateful to the Center for Laboratory Animal Science,
Tohoku Medical and Pharmaceutical University, for their services, to Prof.
Koichi Fukase at Osaka university for helpful suggestions, to K. Miyahara,
N. Okajima, and M. Goto at Inokuchi laboratory for their skillful technicalsupport, and to Dr. S. Anderson for English editing of the manuscript.

Conﬂict of interest statement
The authors declare that they have no conﬂict of interest.

Abbreviations
BMDMs, bone marrow-derived macrophages; ELISA, enzyme-linked
immunosorbent assay; FAs, fatty acids; Gb3Cer, globotriaosylceramide;
Gb4Cer, globotetraosylceramide; GlcCer, glucosylceramide; GSL(s),glycosphingolipid(s); HFD, high-fat diet; LacCer, lactosylceramide; LC-ESIMS/MS, HPLC coupled with electrospray ionization tandem mass
spectrometry; LCFAs, long chain fatty acids; LPS, lipopolysaccharide; ND,

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T Nitta et al.

CURRENT EYE RESEARCH
https://doi.org/10.1080/02713683.2019.1576206

Visual Function in Mice Lacking GM3 Synthase
Miki Hiraokaa, Ei Ohkawab, Akira Abea, Masaki Muratac, Shinji God,e, Jin-ichi Inokuchid, and Hiroshi OhguroaaDepartment of Ophthalmology, School of Medicine, Sapporo Medical University, Sapporo, Japan; bOhkawa Eye Clinic, Iwamizawa, Japan;
Department of Pathology, School of Medicine, Sapporo Medical University, Sapporo, Japan; dDivision of Glycopathology, Institute of Molecular
Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan; eDepartment of Pathophysiology and Metabolism,
Kawasaki Medical University, Kuraishi, Japanc

ABSTRACTARTICLE HISTORY

Purpose: Most complex gangliosides in vertebrates are formed from ganglioside GM3. GM3 deficiency
in humans can result in epilepsy and visual impairment. To investigate whether a deficiency of GM3 isinvolved in visual function, ST3GAL5−/− mice with mutations in the ST3GAL5 gene-coded GM3 synthase
were employed.
Materials and Methods: Sixty mice were employed in this study. The glycosphingolipids of mice retinas
were analyzed through high performance thin layer chromatography. The morphology of the optic
nerves and retinas were evaluated by hematoxylin and eosin staining and immunohistochemical
analysis using an anti-glial fibrillary acidic protein (GFAP) antibody. An electroretinogram (ERG) wasapplied on the eyes of 4, 9, 12, and 14-month-old mice. Also, visual evoked potential (VEP) was applied
on 13-month-old mice.
Results: The GM3 in the retinas was detected in ST3GAL5+/+ mice but not ST3GAL5−/− mice. Also, GM1band GD1α expressions and lactosylceramide accumulation were found in the ST3GAL5−/− mouse retinas.
There was no significant difference in GFAP expression in the retinas or optic discs between ST3GAL5+/+and ST3GAL5−/− mice. Furthermore, the outcome of ERG and VEP analysis showed no disparity between
the two strains in 13 and 14-month-old mice.
Conclusion: In the eye, neither histopathological abnormalities nor abnormal functions of the retina
were found in GM3-deficient mice. Differing from the situation in patients with GM3 deficiency, the lackof GM3 in mice did not lead to optic nerve atrophy.

Received 8 May 2018
Revised 22 January 2019
Accepted 25 January 2019

Introduction
Gangliosides are a class of glycosphingolipids ubiquitously present
in vertebrate plasma membranes. They are located in the outerleaflet of the cell membranes where they form microdomains andparticipate in transmembrane signaling and cell adhesion.1 It is
known that gangliosides are abundant in the central nervous
system, and essential for neural development.2 Among gangliosides, GM3 serves as a common precursor toward the formation ofcomplex ganglioside species.3 Recent studies have shown some of
the biological roles of GM3. For example, GM3 acts as an inducer
for insulin resistance and is involved in the etiology of diabetes andobesity.4–6 Additionally, in the immune system, GM3 is essential
for CD4+ T cell activation.7 In the auditory system, GM3 isnecessary for the development of hair cells in the organ of Corti.8,9The ST3GAL5 gene encodes GM3 synthase, which is thefirst enzyme mediating the biosynthesis of complex a- andb-series gangliosides.3,10 The ST3GAL5−/− mutant mice haveno GM3 synthase activity. The deficiency of GM3 synthaseleads to the a- and b-series gangliosides being replaced byo-series gangliosides. The ST3GAL5−/− mice live normal lifespans with fair fertile ability. The tissues from the brain, liver,and muscles of ST3GAL5−/− mice do not show any

KEYWORDS

Gangliosides; GM3;
electroretinogram (ERG);
visual evoked potential(VEP); anti-glial fibrillary
acidic protein (GFAP); micehistological abnormalities.4 ST3GAL5−/− mice have higher
insulin sensitivity and a low response to a high-fat diet.4
These mice also express mild symptoms of experimental
asthma induction through suppression of CD4+ T cells.7 In
addition, studies indicate that the mice acquire deafness.8,9
A 2004 study examines several patients in a family fromthe Old Order Amish community suffering from infantile
onset epilepsy syndrome with hearing loss and vision loss.11
They expressed an autosomal recessive inheritance pattern
and the plasma samples from affected family members wereGM3-deficient. The genetic screening of the patients revealed
homozygous mutations in the ST3GAL5 gene. An additional
report from France focuses on a family with two members
suffering from early onset epilepsy, deafness, and blindness.12
Those patients were identified to have homozygous mutationsin the ST3GAL5 gene. Yet another report examines GM3deficient patients with progressive epilepsy syndrome withvisual impairment due to optic nerve atrophy.13
In the present study, we investigated ophthalmologicaldifferences between ST3GAL5+/+ mice and ST3GAL5−/−
mice with a C57BL/6 background to understand the role of
GM3 in the visual system.

CONTACT Miki Hiraoka
mikihira@sapmed.ac.jp
Department of Ophthalmology, School of Medicine, Sapporo Medical University, S1W16 Chuo-ku, Sapporo,
Hokkaido 060-8543, Japan
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/icey.
Supplemental data for this article can be accessed here.
© 2019 Taylor & Francis Group, LLC

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M. HIRAOKA ET AL.

Materials and methods
Mice
The study protocols were approved by the Ethics Committee of
the Sapporo Medical University School of Medicine. All procedures involving animals were performed in accordance with the
ARVO Resolution on the Use of Animals in Research.
ST3GAL5+/+ and ST3GAL5−/− with a C57BL/6 backgroundmice were generated as previously described.8 Mice colonies
were maintained on a C57BL/6 background through heterozygous mating to generate a littermate control. Genotyping of
ST3GAL5 alleles was performed by PCR as previously
described.8 C57BL/6 mice were obtained from Hokudo(Sapporo, Japan), and bred in a pathogen-free condition in the
animal resource center under 12-hour light and dark cycles.
Before each experiment, mice were anesthetized with an intraperitoneal injection of a mixture of 0.75 mg/kg of medetomidine,
4 mg/kg of midazolam, and 5 mg/kg of butorphanol. Pupils weredilated with tropicamide and phenylephrine eye drops for electroretinogram (ERG) and visual evoked potential (VEP) study(0.5% each). No mice were used in more than one study.
Lipid extraction
Ten eyes were collected from ST3GAL5+/+ and ST3GAL5−/−
euthanized mice. The tissues (30–50 mg of wet weight) weretransferred into a glass tube and homogenized with 800 μl of
0.25 M sucrose/10 mM Hepes (pH 7.4)/1 mM EDTA on ice. The
homogenate was subjected to a one-phase extraction ina chloroform/methanol/aqueous solution (1:2:0.8, v/v), sonicatedfor 5 min using a sonic bath, and then stood for 1 h at room
temperature. The mixture was centrifuged at 2,330 g for 30 min at
25°C. The resultant supernatant was collected and the pellet was
reextracted in 1 ml of chloroform/methanol (1:1, v/v) by a sonicbath for 2 min. After centrifugation at 2,330 g for 30 min at 25°C,
the supernatant was combined with the first supernatant and kept
at −30°C as a retina lipid extract. A part of the extract was used forthe determination of phospholipid concentration.
Ganglioside analysis
Individual retina lipid extracts (containing 500 nmol of phospholipid) obtained from wild-type and GM3-deficient mice were dried
down under a stream of N2 gas using a water bath, dispersed with
2 ml of chloroform/methanol (1:1, v/v) using a sonic bath, andmixed with 0.45% (w/v) NaCl. After centrifugation at 1,000 g for
5 min at 25°C, the supernatant was collected and the organic phase
was washed with 1 ml of chloroform, 2 ml of methanol, and 2 ml ofwater. After further centrifugation at 1,000 g for 5 min at 25°C, thesupernatant was again collected and the organic phase was washedwith 2 ml of methanol and 2 ml of water. After another process ofcentrifugation at 1,000 g for 5 min at 25°C, the resultant supernatant was collected and the organic phase was washed with 1 mlof methanol and 0.8 ml of water. After a final centrifugation at
1,000 g for 5 min at 25°C, the resultant organic phase was transferred into another tube, dried by N2 gas and kept at −30°C as
a neutral glycolipid fraction. The resultant supernatant was combined with other supernatants and loaded to a Sep–Pak C18
cartridge (Waters, Milford, MA), which had been prewashedwith methanol, chloroform/methanol (1:1, v/v), methanol, andmethanol/water (1:1, v/v). After rinsing with water, gangliosides
were eluted with methanol, dried by N2 gas, and applied to a highperformance thin layer chromatogram (HPTLC) plate (Merck,
Darmstadt, Germany). The plate was developed in a solvent system consisting of chloroform/methanol/0.2% (w/v) CaCl2.2H2
O (55:45:10, v/v). After drying the plate, it was soaked in an
orcinol-sulfuric acid reagent to detect glycolipids. The uniformly
wet plate was briefly dried by a hair dryer and baked for 15 min in
a 100°C oven.
Neutral glycolipid analysis
The Individual neutral lipid fractions obtained in the ganglioside analysis section were dissolved with 500 μl of chloroform and 1 ml of 0.21 N NaOH in methanol, and incubatedfor 1 h at 37°C. The reaction was terminated by the additionof 1 ml of 0.2 N HCl. After centrifugation at 1,000 g for 5 min
at 25°C, the organic phase was transferred into a glass tube
and mixed with 2 ml of methanol plus 800 μl of 0.05 N HCl
containing 25 mM HgCl2. The reaction mixture was incubated for 20 min at 37°C and mixed with 1 ml of chloroform
plus 1.2 ml of water. After centrifugation at 1,000 g for 5 min,the organic phase was washed with 1 ml of methanol and800 μl of 33.3 mM EDTA and then washed twice with 1 ml ofmethanol plus 800 μl of 0.9% (w/v) of NaCl. The resultant
organic phase was transferred into another glass tube, driedby N2 gas and applied to an HPTLC plate. Glycolipids weredetected as described in the ganglioside analysis section.
ERG recording
Two sets of experiments were performed. During the first set,
mice aged 4, 9, and 12 months were employed and rod anddark-adapted maximal response ERG was performed. The
number of eyes is listed in Supplementary data 1 and 2.
During the second set, cone, rod, and dark-adapted maximal
response ERG was performed using 14-month-old mice (fiveeyes for each genotype) (Figure 3).
Mice were dark-adapted overnight before the experiments.
After anesthesia and mydriasis, the mice were placed ona heating pad for the duration of the ERG recordings. For eachmouse, an active contact lens electrode (Mayo, Inazawa, Japan)
was placed on the cornea. A reference electrode with a gold cup
was placed in the mouth, and a ground electrode with a stainlessclip was attached to the tail. The mice were placed in a Ganzfeld
dome (LKC Technologies Inc., Gaithersburg, MD) and the
response was obtained from one eye. The SG-2002 stimulator(LKC Technologies Inc., Gaithersburg, MD) was used, and analyzed with ERG-analysis software (PowerLab Scope version 3.7;
ADInstruments Ltd., Castle Hill, NSW, Australia). According to
the ISCEV standard,14 the a-wave amplitude was determinedfrom the baseline to the negative peak of the a-wave (Figure 2).
The b-wave amplitude was determined from the bottom of thea-wave to the top of the b-wave (Figure 2). For rod ERGs, four
responses were averaged with an interstimulus interval of 2 s andstimulus strength of 0.008 cds/m2. For dark-adapted maximal
response ERGs, the single response was recorded with stimulus
strength of 2.5 cds/m2. After 10 min of light adaptation for mice,CURRENT EYE RESEARCH

cone ERGs were recorded. The responses to 16 flashes wereaveraged with an interstimulus interval of 0.5 s and stimulus
strength of 2.5 cds/m2 presented on a rod saturated backgroundof 30 cd/m2. Neutral density filters were used to reduce thestimulus strength.
VEP recording
Five mice for ST3GAL5+/+ and six mice for ST3GAL5−/− mice,all 13 months old, were employed. One week prior to theexperiment, under anesthesia, a stainless steel screw (1 mmdiameter, 5 mm length) was inserted into the mice throughthe skull into the left visual cortex at the point of 1.5 mm lateral
to the midline and 1.5 mm anterior to the lambda, penetrating
the cortex to 1 mm in depth. For measurements, each screw
was connected to the amplifier as a measuring electrode. The
right eyes were stimulated in order to make the measurements.
VEPs were recorded using the same equipment as for ERG
under dark-adapted conditions. The responses to 128 flashes
were averaged with an interstimulus interval of 1 s and stimulus strength of 2.5 cds/m2. The N1 wave was defined as theinitial negative deflection, and the P1 wave was defined as the
positive deflection following the N1 wave. VEP was measured
twice for each mouse, with the average taken as data.
Immunohistochemical analysis
After euthanasia, mouse eyes were immediately enucleated, fixedin Super Fix (Kurabo, Osaka, Japan), and embedded in paraffin.
The 12-month-old mice were used for anti-glial fibrillary acidic
protein (GFAP) study, and 9-month-old mice were used for PKC
α study. Retinal sections were sagitally cut through the optic nerve
head at 3 μm thickness, mounted on slides, and dried. After the
deparaffinization with graded ethanol and xylene solutions, sections were sequentially incubated with a blocking reagent, andthen with an GFAP monoclonal antibody or anti-PKC α monoclonal antibody (clone MC5, Sigma Aldrich, St. Louis, USA). Afterwashing the slides with PBS, the specific staining by the antibody
was detected by the Histofine mouse stain kit (Nichirei, Tokyo,
Japan) according to the manufacturer’s protocol, and then developed with diaminobenzidine as a chromogen. Sections were counterstained with hematoxylin, dehydrated, and mounted. In thecontrol sections, equivalent concentrations of irrelevant antibodies
were substituted for the primary antibodies. The hematoxylineosin staining was also performed on each eye sample. All thesections were examined under a microscope equipped witha digital camera (Nikon, model ECLIPSE Ni, Tokyo, Japan).
Three eyes for each genotype were examined for GFAP staining.
The GFAP expression at the optic nerve was estimated by NIHImageJ 1.52a.
Statistical analysis
All results are expressed as mean ± SD. The values were processed
for statistical analyses (unpaired Student’s t-test) and significant
differences were considered at p < 0.05. Statistical analysis ofmultiple groups in ERG experiments was performed by using twoway ANOVA followed by the Tukey-Kramer test. P less than 0.05
was considered statistically significant.

3

Results
Glycosphingolipid expression in mouse retinas
The glycosphingolipid expression in mouse retinas fromST3GAL5+/+, ST3GAL5+/−, and ST3GAL5−/− mice was examined. As shown in Figure 1, ST3GAL5−/− mouse retinas had
undetectable GM3, and expressed o-series gangliosides(GM1b and GD1α) and a high content of lactosylceramide(LacCer), which is the proximate substrate of GM3 synthase.
ERG responses of mice
Representive ERG waveforms of 14-month-old mice (second
set experiment), elicited by stimuli of different intensities
recorded under cone, rod, and dark-adapted maximal
response conditions are shown in Figure 2. From each strain
4, 9, 12, and 14-month-old mice were examined. The amplitudes and implicit times of the a-wave and b-wave in
ST3GAL5+/+ and ST3GAL5−/− mice were statistically analyzed. The results of 4, 9, and 12-month-old mice (first set
experiment) are listed in Supplementary data 1 and 2, andthose of 14-month-old mice are listed in Figure 3. In mice at
the middle age of 4 and 9 months old, the implicit time of
the b-wave was delayed in ST3GAL5−/− mice in several
instances. Those delays were not observed in 12- and 14month-old mice.
VEP responses of mice
Representive VEP waveforms of 13-month-old mice areshown in Figure 2. The amplitudes and implicit times of
the N1 peak and P1 peak in ST3GAL5+/+ and ST3GAL5−/−
mice were statistically analyzed. The results are listed inTable 1. There was no significant difference between thetwo strains at amplitude and implicit time.
Histological and immunohistological study of mouse eyes
Through hematoxylin-eosin staining, both ST3GAL5+/+ andST3GAL5−/− mice showed orderly formation of ocular tissues
in the retinas and optic nerves (Figure 4). Optic nerve changes
such as swelling or atrophy were not observed even in elderly
mouse eyes of either strain.
GFAP is a marker of astrocytes and is also known asa marker of glial activation in response to neural injury.15 In
the glaucoma model, the GFAP expression in the optic nerve isknown to increase along with optic nerve damage. In the retina,
GFAP expressions are limited in layers of ganglion cells (GCL)
under normal conditions, but extend to the inner plexiform
layer and inner nuclear layer (INL) under conditions such asuveoretinitis and retinal degeneration. In our study, the GFAP
expression of the optic discs showed a similar intensity between
12-month-old ST3GAL5+/+ and ST3GAL5−/− mice (Figure 4).
The mean arbitrary unit of positive staining intensity in the
optic nerve was 84.6 ± 5.73 × 106 (bit/area) in ST3GAL5+/+ and87.4 ± 4.00 × 106 in ST3GAL5−/− mice (p value = 0.52). In theretina, the GFAP expression was restricted in the GCL in bothstrains (Figure 5). In addition, immunohistochemical analysis

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M. HIRAOKA ET AL.

Figure 1. Glycosphingolipid expression in mice retinas.
HPTLCs of ganglioside fraction (left) and neutral lipid fraction (right) obtained from the retinas of ST3GAL5+/+ and ST3GAL5−/− mice are presented. The ganglioside
fraction and neutral lipid fraction were prepared and developed in HPTLC plates as described in the Materials and Methods. The amount of lipid spotted per lane was
equivalent to 500 nmol retina phospholipid. Std, +/+, and −/− denote standard, ST3GAL5+/+, and ST3GAL5−/− mice, respectively.

Figure 2. Representative ERGs of 14-month-old, and VEP of 13-month-old ST3GAL5+/+ and ST3GAL5−/− mice.
The dark-adapted ERG responses to 2.5 and 0.008 cds/m2 are shown in the upper and lower left panels, respectively. The light-adapted cone response is shown(2.5 cds/m2) in the upper right panel. Representative VEP responses are shown in the lower right panel. The waveform of ST3GAL5+/+ mice (black) and ST3GAL5−/−
mice (red) were plotted.

using an antibody against protein kinase C alpha subunit(PKCα), described in a previous study as being a marker on
ON bipolar cells,16 was performed to examine the distributionof these cells in the retinas.16 The PKCα-positive cells were
found at the side of the INL in the retinas of both strains of 12month-old mice (Figure 5).

Discussion
In the present study, we investigated the ocular features of
ST3GAL5−/− mice. First, the glycosphingolipid profiles were
examined. The retinas of ST3GAL5−/− mice showed
a complete lack of GM3 gangliosides and an increase in lactosylceramide, which is a precursor of GM3. In addition, GM1b

CURRENT EYE RESEARCH

5

Figure 3. ERG intensity-response curves of the mean amplitude and implicit time of mice.
ERGs were elicited by three different stimulus intensities. The amplitudes are shown in the upper panel, and implicit times are shown in the lower panel. The mean
amplitudes of ST3GAL5+/+ (open circles), and ST3GAL5−/− mice (closed circles) were plotted with a SD bar. Five 14-month-old mice for each strain were employed.
There was no difference between the two strains (p > 0.05).

Table 1. VEP amplitudes and implicit times in 13 moths old ST3GAL5+/+ and ST3GAL5−/− mice.
N1 wave
StrainST3GAL5 +/+ST3GAL5 −/−p Value

P1 wave

Eyes

amp1

I.T.1

amp1

I.T.1

(n)
5
6

(μV)
−51.3 ± 19.60−45.0 ± 20.74
0.62

(ms)
43.3 ± 2.50
42.5 ± 5.00
0.76

(μV)
42.2 ± 15.74
40.2 ± 5.34
0.77

(ms)
56.9 ± 3.20
57.2 ± 5.45
0.91

amp: amplitude; I.T: implicit time, 1: Mean ± SD. P value: comparison with ST3GAL5+/+.

Figure 4. Histology of 12-month-old mouse eyes at optic disc area.
Panels A and C represent ST3GAL5+/+ mice and panels B and D represent ST3GAL5−/− mice. Panels A and B show the optic disc area with hematoxylin-eosin staining.
The GFAP expression of the optic disc area is shown in panels C and D.

6

M. HIRAOKA ET AL.

Figure 5. Histology of mouse eyes.
The retinal section of the central area between the peripapillary and peripheral areas are shown. Panels A, C, and E represent ST3GAL5+/+ mice and panels B, D, andF represent ST3GAL5−/− mice. Panels A and B show images with hematoxylin-eosin staining. The GFAP expression is shown in panels C and D. The PKCα expression isshown in panels E and F. A scale bar represents 50 μm.

and GD1α were found to be upregulated in response toa deficiency of GM3 synthase, indicating that the deficiencyof a- and b-series gangliosides are compensated by compensatory o-series gangliosides (GM1b and GD1α) in the retinas of
ST3GAL5−/− mice. Although gangliosides are ubiquitously present in most tissues, the highest concentration is found in thegray matter of the brain.2 Gangliosides are abundant in thecentral nervous system and essential for normal neural development and function. Most of the ganglio-series gangliosides invertebrates are synthesized from GM3, and GM3 synthasecatalyzes the primary step for the synthesis of complex gangliosides from lactosylceramide. GM3 is known to be involved indifferentiation, proliferation, and signal transduction in cells.
The antisense morpholino knockdown of ST3GAL5 expressionin zebrafish embryos results in neural apoptosis.17
Next, the pathophysiological manifestation of GM3-deficient
mice was studied. It has been previously reported that the GM3
synthase deficiency caused by homozygous nonsense mutations in
the ST3GAL5 gene results in infant onset epilepsy syndrome.11,12
Some of those affected family members showed bilateral optic
nerve pallor, but rod and cone ERG waveforms were found to benormal.13 The brain magnetic resonance imaging of these patients
showed diffuse cortical atrophy at older ages. In chick embryos,
GM3 was detected in the retinal pigment epithelium and neuralretina.18 It was assumed that blindness resulted from cortical
atrophy in the visual cortex. In our study, we performed ERG on
normal and GM3-deficient mice to clarify whether the deficiency
of GM3 influences retinal development. In those the age of 4 and9 months, the implicit time of the b-wave was delayed in GM3deficient mice. Since the b-wave is generated by bipolar cells, theexpression of PKCα in the retina was compared between two
strains resulting in a similar outcome. There was no statisticaldifference in ERG results between ST3GAL5+/+ and ST3GAL5−/−
mice at ages 12 and 14 months. In addition, histological andimmunohistological examinations were applied. Abnormalities
or destruction of eyeball structure, either in the anterior or posterior segment of the eyes, were not found in GM3-deficient mice,even those aged 14 months. There was no evidence of GM3
involvement in retinal function in old age. Unlike results seen intrials with humans, optic nerve atrophy was not observed in GM3deficient mice. Our VEP study did not show any influence due tolack of GM3. Additionally, the severe neurological phenotype ofCURRENT EYE RESEARCH

GM3-deficient patients was not seen in ST3GAL5−/− mice. These
disparities might be due to species differences.
In conclusion, the absence of GM3 has no influence on theretinal or optic nerve function of mice.

Acknowledgments
We would like to express our appreciation to Dr. Kimura and
Dr. Yamaguchi in the Division of Morphological Research at Sapporo
Medical University for their histological work, and to Mr. Ito and
Mr. Takeda in the Image and Media Support section at Sapporo
Medical University Hospital for their technical support. Also, we would
like to express our appreciation to Mr. Yoshikawa from the Mayo
Corporation for his technical support on ERG measurements.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
None.

References
1. Hakomori S. Glycosynapses: microdomains controlling carbohydrate-dependent cell adhesion and signaling. An Acad Bras Cienci.
2004;76(3):553–72. doi:10.1590/S0001-37652004000300010.
2. Schengrund CL. Gangliosides: glycosphingolipids essential fornormal neural development and function. Trends Biochem Sci.
2015;40(7):397–406. doi:10.1016/j.tibs.2015.03.007.
3. Kolter T, Proia RL, Sandhoff K. Combinatorial gangliosidebiosynthesis. J Biol Chem. 2002;277(29):25859–62. doi:10.1074/jbc.
R200001200.
4. Yamashita T, Hashiramoto A, Haluzik M, Mizukami H, Beck S,
Norton A, Kono M, Tsuji S, Daniotti JL, Werth N, et al. Enhanced
insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad
Sci U S A. 2003;100(6):3445–49. doi:10.1073/pnas.0635898100.
5. Inokuchi J. GM3 and diabetes. Glycoconj J. 2014;31(3):193–97.
doi:10.1007/s10719-013-9516-4.
6. Tagami S, Inokuchi Ji J, Kabayama K, Yoshimura H, Kitamura F,
Uemura S, Ogawa C, Ishii A, Saito M, Ohtsuka Y, et al.
Ganglioside GM3 participates in the pathological conditions of
insulin resistance. J Biol Chem. 2002;277(5):3085–92. doi:10.1074/
jbc.M103705200.
7. Nagafuku M, Okuyama K, Onimaru Y, Suzuki A, Odagiri Y,
Yamashita T, Iwasaki K, Fujiwara M, Takayanagi M, Ohno I, et al.
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activation. Proc Natl Acad Sci U S A. 2012;109(6):E336–42.
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8. Yoshikawa M, Go S, Takasaki K, Kakazu Y, Ohashi M,
Nagafuku M, Kabayama K, Sekimoto J, Suzuki S, Takaiwa K,
et al. Mice lacking ganglioside GM3 synthase exhibit complete
hearing loss due to selective degeneration of the organ of Corti.
Proc Natl Acad Sci U S A. 2009;106(23):9483–88. doi:10.1073/
pnas.0903279106.
9. Yoshikawa M, Go S, Suzuki S, Suzuki A, Katori Y, Morlet T,
Gottlieb SM, Fujiwara M, Iwasaki K, Strauss KA, et al.
Ganglioside GM3 is essential for the structural integrity andfunction of cochlear hair cells. Hum Mol Genet. 2015;24(10):2796–807. doi:10.1093/hmg/ddv041.
10. Ishii A, Ohta M, Watanabe Y, Matsuda K, Ishiyama K, Sakoe K,
Nakamura M, Inokuchi J, Sanai Y, Saito M. Expression cloning
and functional characterization of human cDNA for gangliosideGM3 synthase. J Biol Chem. 1998;273(48):31652–55.
11. Simpson MA, Cross H, Proukakis C, Priestman DA, Neville DC,
Reinkensmeier G, Wang H, Wiznitzer M, Gurtz K, Verganelaki A,
et al. Infantile-onset symptomatic epilepsy syndrome caused bya homozygous loss-of-function mutation of GM3 synthase. Nat
Genet. 2004;36(11):1225–29. doi:10.1038/ng1460.
12. Fragaki K, Ait-El-Mkadem S, Chaussenot A, Gire C, Mengual R,
Bonesso L, Beneteau M, Ricci JE, Desquiret-Dumas V,
Procaccio V, et al. Refractory epilepsy and mitochondrial dysfunction due to GM3 synthase deficiency. Eur J Hum Genet. 2013;21(5):528–34. doi:10.1038/ejhg.2012.202.
13. Farukhi F, Dakkouri C, Wang H, Wiztnitzer M, Traboulsi EI.
Etiology of vision loss in ganglioside GM3 synthase deficiency.
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862626.
14. McCulloch DL, Marmor MF, Brigell MG, Hamilton R,
Holder GE, Tzekov R, Bach M. ISCEV Standard for full-fieldclinical electroretinography (2015 update). Doc Ophthalmol.
2015;130(1):1–12. doi:10.1007/s10633-014-9473-7.
15. Wang L, Cioffi GA, Cull G, Dong J, Fortune B. Immunohistologic
evidence for retinal glial cell changes in human glaucoma. Invest
Ophthalmol Vis Sci. 2002;43:1088–94.
16. Grunert U, Martin PR, Wassle H. Immunocytochemical analysis of bipolar cells in the macaque monkey retina. J Comp
Neurol. 1994;348(4):607–27. doi:10.1002/cne.903480410.
17. Boccuto L, Aoki K, Flanagan-Steet H, Chen CF, Fan X, Bartel F,
Petukh M, Pittman A, Saul R, Chaubey A, et al. A mutation ina ganglioside biosynthetic enzyme, ST3GAL5, results in salt &pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation. Hum Mol Genet. 2014;23(2):418–33. doi:10.1093/hmg/ddt434.
18. Daniotti JL, Landa CA, Maccioni HJ. Regulation of gangliosidecomposition and synthesis is different in developing chick retinal
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Biochemical and Biophysical Research Communications 505 (2018) 36e39

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Regulation of membrane raft recruitment of the bradykinin B2
receptor by close association with the ATP/UTP receptor P2Y2
Tetsuto Nakagawa, Chihiro Takahashi, Hitomi Matsuzaki, Yoshiyuki Kuroda,
Hideyoshi Higashi*
Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi,
981-8558, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 4 September 2018
Accepted 12 September 2018
Available online 17 September 2018

Several G protein-coupled receptors are present in lipid rafts. We have shown that most of the P2Y2
receptor (P2Y2R) protein is fractionated into lipid rafts in COS 7 cells. In the same cells, about 25e30% of
the bradykinin B2 receptor (B2R) protein is also fractionated into lipid rafts. When both P2Y2R and B2R
are co-expressed, the distribution of P2Y2R remained unchanged, but more B2R shifted into the raft
fraction. This indicates that the interaction between both receptors recruited B2R into the lipid rafts.
After 15 min of UTP stimulation, both receptors almost completely disappeared from the cell surface byendocytosis as observed with a confocal ﬂuorescence microscope. Furthermore, with bradykinin stimulation for 15 min, portions of both receptors disappeared from the cell surface and were endocytosed. As
we reported previously with both CHO-K1 cells and HEK 293 cells, continuous stimulation of COS7 cellswith GT1b and CSC resulted in the disappearance of both P2Y2R and B2R from the cell membrane surface.
Thus, both P2Y2R and B2R migrate into membrane rafts and are endocytosed in parallel with signal
crosstalk, clearly indicating that both closely interact on membrane rafts. The P2Y2R N-glycosylationdeﬁcient mutant does not migrate to the cell surface. It remains predominantly in the endoplasmic
reticulum and is fractionated into raft fractions. In the presence of this glycosylation mutant, most of B2R
remains in the endoplasmic reticulum, and is fractionated into the raft fraction. These ﬁndings
demonstrate that in the membrane rafts of the endoplasmic reticulum, both receptors are already closely
associated, and B2R shifts into the rafts by afﬁnity with P2Y2R.
© 2018 Elsevier Inc. All rights reserved.

Keywords:
GPCR-GPCR interaction
Lipid rafts
N-linked glycosylation
B2 receptor
P2Y2 receptor

1. Introduction
P2Y2R, which uses ATP/UTP as a ligand, and B2R, which uses
bradykinin as a ligand, are G protein-coupled receptors (GPCRs).
Both receptors are coupled with Gaq/11 and are involved in physiological functions, such as blood pressure reduction, inﬂammation,and pain. In our previous studies using both Ca2þ imaging and theb-galactosidase a-complementation assay, we showed that both
receptors are associated closely in desensitization and internalization, are co-immunoprecipitated, and are involved in signal crosstalk with each other [1].
A lipid raft, a membrane microdomain on the cell membrane, isrich in both sphingolipids and cholesterol. Lipid rafts have characteristic receptor proteins and signaling molecules and play

important roles in signal transduction through membranes, bacterial and viral infections, cell adhesion, intracellular vesicular
trafﬁcking, and intracellular polarity [2,3]. Experimentally, lipid
rafts are insoluble in nonionic surfactants at low temperature, andthus can be distinguished from other membrane regions. We
recently showed that most P2Y2R molecules localize to lipid rafts in
COS 7 cells [4]. On the other hand, B2R was distributed in both raftand non-raft fractions. Therefore, we examined whether theinteraction between the two receptors affects their distribution
into rafts. The co-endocytosis of both receptors was observed byimmunoﬂuorescence microscopy even when both molecules wereseparately stimulated.

2. Materials and methods* Corresponding author.
E-mail address: hhigashi@tohoku-mpu.ac.jp (H. Higashi).
https://doi.org/10.1016/j.bbrc.2018.09.072
0006-291X/© 2018 Elsevier Inc. All rights reserved.

Preparation of Plasmids. Plasmids encoding human P2Y2R andhuman short variant B2R, starting at the third initiating ATG wereT. Nakagawa et al. / Biochemical and Biophysical Research Communications 505 (2018) 36e39

37

Immunocytochemistry. After one day of culture, COS-7 cells weretransfected and cultured for an additional day on glass coverslips in
24-well culture plates. The cells were exposed to the following
stimulants: either 100 mM UTP for 15 min, 1 mM bradykinin (BK) for
15 min, or 100 ng/mL GT1b ganglioside for 15 min followed by
10 mg/mL chondroitin sulfate C (CSC) for 15 min. After removing the
medium, the cells were ﬁxed with 3% paraformaldehyde. After
blocking with 5% skim milk in PBS, P2Y2R-Myc and B2R-HA weredetected using anti-Myc monoclonal antibody (71D10 rabbit mAb;
Cell Signaling Technology) and anti-HA monoclonal antibody 3F10,
respectively, followed by Alexa Fluor 546 conjugated goat antirabbit IgG and Alexa Fluor 488 conjugated goat anti-rat IgG(Thermo Fisher Scientiﬁc), respectively. The cells were observed
using confocal laser scanning microscopy (FV1000; Olympus).
Fig. 1. Effects of co-expression on B2R distribution in the lipid raft fraction of both wild
type (WT) and N-linked sugar chain-deﬁcient mutant P2Y2R (N9Q/N13Q). HA-tagged
B2R was expressed either alone or with P2Y2R, and the detergent insoluble (Insol)
membrane fraction and the detergent soluble (Sol) membrane fraction were analyzed
by Western blotting using an anti-HA antibody.

prepared by insertion of each cDNA between the EcoRI and BamHI
sites of pIRES2-EGFP (Clontech). The N-glycan deﬁcient N9Q/N13Q
P2Y2R mutant was prepared as described previously [4]. Human
P2Y2R genes with a C-terminal c-Myc epitope tag and human B2R
genes with a C-terminal HA epitope tag were prepared as described
previously [1,4].
Cell culture. COS-7 cells were grown in D-MEM supplemented
with 10% fetal calf serum and kept in a humidiﬁed incubator with
5% CO2 at 37  C. COS-7 cells were transiently transfected using
Viafect transfection regent (Promega).
SDS-PAGE and Western blotting. Membrane fractions and protein
samples for SDS-PAGE was prepared as described previously [4].
SDS-PAGE was performed using the Novex NuPAGE SDS-Gel systemwith 4e12% Bis-Tris gels (Thermo Fisher Scientiﬁc), and proteins
were electrophoretically transferred to an Immobilon PVDF membrane (Merck Millipore) using a semi-dry blotting system. After
blocking with 5% skim milk in PBS, P2Y2R-Myc was stained using
anti-Myc mouse mAb 9E10 (Roche Diagnostics) followed byhorseradish peroxidase-conjugated anti-mouse IgG (PIERCE). B2RHA was stained using anti-HA rat mAb 3F10 antibody (Roche Diagnostics), followed by horseradish peroxidase-conjugated anti-rat
IgG (#16549, Invitrogen). Then, the bound peroxidase was detectedwith Luminata Forte Western HRP Substrate (Merck Millipore).

3. Results
3.1. Effect of P2Y2R on B2R membrane microdomain distribution
Since B2R and P2Y2R interact and P2Y2R localizes in lipid rafts,
the interaction between both receptors was expected on lipid rafts.
When B2R-HA was expressed in COS7 cells and immunoblottedwith anti-HA antibody after SDS-PAGE, about 25e30% of the protein was localized to the lipid rafts and the remainder of the protein
fractionated to non-raft fractions. When P2Y2R was co-expressed,
80% or more of B2R was fractionated into the raft fraction (Fig. 1).
This result shows that the interaction between the two receptors
promotes the transition of B2R into lipid rafts by P2Y2R. The sameﬁnding was also observed when an N-linked sugar chain deletionmutant of P2Y2R (N9Q/N13Q) was co-expressed. This indicated that
the N-linked sugar chain of P2Y2R was not involved in association
with B2R. In addition, the N9Q/N13Q mutant is not expressed on
the cell surface and remains in the endoplasmic reticulum [4]despite its association with B2R. The intracellular localization ofboth receptors was observed with a confocal ﬂuorescence microscope. The staining of N9Q/N13Q-P2Y2R in the endoplasmic reticulum was consistent with some of the B2R staining (Fig. 2).
Therefore, it was demonstrated that both receptors were alreadyassociated in the endoplasmic reticulum.
Almost all of P2Y2R is fractionated into raft fractions in both wild
type and the N9Q/N13Q mutant. The inﬂuence of B2R coexpression on the raft distribution of these P2Y2R molecules wasinvestigated. Both P2Y2R and its mutant fractionated into raft
fractions, and no migration to non-raft fractions was observed

Fig. 2. Confocal ﬂuorescence microscopic images of N9Q/N13Q-P2Y2R and B2R. Myc-tagged P2Y2R mutant and HA-tagged B2R were expressed in COS 7 cells and observed with aconfocal microscope. Anti-Myc rabbit mAb and anti-HA rat mAb were used as the primary antibodies, respectively, and Alexa Fluor 546 goat anti-rabbit IgG and Alexa Fluor 488 goatanti-rat IgG were used as the secondary antibodies, respectively."P2Y2R00 is the ﬂuorescence (red) of the secondary antibody bound to the anti-Myc antibody and "B2R00 is theﬂuorescence (green) of the secondary antibody bound to the anti-HA antibody. "Merge" is both ﬂuorescent images superimposed. Scale bar indicates 10 mm.

38

T. Nakagawa et al. / Biochemical and Biophysical Research Communications 505 (2018) 36e39

Fig. 3. Distribution of wild type (WT) and N9Q/N13Q-P2Y2R into lipid rafts during B2R
co-expression. Wild type and mutant of Myc-tagged P2Y2R was co-expressed either
individually or with B2R, and the detergent insoluble (Insol) membrane fraction andthe detergent soluble (Sol) membrane fraction were analyzed by Western blotting
using an anti-Myc antibody.

(Fig. 3). These data demonstrate that P2Y2R was fractionated into
lipid rafts with or without B2R co-expression.

3.2. Intracellular dynamics of P2Y2R and B2R
The data suggest that B2R and P2Y2R directly interact andfunction together. The effects of stimulants on the localization ofboth receptors were investigated with a confocal ﬂuorescence microscope in cells co-expressing both P2Y2R and B2R (Fig. 4). Whenstimulated with 100 mM UTP for 15 min, most of P2Y2R disappeared
from the cell membrane surface by endocytosis. B2R was involvedin the endocytosis of P2Y2R, and most of the B2R disappeared fromthe cell membrane surface. On the other hand, when the cells were
stimulated with 1 mM BK for 15 min, only a portion of both B2R and
P2Y2R was endocytosed (Fig. 4). When the stained images after UTP
stimulation were merged, it was shown that P2Y2R and B2R were

present in different granules, suggesting that these receptors did
not interact with each other after endocytosis.
Since we have found that desensitization of B2R is caused bycontinuous stimulation of both GT1b and CSC [5] and that P2Y2R is
also desensitized at that time [1], we investigated the intracellular
localization of both receptors. After incubation with 100 ng/mL
GT1b for 15 min followed by incubation with 10 mg/mL CSC for
15 min, most of both receptors disappeared from the cell membrane surface. In this case too, it was shown that many of the both
receptors were present in different granules (Fig. 4).
4. Discussion
We have previously demonstrated signal crosstalk between
P2Y2R and B2R. Both receptors have a close relationship and agonist
stimulation of one receptor results in activation of both receptors[1]. Since most of the P2Y2R protein is fractionated into lipid rafts[4], interaction between both receptors was expected to occur inthe lipid rafts. P2Y2R fractionated into lipid rafts regardless of either
the presence or absence of its component N-linked sugar chain or ofco-expression with B2R. B2R was fractionated into lipid rafts whenco-expressed with P2Y2R, but when B2R was not co-expressed with
P2Y2R, more of the B2R protein was localized in non-raft fractions.
Since the localization of B2R into lipid rafts was affected by theexpression of P2Y2R, additional evidence was obtained that a
portion of the B2R protein was associated with P2Y2R. These ﬁndings demonstrate that B2R localizes to lipid rafts due to its afﬁnity
for P2Y2R. Both receptors signal cross talk probably by forming ahetero-oligomer [1] in the rafts. Moreover, this association also
occurred between B2R and the N-linked sugar chain-deﬁcientFig. 4. Confocal ﬂuorescence micrograph of wild-type P2Y2R and B2R. Myc-tagged wild-type P2Y2R and HA-tagged B2R were expressed in COS7 cells and observed with a confocal
microscope as in Fig. 2. Scale bar indicates 10 mm.

T. Nakagawa et al. / Biochemical and Biophysical Research Communications 505 (2018) 36e39

P2Y2R mutant that is not expressed on the cell surface [4]. In thiscase, both receptors remained in the endoplasmic reticulum.
After a 15 min stimulation with UTP, P2Y2R is endocytosed, andB2R was also endocytosed (Fig. 4). In addition, after continuous
stimulation with GT1b/CSC, B2R is desensitized, and co-existing
P2Y2R is also desensitized [1,5]. Thus, after desensitization ofeither receptor, both receptors disappeared from the cell membrane surface (Fig. 4). With both UTP and continuous GT1b/CSC
stimulation, many of the receptors were localized in different
granules. This may be due to the fact that either P2Y2R and B2R
cooperate and follow different routes after being transported into
the cell or the two receptors have different transport speeds fromthe early endosome to the recycling endosome.
A membrane raft is a signaling domain in the cell membrane,
and the signals of some GPCRs, such as P2Y2R [6,7] andgonadotropin-releasing hormone (GnRH) [8] are transmitted
downstream by localizing into rafts. Also, B2R is transferred by
activation in smooth muscle cells by caveolae, a type of raft [9]. In
both adrenal chromafﬁn cells and PC 12 cells, a raft is involved insensitization of the EGFR-inducing signal by B2R [10]. In smoothmuscle cells, P2Y2R also shifts into rafts by agonist stimulation [11].
In addition, there are chemokine receptor GPCRs that transmit
different signals through inside or outside rafts [12]. Kaiser et al. [7]reported that whereas lipid rafts are required for the activation of
P2Y2R, it is not necessary for the activation of B2R. However, lipid
rafts are required for both receptors to exert the relaxing action on
guinea pig endothelium. Our data demonstrate that the downstream B2R signal is regulated by the presence of P2Y2R since B2R
associates with P2Y2R and enters into the rafts.
In summary, we demonstrated that P2Y2R recruits B2R into lipid
rafts due to the close interaction between these molecules. Bothreceptors disappear from the cell membrane surface after stimulation of either receptor. In the future, it should be determined
whether this process after endocytosis is carried out independently
or not.

Transparency document
Transparency document related to this article can be foundonline at https://doi.org/10.1016/j.bbrc.2018.09.072.

39

Acknowledgements
Funding
This study was supported by the Strategic Study Base Formation
Support Project for Private Universities (S1201031), Japan.

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K.T. Kim, Sensitization of epidermal growth factor-induced signaling by bradykinin is mediated by c-Src. Implications for a role of lipid microdomains,
J. Biol. Chem. 279 (2004) 5852e5860.
[11] A. Norambuena, F. Palma, M.I. Poblete, M.V. Donoso, E. Pardo, A. Gonzalez,
J.P. Huidobro-Toro, UTP controls cell surface distribution and vasomotor activity of the human P2Y2 receptor through an epidermal growth factor
receptor-transregulated mechanism, J. Biol. Chem. 285 (2010) 2940e2950.
[12] R. Shamri, V. Grabovsky, S.W. Feigelson, O. Dwir, Y. Van Kooyk, R. Alon,
Chemokine stimulation of lymphocyte alpha 4 integrin avidity but not of
leukocyte function-associated antigen-1 avidity to endothelial ligands undershear ﬂow requires cholesterol membrane rafts, J. Biol. Chem. 277 (2002)
40027e40035.

J. Biochem. 2018;163(6):475–480

doi:10.1093/jb/mvy010

Homodimer formation by the ATP/UTP receptor P2Y2
via disulfide bridges
Received September 20, 2017; accepted December 20, 2017; published online January 18, 2018

Maya Abe, Kanae Watanabe,
Yoshiyuki Kuroda, Tetsuto Nakagawa and
Hideyoshi Higashi*
Division of Glyco-Signal Research, Institute of Molecular
Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, Sendai, Miyagi 981-8558, Japan*Hideyoshi Higashi, Division of Glyco-Signal Research, Institute of
Molecular Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, Sendai, Miyagi 981-8558, Japan.
Tel: +81-22-727-0119, Fax: +81-22-727-0077,
email: hhigashi@tohoku-mpu.ac.jp

Many class C G-protein coupled receptors (GPCRs)
function as homo- or heterodimers and several class
A GPCRs have also been shown to form a homodimer.
We expressed human P2Y2 receptor (P2Y2R) in cultured cells and compared SDS-PAGE patterns under
reducing and non-reducing conditions. Under non-reducing conditions, approximately half of the P2Y2Rs were
electrophoresed as a dimer. We then produced Cys to
Ser mutants at four sites (Cys25, Cys106, Cys183 andCys278) in the extracellular domains of P2Y2R andexamined the effect on dimer formation and receptor
activity. All single mutants formed dimers similarly to
the wild-type protein, but C25S, C106S and C183S
P2Y2R lost activity, while C278S P2Y2R maintained
weak activity. Coexpression with wild-type P2Y2R recovered the activity of the C25S mutant. These results
show that Cys106 and Cys183 are required for monomer or homodimer activity; Cys25 is required formonomer activity, but it is not needed in one protomer
for homodimer activity; and Cys278 can be replaced in
the monomer and homodimer. Approximately, half of
C25S/C278S double mutants were electrophoresed as a
dimer, similarly to the wild-type and single mutants,
and dimers with the wild-type protein were active.
These results suggest involvement of Cys106 andCys183 in disulﬁde bonding between protomers inhomodimer formation.
Keywords: a-complementation assay; arrestin recruitment; disulﬁde bond; GPCR homodimer; GPCR
signal cross-talk.
Abbreviations: A1R, adenosine A1 receptor; B2R,
bradykinin receptor B2; BSS, balanced salt solution;
GPCR, G-protein coupled receptor; HEPES, 4-(2hydroxyethyl)-1-piperazi- neethanesulfonic acid;P2Y2R, P2Y2 receptor; PBS, phosphate bufferedsaline; PCR, polymerase chain reaction; SDS-PAGE,
sodium dodecyl sulphate polyacrylamide gelelectrophoresis.

Some G-protein coupled receptors (GPCRs) form
homodimer or heteromers. Among the many class A
GPCRs, rhodopsin (1), bradykinin receptor B2 (B2R)(2), purinergic adenosine A1 receptor (A1R) and P2Y2
receptors (P2Y2R) (3) form homodimers. Disulﬁdebridges are present in B2R (4), but the bridging details
are unknown and the quaternary structure has not been
determined. All class A GPCRs except S1P1 have preserved Cys residues in the ﬁrst and second extracellular
loops and disulﬁde bridges are formed between two Cys
residues in monomers, as shown by structural analysis ofseveral GPCRs, including rhodopsin (5), and this structural motif is presumed to be similar in other GPCRs.
P2Y2R is a GPCR activated by ATP/UTP that couples to Gaq/11 (G protein) and is involved in inductionof inﬂammation, hypotension and pain. P2Y2R forms
hetero-oligomers or has cross-talk with other class A
GPCRs, such as b2-adrenergic receptor (6), CXC chemokine receptor 2 (7), A1R (8) and B2R (9). Given that
homodimer formation by disulﬁde bridges was found in
SDS-PAGE, we examined how four extracellular Cys
residues in P2Y2R (Cys25, Cys106, Cys183 and Cys278)
form bridges in the monomer and dimer. Hillmann et al.
proposed Cys25Cys278 and Cys106Cys183 disulﬁdebridges based on the effects of mutation of Cys106 andCys278 to Ser, homologous residues in rhodopsin, and asimilar structure to P2Y1 receptor (10), in which there isa disulﬁde bond between Cys residues in N terminal
domain and third extracellular loop (11). In the current
study, we produced point mutants with each of the four
Cys residues replaced with Ser. These proteins wereused to examine disulﬁde bridge formation using SDSPAGE under reducing and nonreducing conditions, andformation of functional homodimers based on UTPinduced activation. Our results show that P2Y2R canfunction as a monomer or homodimer and that disulﬁdebonds contribute to homodimer formation.

Materials and Methods
Cell culture
HEK293T and 1321N1 cells were grown in Dulbecco’s Modiﬁed
Eagle’s Medium with 25 mM glucose supplemented with 10% (v/v)
fetal calf serum and kept in a humidiﬁed 10% CO2/90% air atmosphere at 37 C. The cells were transiently transfected with plasmids
using Lipofectamine 2000 (Invitrogen) for western blotting andCa2+ imaging. Cell membranes for western blotting were preparedfrom cells 48 h after transfection.
Plasmids and constructs
Plasmids encoding human P2Y2R were prepared by inserting P2Y2R
cDNA between the EcoRI and BamHI sites of pIRES2-EGFP(Clontech). P2Y2Rs with Ser substitution at Cys25, Cys106,
Cys183, Cys278 and Cys25/278 were prepared by PCR. The C-terminal of P2Y2R was fused to the H31R-substituted a donor peptideß The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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M. Abe et al.
of a LacZ b-galactosidase reporter enzyme (P2Y2R-a) or Myc
epitope. The C-terminal of b-arrestin-2 was fused to the M15 acceptor deletion mutant of b-galactosidase (the o peptide, arrestin-o).
Plasmids encoding fusion proteins were generated by subcloning
PCR products into pAlpha-N1 or pOmega-N1 vectors, which wereconstructed from pAcGFP-N1 vector (Clontech) (9).
Preparation of membrane fractions, SDS-PAGE and western
blotting
Preparation of membrane fractions (12), SDS-PAGE and western
blotting (9) were performed as described previously.
b-galactosidase complementation assay
The interaction between activated GPCR and b-arrestin (arrestin recruitment) was measured using complementation between the a and odomains of LacZ b-galactosidase (9, 13). Since P2Y2R was activatedwith autocrine ATP generated by physical stimulus during cell cultivation in our original method (9), we modiﬁed the procedure to avoid
autocrine effects by cultivating the cells 24 h before the assay, as
described below. HEK293T or 1321N1 cells were cultured in 10 cm
f cell culture dishes to 80% conﬂuence. Cells were washed once withphosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM
KH2PO4, and 8.1 mM Na2HPO4) and harvested by digestion with
2 mL of 0.05% trypsin/EDTA for 30 s at room temperature.
Digestion was stopped by adding 2.5 mL of culture medium. The harvested cells were spun down (700 g, 5 min) and washed with 3 mL of
PBS. The pelleted cells were suspended in 36 mL of culture medium
and dispensed as 3 mL aliquots. Cell suspensions were mixed withtransfection reagents, 0.4 mg of each plasmid DNA coding for
GPCR-a and arrestin-o in 200 mL Opti-MEM, and 1.6 mL of
Lipofectamine 2000 (Invitrogen) in 200 mL of Opti-MEM. Then, a
180 mL aliquot of the mixture was seeded on a white tissue culture
96-well plate (Corning 3917) and cultured at 37 C for 24 h. After stimulation of the cells with 20 mL of 10 concentrated UTP for 1 h at 37 C,
150 mL of the supernatant was discarded and 50 mL of Gal Screen (B,Applied Biosystems) was added, followed by incubation at room temperature. Luminescence of the wells was read after about 40 min.
Ca2+ imaging
Cells cultured on glass-bottom dishes were loaded with the Ca2+
indicator Fura-2 and exposed to stimulants using the bath application method. Images were acquired and analyzed using balanced salt
solution (pH 7.3; 130 mM NaCl, 5.4 mM KCl, 20 mM HEPES,
5.5 mM glucose, 0.8 mM MgSO4, and 1.8 mM CaCl2) as theextracellular media (1416).
Statistical analysis
Results are presented as means ± SEM. Differences between groups
were assessed by two-way ANOVA followed by a Dunnett’s multiple
comparison test, using GraphPad software (San Diego, CA).
P50.05 was considered signiﬁcant.

Results
Homodimer formation by P2Y2R and effect of mutation of Cys residues

P2Y2R with a C-terminal Myc tag was expressed in
HEK293T cells. Western blotting was performed
after SDS-PAGE under reducing and non-reducing
conditions. Under non-reducing conditions, western
blot analysis showed bands at the monomer position
and at a position consistent with a dimer (Fig. 1B). Cys
to Ser mutants were examined similarly, and all mutants, including the C25S/C278S double mutant,formed bands with molecular masses consistent withthe monomer and dimer. However, most dimer bands
transferred to the monomer position under reducing
conditions (Fig. 1A), which suggests that P2Y2R
forms a homodimer with disulﬁde bonds. The ratios
of monomer and dimer forms of wild-type and mutant
P2Y2Rs were estimated from Fig. 1B. About 60% of
476

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wild-type P2Y2R formed a dimer, while less than 40%of the C278S mutant formed a dimer (Table I). We
previously showed that most P2Y2R molecules in
COS7 cells are present in lipid rafts (12), and a similar
result was found in HEK293T cells, with most wildtype and mutant P2Y2Rs in raft component fractions.
Effect on arrestin recruitment of homodimer
formation by wild-type and Cys-mutant P2Y2R

Using P2Y2R with the C-terminal attached to the LacZ
a-domain, UTP-stimulated arrestin recruitment wasdetermined based on reactivity to the arrestin attachedto the LacZ o-domain. In HEK293T cells, C25S, C278S
and C25S/C278S mutants had weaker activity than thewild-type protein but were activated by UTP, whereas
C106S and C183S mutants were not activated (Fig. 2A).
Since endogenous P2Y2R is expressed in HEK293T
cells, the assay was also conducted in 1321N1 cells,
which do not have endogenous expression of P2Y2R.
In these cells, the activity of the C25S and C25S/C278S
mutants disappeared (Fig. 2B). The C278S mutant
maintained some activity, but this was weaker thanthat of wild-type P2Y2R. To conﬁrm the effect ofdimers, both wild-type P2Y2R and P2Y2R mutant-a
proteins were expressed in 1321N1 cells. Under these
conditions, results similar to those observed in
HEK293T cells were obtained (Fig. 2C). Combinationof wild-type P2Y2R with C25S, C278S or C25S/S278S
mutants gave activity of approximately 25% that of thewild-type homodimer. C25S-a was activated in the presence of wild-type or C278S P2Y2R but was not activated in the presence of C25S/C278S mutant (Fig.
2D). This result implies that the activation signal fromwild-type or the C278S mutant was transmitted to
C25S-a and shows that activation signals from wildtype are not transmitted to C106S-a or C183S-a.
Examination of G-protein activation

The above results show that Cys25, Cys106 andCys183 are required for recruitment of arrestin.
Further assays were conducted to examine whether
mutation of these Cys residues inhibited G-protein activity, an upstream reaction in 1321N1 cells, using
intracellular Ca2+ imaging. None of the C25S,
C106S and C183S mutants caused an increase inCa2+, in contrast to wild-type P2Y2R (Fig. 3; resultsfor C106S and C183S P2Y2R are not shown but weresimilar to those for C25S P2Y2R). Expression of thethree mutants with pIRES2-EGFP resulted in less expression of EGFP than with wild-type P2Y2R, for areason that is unclear.

Discussion
Extracellular Cys residues in proteins located on thesurface of cell membranes maintain protein structure
via disulﬁde bridging. Among GPCRs, rhodopsin has
four extracellular Cys residues that form disulﬁdecrosslinks between two central and two terminal
residues (cf. Fig. 4A). The activity of GPCRs isregulated by formation of homodimers and heterooligomers, which leads to signaling cross-talk. Many class
C GPCRs form homo- or heterodimers, and mGluRs

P2Y2 receptor homodimer via disulﬁde bridges

A

B

kDa
100
Di

75
48

Mon
35
WT

C25S C106S C183S C278S

C25S/
C278S

WT

C25S C106S C183S C278S

C25S/
C278S

Fig. 1 Wild-type (WT) P2Y2R-Myc and Cys to Ser mutants were expressed in HEK293T cells and detected by Western blotting. Results
are shown for detergent-resistant membrane fractions under reducing (A) and nonreducing (B) conditions. Almost no expressed protein was
fractioned in the detergent-soluble fraction. Di, dimer; Mon, monomer.

Table I. Ratio of monomer and dimer forms of P2Y2R and its Cys
to Ser mutantsa
P2Y2R

Monomer (%)

Dimer (%)

Wild type
C25S
C106S
C183S
C278S
C25S/C278S

38
48
42
53
69
62

62
52
58
47
31
38

a

Ratios were calculated from scanned image of western blot in Fig. 1B.

(17) and Ca2+ sensing receptor (18) have disulﬁdebonds that contribute to dimer formation. In
mGluRs, Cys residues in the long N terminal extracellular domain contribute to homodimer formation (19).
Several class A GPCRs also form homodimers, including rhodopsin (1), A1R and P2Y2R (3), B2R (2, 4),
D2R (20), b2AR (21) and 5-HT2C (22). Many class
A GPCRs have Cys residues in N terminal domain
and the ﬁrst, second and third extracellular loops,
but the majority view is that homodimer formation
depends on interactions between transmembrane domains. Only B2R (4) and P2Y2R in this study havebeen shown to have disulﬁde bonds involved in homodimer formation and it is still unclear how these Cys
residues are bridged.
Fusion of a GPCR with an a domain to facilitate a
LacZ a complementary reaction permits evaluation of
signaling cross-talk by GPCR homodimers and heterooligomers (9). In this study, we found that someP2Y2Rs formed dimers under non-reducing conditions(Fig. 1). The dimers were formed with all of the mutants examined. We examined whether extracellular
Cys residues at four sites contributed to ‘functional’
dimer formation by evaluating signaling cross-talk of
Cys to Ser mutants expressed alone or with wild-type
P2Y2R (Fig. 2). All mutants formed dimers with eachother or with wild type. The C25S mutant received
active signal from wild type revealing arrestin recruitment activity. That is, it formed a ‘functional’ dimer
capable of cross-talk. The C106S and C183S mutants
were able to form dimers, but they were not activatedalone and could not engage in signal cross-talk from
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wild type. This indicates that these mutants cannot
form a ‘functional’ dimer, even though they wereable to form a dimer with wild type. The C278S
mutant showed somewhat diminished dimer formation
but had activity itself and was able to transfer a signal
to the C25S mutant. Based on these results, a disulﬁdebridge pattern can be proposed for P2Y2R (Fig. 4).
Cys106 and Cys183 are positioned in the ﬁrst andsecond extracellular loops, and mutation of either ofthese residues to Ser eliminated receptor activity when
expressed alone and in coexpression with wild-type
P2Y2R. This suggests that a disulﬁde bond is formed
between these two residues, as Hillmann et al. proposed based on homology to rhodopsin (11).
Hillmann et al. indicated that C106S P2Y2R was not
expressed on the cell surface in 1321N1 cells, as thebinding of antibodies to the HA-tag added to the
N-terminus is undetectable (11). In our observation,
the level of protein expression did not differ between
wild-type and mutant proteins in both HEK293T(Fig. 1) and 1321N1 cells (unpublished observation).
Thus, C106S mutant protein is expressed but its sorting to the cell surface is prohibited. It is probable thatbridging with an appropriate disulﬁde bond between
Cys106 and Cys183 is sufﬁcient for the cell surface
expression of the receptor.
Cys106 and Cys183 are also probably involved in adisulﬁde bridge between protomers in homodimer formation (Fig. 4C). Ser mutations at each of these sites
formed dimers in SDS-PAGE under nonreducing conditions. In the C25S/C278S double mutant, dimer
bands also occurred and were changed to monomers
by reduction. Therefore, an inter-protomer disulﬁdebridge between Cys106 and Cys183 is also involvedin homodimer formation (Fig. 4C).
Mutation of Cys25 in N terminal domain to Ser did
not prevent activation in coexpression with wild-type
P2Y2R. If molecules forming homodimers are equivalently expressed, the ratio of homodimer formation
with wild-type P2Y2R is approximately half; however,
the activity was less than half. Mutation of Cys278 in
the third extracellular loop to Ser attenuated activity,
but some was maintained. Therefore, Cys278 seems tobe less important for monomer and homodimer
formation. The C25S/C278S double mutant lost477

M. Abe et al.

UTP ( M)
2.5
**

2.0

Fold induction( -galactosidase activity)

*
**

1.5

B

****
**

1.0
0.5
0.0

P2Y2R-

C

0
1
10
100

6

WT

C25S/
C25S C106S C183S C278S
C278S

0
1
10
100

4

****

****

***

***

2

0
WT

WT

0
1
10
100

****

2

P2Y2R

WT

WT

P2Y2R-WT

C25S C106S C183S C278S

***
**

0
P2Y2R None

None

P2Y2R-C25S/
C25S C106S C183S C278S
C278S

WT

None

None

UTP ( M)
2.5 ****
2.0

None

None

0
1
10
100

****

1.5
1.0
0.5
0.0

WT

P2Y2R

WT

C278S

C25S/
C278S

C25S/
C278S

P2Y2R-

C25S

C25S

C25S

WT

****

1

D

UTP ( M)****

UTP ( M)
3

Fold Induction( -galactosidase activity)

Fold Induction( -galactosidase activity)****

Fold Induction( -galactosidase activity)

A

Fig. 2 Difference in reaction of Cys to Ser mutants with and without wild-type P2Y2R. P2Y2R-a or mutant-a in HEK293T cells with endogenous wild-type P2Y2R (A) and in 1321N1 cells that do not express wild-type P2Y2R (BD). Wild-type or mutant P2Y2R was coexpressedwith arrestin-o and stimulated by UTP, after which arrestin recruitment activity was determined by an a complementary reaction. Activity isexpressed as a multiple of the control activity measured without the reagents. n = 3. ****P50.0001; ***P50.001; **P50. 01; *P50. 05.

Fig. 3 Intracellular Ca2+ changes for wild-type and C25S P2Y2R in 1321N1 cells after UTP addition. Wild-type (A) or C25S (B) P2Y2R was
expressed in 1321N1 cells and 15 cells each were stimulated by 10 mM UTP, followed by stimulation with 1 mM BK. C106S and C183S mutants
gave similar results to those for C25S P2Y2R. (C) Non-transfected 1321N1 cells were stimulated as above and served as controls (n = 21).

478

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P2Y2 receptor homodimer via disulﬁde bridges

A

B
25

25 -S-S- 278

106 -S-S- 183

278

S
S

S
S
106 -S-S- 183278

183 -S-S- 106

25

25

106

183

278

S
S

S
S

S
S

S
S

278

183

106

25

C

Fig. 4 Proposed disulﬁde bridges in P2Y2R monomers and homodimers. In monomers, similarly to rhodopsin, Cys25 and Cys278, and Cys106
and Cys183 are likely to be bridged (A). When homodimers are formed, Cys25 is bridged with Cys278 in the other protomer. Consequently, it ispossible that bridges between Cys106 and Cys183 occur within one protomer (B) and between two protomers (C).

activity, but this was recovered in coexpression
with wild-type P2Y2R. These results are likely to be
due to homodimer formation and thus support theconclusion that Cys106 and Cys183 are involved indisulﬁde bridges with wild-type P2Y2R; i.e. they are
involved in bridging between molecules. Therefore, a
Cys106-Cys183 intra- or interprotomer disulﬁde bondis essential for receptor expression on the cell surface
and activation (Fig. 4). This hypothesis is also supported by the activation of C25S-a in the presence of
C278S P2Y2R (Fig. 2D). Signals were transduced from
C278S to C25S P2Y2R but not from C25S/C278S
P2Y2R (Fig. 2D). This suggest that Cys25 andCys278 form a disulﬁde bond, but that this disulﬁdebond is not essential for homodimer formation, giventhat signals were transduced from wild-type P2Y2R to
the C25S/C278S mutant (Fig. 2A and C).
In COS7 cells, most P2Y2R molecules are found in
fractions corresponding to lipid raft components (12).
The results of this study showed that most P2Y2R
molecules also fractionated to lipid raft components
in HEK293T cells. Only some of these molecules
formed dimers, which shows that dimer formation
has no relationship with distribution of P2Y2R toraft fractions. Also, in the presence of wild-type
P2Y2R, Cys mutants had no effect on raft distribution.
Formation of B2R homodimers with disulﬁde bonds
is promoted by agonist binding (2). However, no agonist was necessary for P2Y2R to form homodimers.
P2Y2R shows cross-reaction with B2R (9) and crosstalk with b2-adrenergic receptor (6), CXC chemokine
receptor 2 (7), and A1R (8). Thus, P2Y2R may form
heteromers with other GPCRs, but it is unknown how
homodimer formation is related to the occurrence ofheteromers. There were many smear bands up to
100 kDa in non-reducing SDS-PAGE (Fig. 1B).
Bands above 100 kDa often appear in non-reducing
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SDS-PAGE, and some of those seen in Fig. 1B may
be homo-oligomers or hetero-oligomers with other
GPCRs.
Formation of a dimer or oligomer greatly affects thebinding kinetics, signal enhancement and termination
thereof, as it regulates the density of molecules
involved in GPCR signal transduction (1). The monomer and dimer are rapidly interconvertible (23), andinvolvement of a disulﬁde bridge is likely to prolong
the presence of the dimer.

Funding
This study was supported by the Strategic Study Base
Formation Support Project for Private Universities,
Japan.
Conflict of Interest
None declared.

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cro

ARTICLEA complex between phosphatidylinositol 4-kinase II␣ andintegrin ␣3␤1 is required for N-glycan sialylation incancer cells
Received for publication, August 19, 2018, and in revised form, January 8, 2019 Published, Papers in Press, January 18, 2019, DOI 10.1074/jbc.RA118.005208

Tomoya Isaji‡1, Sanghun Im‡1, X Akihiko Kameyama§, Yuqin Wang¶, Tomohiko Fukuda‡, and Jianguo Gu‡2
From the ‡Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai Miyagi 981-8558, Japan, the §Department of Life Science andBiotechnology, National Institute of Advanced Industrial Science and Technology, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568,
Japan, and the ¶Department of Pharmacology, Pharmacy College, Nantong University, Nantong, Jiangsu 226001, China

Aberrant N-glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic
potential, which includes cell adhesion, migration, and growth.
Recently, phosphatidylinositol 4-kinase II␣ (PI4KII␣), which is
localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KII␣ andhelps anchor sialyltransferases and thereby regulates sialylationof cell surface receptors. However, how PI4KII␣-mediated sialyation of cell surface proteins is regulated remains unclear. In
this study, using several cell lines, CRISPR/Cas9-based geneknockout and short hairpin RNA–mediated silencing, RT-PCR,
lentivirus-mediated overexpression, and immunoblotting methods, we confirmed that PI4KII␣ knockdown suppresses the sialylation of N-glycans on the cell surface, in Akt phosphorylation
and activation, and integrin ␣3-mediated cell migration of
MDA-MB-231 breast cancer cells. Interestingly, both integrin␣3␤1 and PI4KII␣ co-localized to the trans-Golgi network,where they physically interacted with each other, and PI4KII␣
specifically associated with integrin ␣3 but not ␣5. Furthermore, overexpression of both integrin ␣3␤1 and PI4KII␣
induced hypersialylation. Conversely, integrin ␣3 knockout significantly inhibited the sialylation of membrane proteins, suchas the epidermal growth factor receptor, as well as in total celllysates. These findings suggest that the malignant phenotype ofcancer cells is affected by a sialylation mechanism that is regulated by a complex between PI4KII␣ and integrin ␣3␤1.

Alteration of the sialylation of glycoproteins has often been
observed in several types of malignant tumors, such as thosefound in breast, ovary, and colorectal cancers (1–3). Sialylation
is linked via either an ␣2,3 or an ␣2,6 bond to Gal/GalNAc andvia an ␣2,8 bond to sialic acid in glycoproteins through a groupof sialyltransferases. The up-regulation of cell surface sialic acidis thought to control cell phenotypes such as cell adhesion,
migration, immune response, apoptosis, and cell epithelial–mesenchymal transition (EMT).3 Sialylation is also believed tobe essential for the differentiation potential of human mesenchymal stem cells (4, 5). The sialylation levels of glycoproteinson the cell surface are mainly determined by sialyltransferase,sialidase, and substrate expression (6, 7). Some transcription
factors are critical for transcriptional activation of sialytransferases in cancer cells. For example, the expression level of␣2,6-galactoside sialyltransferase 1 (ST6GAL1) is up-regulatedby the RAS oncogene and increases the ␣2,6 sialylation of ␤1
integrins, which promotes integrin-mediated cell migration,adhesion, and cell proliferation (8, 9).
As described above, sialylation levels are mainly dependent
on their gene expression levels, whereas other mechanisms forregulation are usually neglected. However, Popoff and co-workers(10) and our group (11) independently reported that Golgi phosphoprotein 3 (GOLPH3), which has been identified as an oncogenic protein and is increased in several human solid tumors (12),could anchor sialyltransferases to regulate sialylation on cell surface receptors without regulating the gene expression levels of sialyltransferases. In particular, suppression of GOLPH3 attenuatedthe levels of cellular sialylation and integrin-dependent cell migration. Furthermore, tumor formation was significantly reduced inmice implanted with GOLPH3 shRNA-expressing cells (11–13).
GOLPH3 has multiple cellular functions in vesicle trafficking andin support of Golgi apparatus structure, which has specific affinity
for phosphatidylinositol 4 (PI4P) (14, 15). PI4P is mainly catalyzed
by phosphatidylinositol 4-kinase II␣ (PI4KII␣), which is localized
to the trans-Golgi network (TGN) (16). Given the importance of3This work was supported in part by Grants-in-Aid for Scientific Research
15H04354 (to J. G.) and JP17H03808 (to A. K.) from the Japan Society for the
Promotion of Science, National Natural Science Foundation of China Grant
31670807, and Grant-in-Aid for Scientific Research on Innovative Areas
18H04868 (to J. G.) from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan. The authors declare that they have no conflictsof interest with the contents of this article.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed. Tel.: 81-22-727-0216; Fax:
81-22-727-007; E-mail: jgu@tohoku-mpu.ac.jp.

The abbreviations used are: EMT, epithelial–mesenchymal transition;
shRNA, short hairpin RNA; PI4P, phosphatidylinositol 4-phosphate; PI4K,
phosphatidylinositol 4-kinase; TGN, trans-Golgi network; ECM, extracellular matrix; EGF, epidermal growth factor; PH, pleckstrin homology; DOX,
doxycycline; KD, knockdown; SNA, Sambucus nigra agglutinin; WGA,
wheat germ agglutinin; PA, pyridilaminated; SSA, Sambucus sieboldiana
agglutinin; MAM, Maackia amurensis mitogen; ConA, concanavalin A; KO,knockout; EGFR, epidermal growth factor receptor; DMEM, Dulbecco’s
modified Eagle’s medium; FBS, fetal bovine serum; cDNA, complementary
DNA; mRFP, monomeric red fluorescent protein; SALSA, sialic acid linkage–
specific alkylamidation; Ctrl, control.

J. Biol. Chem. (2019) 294(12) 4425–4436© 2019 Isaji et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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Importance of the PI4K-integrin ␣3␤1 complex for sialylation

Results
We recently reported that the expression of GOLPH3 upregulated cell surface sialylation and cell migration (11).
However, the molecular mechanism of posttranslational
modification of sialylation on the cell surface remains unclear. Considering that GOLPH3 exhibits a highly selective
affinity for PI4P that is similar to the canonical pleckstrin
homology (PH) domain in the TGN (33), we hypothesized thatthe expression of PI4P in the TGN could affect sialylation andcell properties. PI4P is produced mainly by PI4Ks, which areclassified as type II and III according to their sensitivity toinhibitors (30). PI4KIII␤ is enriched in early Golgi compartments, whereas PI4KII␣ is localized mainly in the TGN and in
the endosome (16), where sialyltransferases also are localized(34). Furthermore, PI4KII␣ is a dominant PI4K in mammaliancells (35). Therefore, we chose PI4KII␣ for functional analysis,4426 J. Biol. Chem. (2019) 294(12) 4425–4436

and established a line of doxycycline (DOX)– controlled
PI4KII␣ silencing in MDA-MB-231 breast cancer cells.
Knockdown of PI4KII␣ affected the sialylation of N-glycans
First, we verified the expression levels of PI4KII␣ and sialyltransferases (ST3GAL3, ST3GAL4, and ST6GAL1) that involve
major sialylated N-glycans (36). RT-PCR analysis showed thatthe expression levels of PI4KII␣ mRNA were significantly decreased in knockdown cells, whereas the expression levels of
ST6GAL1 for ␣2,6 sialylation and those of ST3GAL3 andST3GAL4 for ␣2,3 sialylation were all similar in both cells (Fig.
1A). Second, we verified the expression levels and the distributions of PI4P in KD cells. The PH domain of four-phosphate
adaptor protein 1 (FAPP1), which preferentially recognizes
PI4P, is known for its use as a monitor for PI4P distribution(37). In control cells, PI4P was localized near the medial Golgi
marker GM130, as expected (Fig. 1B). In contrast, PI4P staining
was significantly decreased in knockdown (KD) cells (Fig. 1B).
These data confirmed that PI4KII␣ is mainly involved in thesynthesis and localization of PI4P in the TGN, which agrees
with previous reports (35).
Next, we tested whether decreased PI4P affects sialylation
expression on the cell surface. As shown in Fig. 1C, top panels,
the intensities of the staining in KD cells with Sambucus nigra
agglutinin (SNA) lectin, which preferentially recognizes ␣2,6
sialylation of glycoproteins, was weaker than that in control
cells. However, the intensities of staining with WGA lectin,
which preferentially recognizes GlcNAc residues and hybridtype and lactohexose N-glycans (38), were almost equal in both
cells (Fig. 1C, bottom panels). These data suggested that theexpression levels of sialylated glycans were decreased in KD
cells. Furthermore, the decrease was confirmed by quantitative
analysis using HPLC. Total pyridylaminated (PA) N-glycans ofcells were examined via anion exchange chromatography, andthe ratios of sialylated N-glycans versus total N-glycans were
calculated. The expression levels of sialylated N-glycans weresignificantly suppressed in KD cells (Fig. 1D). A similar changein ␣2,6 sialylation on specific membrane proteins such as integrin ␤1 was also consistently observed (Fig. 1E) using Sambucus
sieboldiana agglutinin (SSA)–agarose. These data suggest thatthe expression levels of PI4P in the TGN are important for
sialylation.
Knockdown of PI4KII␣ significantly inhibited cell migration
and Akt phosphorylation
Alterations in N-glycosylation affect many cellular events
involved in cellular signaling and cell migration. Here we examined the effects of PI4KII␣ KD on cell migration and Akt activation. As shown in Fig. 2A, cell migration on laminin-332 in a
Transwell assay was significantly reduced in KD cells comparedwith control cells. The expression levels of integrin ␣3␤1, a
major receptor for laminin-332 and ␣5␤1 on the cell surface
were similar in both cells (Fig. 2B). Activation of Akt is requiredfor integrin-mediated cell migration (39). As shown in Fig. 2C,
the level of phosphorylated Akt was apparently decreased in KD
cells compared with control cells. These data suggest that PI4P
in the TGN plays an important role in Akt activation and inintegrin ␣3–mediated cell migration.

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GOLPH3 in sialylation and cell functions, it is plausible that PI4P
expression at the TGN may influence sialylation.
Integrins are heterodimeric cell surface adhesion receptors
and major carriers of sialyation. The interaction between integrin and the extracellular matrix (ECM) is essential for cell adhesion, migration, viability, and proliferation (17, 18). In fact, glycosylation is a key regulator that plays important roles inmodulating integrin functions. For example, integrin ␣5␤1
binding to fibronectin and integrin-mediated cell spreading
and migration are modulated by overexpression of glycosyltransferase genes such as N-acetylglucosaminyltransferase III
and V or ST6GAL1 (19 –21). In addition, sialylated N-glycans
on the membrane-proximal domain of integrin ␤1 play crucial
roles in integrin activation and in complex formation between
integrin and EGF receptors and syndecan-4 to regulate cell
migration and proliferation (22).
In contrast to the regulation of integrin functions from theextracellular domain, it is well known that integrin function isregulated by its association with cytoplasmic molecules such as
focal adhesion kinase and phosphatidylinositol 3-kinase (23,24). Other studies have detected PI4K activity in the immune
complex with integrin ␤1, suggesting that integrin might also
regulate the biosynthesis of PI4P (25–27). Mammalian PI4Ks
are classified as types II and III (28). PI4KII␣ plays importantroles in clathrin-dependent molecular sorting and associateswith TGN membranes (29 –31), whereas PI4KIII␤ is enrichedin the cis-medial Golgi in breast cancer cells (32).
In this study, to further understand the underlying mechanism for GOLPH3 expression on sialylation and cell functions,
we investigated the effects of PI4KII␣, which is one of the regulators of GOLPH3 in breast cancer MDA-MB-231 cells. We
found that the sialylation on integrins, Akt phosphorylation,and integrin ␣3-mediated cell migration all were significantly
inhibited in PI4KII␣ knockdown cells. It was interesting thatoverexpression of both PI4KII␣ and integrin ␣3 greatly
increased sialylation. Conversely, knockout of integrin ␣3 significantly inhibited sialylation in membrane proteins. Thesefindings suggest a novel mechanism for sialylation, which suggests a new concept for the regulation of glycosylation in cellbiology.

Importance of the PI4K-integrin ␣3␤1 complex for sialylation

Complex formation between integrin ␣3 and PI4KII␣ isimportant for sialylation
As described above, PI4KII␣ regulated sialylation and theintegrin ␣3–mediated phenotype. Therefore, we examined theinteraction between PI4KII␣ and integrin ␣3 in the Golgi apparatus. First, we used immunostaining to assess the intracellular
distributions of PI4KII␣ and ␣3. Immunostaining showed that
PI4KII␣ was localized in the vicinity of GM130 and that it was
extensively co-localized with integrin ␣3 (Fig. 3A). Notably, coimmunoprecipitation with anti-integrin ␤1 showed that it was
␣3, rather than ␣5, that interacted with PI4KII␣ in 293T cellstable expression of PI4KII␣ and/or either integrin ␣3 or ␣5(Fig. 3B). The interaction between PI4KII␣ and ␣3 was further
reciprocally confirmed by immunoprecipitation with either
anti-␣3 antibody (Fig. 3C) or anti-GFP antibody (Fig. 3D).
Next we wondered whether this interaction was required for
efficient sialylation. To address this question, we precipitatedthose cell lysates with SSA or Maackia amurensis mitogen(MAM) agarose, which preferentially recognize ␣2,6-sialylatedand ␣2,3-sialylated N-glycans, respectively, and then performed
Western blotting against integrin ␤1. Interestingly, both sialylated
N-glycans were up-regulated on integrin ␤1 in cells that
expressed both PI4KII␣ and integrin ␣3 but not in those that
expressed PI4KII␣ and ␣5 (Fig. 3E, top and center panels). In
contrast to SSA or MAM lectin blotting, ConA lectin blots
showed a similar level in both integrin ␣3- and ␣5-expressing
cells (Fig. 3E, bottom panel). ConA lectin equally recognizedboth mature and immature forms of integrin ␤1 in ␣3-expressing cells but only the most immature form of ␤1 in ␣5-expressing cells. These data further suggest the importance of ␣3 forsialylation. Data obtained from flow cytometry analysis via
MAM lectin consistently supported this observation, although
SSA lectin showed no significant changes in these cells (Fig. 3F).
Therefore, these results clearly suggest that intracellular comJ. Biol. Chem. (2019) 294(12) 4425–4436

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Figure 1. Effects of PI4KII␣ gene knockdown on the sialylation of N-glycans. A, RT-PCR using total RNA extracted from the control (Ctrl) and PI4KII␣ KD of
MDA-MB-231 cells were carried out to examine the expression levels of PI4KII␣ and sialyltransferases that involve sialylated N-glycans. The expression level ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ST3GAL, ␤-galactoside ␣2,3-sialyltransferase. B, the amounts and distribution of PI4P in KD and Ctrl cells were examined by expressing mRFP-FAPP, which specifically binds to PI4P. These cells were also immunostained with
anti-GM130 antibody, which is a medial Golgi marker. The immunostaining intensities for PI4P were quantified using ImageJ software, averaged by cellnumbers, and normalized to the control values (percent). Values represent the means ⫾ S.E. (n ⫽ 10). **, p ⬍ 0.01 (Welch’s t test). Scale bar ⫽ 10 ␮m. C, Cells wereincubated with biotin-conjugated SNA or WGA and then fixed and visualized by Alexa Fluor–streptavidin conjugates. Scale bar ⫽ 20 ␮m. D, preparation of PA
oligosaccharides and calculation of ratios of sialylated N-glycans versus total N-glycans as described under “Experimental procedures.” Data were normalized
to the control. E, equal amounts of cell lysates from Ctrl and KD cells were precipitated by SSA-agarose (preferentially recognizes the ␣2,6 sialylation) andprobed with anti-␤1 antibody (top panel). The expression levels of integrin ␤1 in total cell lysates were verified by Western blotting with anti-␤1 antibody (center
panel). ␣-Tubulin was used as a loading control. KD and Ctrl cells refer to transfectants with DOX-inducible knockdown of PI4KII␣ cultured with or without DOX,
respectively. IB, immunoblot.

Importance of the PI4K-integrin ␣3␤1 complex for sialylation

plex formation between PI4KII␣ and ␣3 regulates the biosynthesis of sialylated N-glycans.
Expression of integrin ␣3 is important for efficient sialylation
To further verify the importance of complex formation
between PI4KII␣ and integrin ␣3 for cellular sialylation, weused the CRISPR/Cas9 system to construct an integrin ␣3
knockout (KO) MDA-MB-231 cell line. Flow cytometric analysis revealed a KO efficiency of more than 95% (Fig. 4A), which
also was confirmed by Western blotting with anti-integrin ␣3(Fig. 4B). Notably, the band mobility of integrin ␤1 on SDSPAGE in KO cells was faster than in the control cells, suggestingthat integrin ␣3 deficiency leads to an accumulation of theimmature ␤1 form without sialylation. It is well known that
integrin ␣3 is important for laminin-mediated cell adhesion,
migration, and several forms of cellular signaling (40 –42). Consistent with previous reports (42, 43), expression of ␣3␤1
regulated cell morphology and that of the actin cytoskeleton
by promoting lamellipodium and filopodium formation onlaminin-332, a specific ligand for integrin ␣3, whereas lack of ␣3
blocked their formation (Fig. 4C). There were no significant
differences between two cells spread on collagen, which is a
specific ligand for integrin ␣1 and ␣2. Furthermore, cell spreading and migration on laminin-332 were inhibited in KO cells
compared with control cells (Fig. 4, D and E).
Next, we compared total sialylated N-glycan levels between
KO and control cells, which were quantified using HPLC. Consistent with the data from PI4KII␣ KD cells, the ratios of sialylated N-glycans versus total N-glycans were greatly suppressedin KO cells (Fig. 5A). Because N-glycans of integrin ␤1 could bedirectly influenced by ␣3 knockout (Fig. 4B, center panel), here
we also analyzed sialylation on the EGF receptor, which has
been reported to affect receptor activation (44). As expected,

4428 J. Biol. Chem. (2019) 294(12) 4425–4436

sialylation of EGF receptors obtained from KO cells was also
decreased compared with control cells (Fig. 5B). To investigate
whether the phenomenon also occurred in other cell lines,
we examined HeLa cells. The knockout efficiency of ␣3 wasassessed via flow cytometry analysis (Fig. 5C). Interestingly, asimilar tendency of sialylation decline was observed in ␣3 KO
HeLa cells compared with control cells (Fig. 5D). To thoroughly
examine the glycosylation states caused by knockout of integrin
␣3 or knockdown of PI4KII␣, we utilized MS-based approaches
to identify the N-glycan and O-glycan structures (Fig. 6, A and
B). Consistently, hyposialylation in N-glycans was observed byattenuation of either integrin ␣3 or PI4KII␣ (Fig. 6A), whichfurther suggests the importance of this pathway in the biosynthesis of sialylated N-glycans. In contrast to N-glycans, there
were no significant changes in the sialylation of O-glycans
among these cells (Fig. 6B). It was also notable that a decrease in␣2,3 sialylation in N-glycans could slightly increase ␣2,6 sialylation in total cell lysates of ␣3 KO and PI4KII␣ KD cells (Fig.
6A), which seems contrary to the observation for integrin ␤1(Fig. 3E). Further study is needed to fully elucidate the mechanism. Nevertheless, these data suggest that complex formationof ␣3 and PI4KII␣ could act as a novel regulator for the sialylation of N-glycans.

Discussion
In this study, we found that PI4KII␣ expression played an
important role in ␣3␤1 integrin-mediated cell migration, cellular signaling, and the expression of sialylation; PI4KII␣ could
specifically associate with ␣3 but not ␣5, and complex formation between PI4KII␣ and ␣3 enhanced sialylation; and deletionof integrin ␣3 significantly inhibited not only cell adhesion andmigration but also sialylation. These observations are the firstto directly demonstrate a novel regulatory mechanism for sia-

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Figure 2. Comparison of cell migration and cellular signaling between Ctrl and KD cells. A, cell migration toward laminin-332 was determined by aTranswell assay as described under “Experimental procedures.” The quantitative data were obtained by counting the cell number of five random fields. The pvalues were calculated using Welch’s t test. Values represent the means ⫾ S.E. **, p ⬍ 0.01. B, comparison of the expression levels of ␣3␤1 and ␣5␤1 integrins
on the cell surface between Ctrl and KD cells. Both Ctrl and PI4KII␣ KD cells were incubated with (bold line) or without (gray shadow) antibodies against integrin
␣3, ␣5, or ␤1 and subjected to FACS analysis. C, comparison of Akt activation upon FBS stimulation between Ctrl and KD cells. After starvation, cells were
supplied with fresh medium with (ⴙ) or without (⫺) 3% FBS. The cell lysates were then analyzed by Western blotting with anti-p-Akt or total Akt antibody.

Importance of the PI4K-integrin ␣3␤1 complex for sialylation

lylation, which may also partially explain the previous observation that GOLPH3 is a special regulator in the sialylation of
N-glycans and a part of the signaling events that could influence
mTOR signaling and tumor progression (11).
Many cancers are associated with sialylated structures suchas sialyl Tn, sialyl Lewis antigen (sLe), ␣2,6-sialylated lactosamine, polysialic acid, and gangliosides (45–48). The alteredexpression of these structures in cancer cells could be the resultof multiple mechanisms. Loss of expression or excessive expression of certain sialyltransferases is frequently observed. For
example, up-regulated expression of ␣2,3 and ␣2,6 sialyltransferases has been observed in many cancers, such as colon cancer, breast cancer, liver cancer, cervical cancer, choriocarcinomas, acute myeloid leukemias, and some malignancies of thebrain (49), and this type of change can affect the structures andfunctions of some important target N-glycoproteins such as
integrin ␤1(50), EGFR (44), and platelet endothelial cell adhesion molecule (51).
Recent studies have suggested that activation of the EMT
programs serves as a major mechanism for generating cancer

stem cells (52). Interestingly, high expression of ST6GAL1 has
been correlated with human-induced pluripotent stem cells
and cancer stem cells, indicating that sialylation may be involved in maintaining some aspects of stem cell behavior (4, 5,
53). In fact, ST6GAL1 expression is required for transforming
growth factor ␤–induced EMT. Knockdown of ST6GAL1 prevented a transforming growth factor ␤–induced increase in cell
migration (54).
Given the accumulating evidence of the importance of sialylation in cancer progression, much attention has been paid
to elucidating the regulatory mechanisms of its expression. The
expression of sialylation on a tumor cell surface can be modulated at different levels (49). The most frequently observed
mechanism is modulation of transcription. For example,
ST6GAL1 expression is induced by the ras oncogene in
NIH3T3 cells via its promoter (8). Beyond its promoter activity,in this study, we clearly showed that sialylation could also be
regulated by complex formation of PI4KII␣ and integrin ␣3␤1
on a posttranscriptional level. In fact, the PI4P-binding Golgi
protein GOLPH3, which functions in secretory trafficking in
J. Biol. Chem. (2019) 294(12) 4425–4436

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Figure 3. Interaction between integrin ␣3␤1 and PI4KII␣ and the effect on sialylation. A, MDA-MB-231 cells overexpressed with GFP-PI4KII␣ were fixed
and then stained with either anti-GM130 (top panel) or anti-integrin ␣3 (bottom panel) antibody, followed by detection using anti-mouse Alexa 647 conjugates.
Scale bar ⫽ 20 ␮m. B, three overexpressed 293T cells were established by lentivirus infection: PI4KII␣ alone; PI4KII␣, GFP-tagged ␣3 and ␤1; and PI4KII␣,GFP-tagged ␣5 and ␤1 integrin. Equal amounts of cell lysates were immunoprecipitated (IP) with anti-integrin ␤1 antibody, followed by detection with theindicated antibodies. The cell lysates were used as input to show similar expression levels of PI4KII␣, integrin ␣3␤1, or ␣5␤1 among these cells. C and D,
reciprocal immunoprecipitation was performed using either anti-␣3 antibody (C) or anti-GFP for tagged PI4KII␣ (D) using cell lysates from 293T cells stably
overexpressed with or without GFP tagged PI4KII␣, which were further transiently transfected with or without ␣3 and ␤1 integrin. The cell lysates were used
as input to show similar expression levels of integrin ␣3 and GFP-PI4KII␣. E, equal amounts of cell lysates were precipitated with either SSA-, MAM-, or
ConA-conjugated agarose, followed by detection with antibody against integrin ␤1. F, 293T cells expressing the indicated genes were incubated with (boldline) or without (gray shading) biotin-conjugated MAM or SNA and subjected to FACS analysis.

Importance of the PI4K-integrin ␣3␤1 complex for sialylation

Figure 5. Deficiency of integrin ␣3 decreased sialylation of N-glycans indifferent cell lines. A, N-glycans released from both Ctrl and ␣3 KO MDA-MB231 cells were pyridylaminated and verified as populations of sialylated
N-glycans. The relative ratios of sialylated N-glycans versus total N-glycans
were calculated by subtracting the neutral portions from the total and thendividing by the total. The control data were set as 1. B, equal amounts of celllysates for control and ␣3 knockout cells were precipitated via SSA-agaroseand probed with anti-EGFR antibody (top panel). The same cell lysates werealso probed with anti-EGFR antibody as a loading control (bottom panel). IP,
immunoprecipitation. C, ␣3 KO of HeLa cells was established using the
CRISPR/Cas9 system and verified via flow cytometric analysis. D, the ratios of
sialylated N-glycans were analyzed as described in A.

the Golgi, is also important for the localization of several glycosyltransferases (10, 55, 56). Our previous study showed thatknockdown of GOLPH3 leads to down-regulation of both ␣2,3
and ␣2,6 sialylation but has no effect on the transcription of

4430 J. Biol. Chem. (2019) 294(12) 4425–4436

sialyltransferases. Therefore, regulation of biosynthetic glycan
is not only dependent on the expression levels of glycosyltransferases but also on those of substrates, chaperones, and on theenvironment of the Golgi apparatus.
Considering that PI4P is a relatively abundant phosphoinositide that is required for the maintenance and function of
the Golgi apparatus, which includes intracellular trafficking,it is plausible that PI4K could be involved in membranetransport from the trans-Golgi network to the plasma membrane. In fact, PI4KII␣ is known to associate with several
cellular receptors such as EGFR, E-cadherin, LDL receptorrelated protein, and the Fas receptor (57–60). In addition,
alteration of the N-glycosylation of such receptors also regulates the functions of these receptors. For example, N-glycosylation is required for EGFR trafficking and has an effecton its endocytosis (61, 62). N-glycans of the LRP ectodomain
also regulate the conformation and bending angle of thereceptor (63). Cell surface sialylation protects Fas ligand–induced apoptosis by modification of the Fas ligand receptor(64), which could partially explain why enhancement of apoptosis has been observed in PI4KII␣ knockdown cells (60).
Thus, the various phenotypes associated with PI4P andmembrane proteins might be partially due to alteration of
glycosylation.
The expression level of integrin ␤1 is negatively correlatedwith the survival rates of patients with invasive breast cancer(65, 66), particularly ␣3␤1 expression in breast carcinoma associated with metastasis (41). During tumor progression, PI4KII␣
is also significantly up-regulated along with tumor growth compared with corresponding normal tissue (67). Given the importance of sialylation as described here, it is reasonable to expect
up-regulation of both ␣3␤1 and PI4KII␣ in cancer cells.

Downloaded from http://www.jbc.org/ at Tohoku Medical and Pharmaceutical University on April 22, 2019

Figure 4. Effects of integrin ␣3 deficiency on cell spreading and migration. Establishment of integrin ␣3 KO MDA-MB-231 cells using the CRISPR/Cas9
system is described under “Experimental procedures.” The efficiency of ␣3 KO was assessed via flow cytometric analysis (A) and Western blotting (B). A, Both ␣3
KO and Ctrl cells were stained with (bold line) or without (gray shading) anti-␣3 or ␤1 integrin antibody and subjected to FACS analysis. B, the same amount ofcell lysate was subjected to Western blotting to detect the indicated antibodies. C, cells were replated on coverslips coated with the indicated ECM, followed
by incubation for 30 min, and were then fixed and stained with Alexa Fluor– conjugated phalloidin. Representative images are shown. Scale bar ⫽ 10 ␮m. D,
cells were detached and then replated on laminin 332-coated dishes. After incubation for 15 min, the cells were fixed. The ratios of cell spreading were
calculated. Cell spreading was defined as a cell spread of more than 0.025 ␮m2. Values represent the means ⫾ S.E. (n ⫽ 5). *, p ⬍ 0.05 (Welch’s t test). E, cell
migration toward laminin-332 was determined using a Transwell assay. Scale bar ⫽ 100 ␮m. The quantitative data were obtained from three independent
experiments. Values represent the means ⫾ S.E. (n ⫽ 6). *, p ⬍ 0.05 (Welch’s t test).

Importance of the PI4K-integrin ␣3␤1 complex for sialylation

These facts raise the question of why the interaction of
PI4KII␣ with integrin ␣3␤1 but not of ␣5␤1 regulates sialylation. So far, the association underlying the mechanism between
␣3␤1 and PI4KII␣ remains unclear. However, the specificity of␣3␤1 could be due to its interaction with the tetraspanin family,such as CD151, CD63, and CD9. In fact, other studies havereported that integrin ␣3␤1 and the tetraspanin family caninteract with type II of PI4K (25–27). In addition, palmitoylation could also be a plausible factor because PI4Ks are proteins
with membrane association and activity that are highly dependent on such a modification (68, 69), and palmitoylation also

plays an important role in the association between integrin andseveral molecules, such as tetraspanin and c-Met (70, 71). We
assume that the association between PI4KII␣ and ␣3, but ␣5,
might also relate to tetraspanin or palmitoylation.
It is noteworthy that both cell spreading and migration onlaminin 332 of integrin ␣3 KO cells were significantly suppressed (Fig. 4), which further supports the notion that ␣3␤1 isa major receptor of lamin-332 compared with integrin ␣6␤1
and ␣6␤4 in epithelial cells (43, 72). Accumulating evidenceshows that integrin ␣3␤1 is important for tumor metastasis in
human breast and prostate cancer cell lines (40, 41) and sialyJ. Biol. Chem. (2019) 294(12) 4425–4436

4431

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Figure 6. MS spectra of glycans obtained from cultured cells. A, MS spectra of N-linked glycans. The reducing ends of the N-linked glycans were derivatizedwith aoWR (86). The sialic acids of the N-linked glycans were differentially amidated with methylamine (⫹13.0 Da) for ␣2,3-sialic acids and isopropylamine(⫹41.1 Da) for ␣2,6-sialic acids via the SALSA method (87). MDA-MB-231 and ␣3 KO cells refer to parent and ␣3 knockout cells, respectively. KD and control cellsof MDA-MB-231 cell line refer to transfectants with DOX-inducible knockdown of PI4KII␣ cultured with or without DOX, respectively. B, MS spectra of O-linked
glycans. The O-linked glycans were obtained as the corresponding alditols and then permethylated.

Importance of the PI4K-integrin ␣3␤1 complex for sialylation

Experimental procedures
Cell lines and cell culture
The HeLa and 293T cells were obtained from the RIKEN
Bioresource Research Center (Japan). The MDA-MB-231 cells
were purchased from the ATCC. All cell lines were maintained
at 37 °C in DMEM containing 10% FBS in a humidified atmosphere of 5% CO2.
PCR for mRNA expression analysis
Total RNA was prepared with TRIzol (Invitrogen), and 1.0
␮g of RNA was reverse-transcribed using the PrimeScript RT
reagent kit (Takara Bio Inc.) according to the manufacturer’s
instructions. PCR primers against ST3GAL3, ST3GAL4,ST6GAL1, and GAPDH have been described previously (54).
The following primers were used for PI4KII␣: 5⬘-CTCCAGCGGAAGCTACTTCG-3⬘ and 5⬘-TCCACTTAGGATTAAGATGCCCA-3⬘.
shRNA-mediated silencing of PI4KII␣ in MDA-MB-231 cells
Conditional knockdown of the target gene was achieved
using the DOX-inducible CS-RfA-ETBsd lentivirus vector(RIKEN) with minor modifications (11, 54). The following
oligonucleotides were inserted into pENTR/H1/TO (Invitrogen): 5⬘-CACCAGAAGCAGAACCTCTTCCTGATGATATGTGCATCAGGAAGAGGTTCTGCTTCT-3⬘ and5⬘-AAAAAGAAGCAGAACCTCTTCCTGATGCACATATCATCAGGAAGAGGTTCTGCTTCT-3⬘ (29). The use of
LR Clonase allowed the inserted oligo to be transferred
to CS-RfA-ETBsd, which is an encoding DOX-dependenttransactivator for shRNA expression. The resultant vector
was then transfected into 293T cells with packaging plasmids of calcium phosphate for the preparation of viruses.
MDA-MB-231 cells were then infected with the obtained
viruses and selected for stable integration with 1 ␮g/ml blasticidin. shRNA-mediated silencing of PI4KII␣ was induced
by addition of 1 ␮g/ml DOX for 72 h in the established cellline, and cells cultured in DOX-free medium were used as acontrol.

4432 J. Biol. Chem. (2019) 294(12) 4425–4436

Gene introduction using the lentivirus system
The cDNA sequences for the PH domain of human FAPP1,
PI4KII␣, and integrin ␣3 were cloned from HeLa cells andinserted into pENTR vectors (pENTR/D-TOPO cloning kit,
Invitrogen). To obtain the N-terminal GFP-tagged PI4KII␣,
C-terminal mRFP-tagged FAPP1 and a C-terminal GFP-tagged
integrin ␣3, in-fusion enzyme (Clontech) was used with standard PCR protocols. The linkers for GFP-PI4KII␣, mRFPFAPP1, and ␣3-GFP were 5⬘-GGGGS-3⬘, 5⬘-KNPPVAT-3⬘(37), and 5⬘-LELKLRILQSTVPRARDPPVAT-3⬘, respectively.
The resultant cDNAs were confirmed by DNA sequencing
using an ABI Prism 3130 sequencer (Applied Biosystems Japan
Ltd., Tokyo, Japan). The subcloned cDNAs were transferred
into CSII-EF-Rfa (11) via LR Clonase (Invitrogen) for lentivirus
production. The resultant vectors (CSII-EF-GFP-PI4KII␣/mRFP-FAPP1/␣3-GFP) and the previously constructed integrins of either ␣5-GFP- or ␤1-overexpressed lentiviral vectors(CSII-EF-␣5GFP (77)/␤1 (78)) were then transfected into 293T
cells with packaging plasmids via calcium phosphate during the
preparation of viruses. Either MDA-MB-231 or 293T cells werethen infected with the obtained viruses for further experiments.
Cell migration (Boyden chamber assay)
Cell Migration was performed as described previously withminor modifications (11, 79). Each Transwell (BD BioCoat control inserts, 8.0-␮m inserts; BD Biosciences) was coated only on
the bottom side with 1 ␮g/ml laminin-332 (Oriental Yeast Co.,
Ltd.) in PBS containing 0.1% BSA at 4 °C for 24 h and thenblocked with 5% BSA in DMEM at 37 °C for 1 h. Cells weretrypsinized and suspended in DMEM containing 1% FBS. The
suspended cells were centrifuged, and the cell pellets were
resuspended in an assay medium (0.1% BSA in DMEM containing 1% FBS) and diluted to 3 ⫻ 105 cells/ml; cell viability wasconfirmed by trypan blue staining. Cell suspensions were thenadded to the Transwell. Following incubation, the membranes
were fixed with 4% paraformaldehyde and stained with 0.5%
crystal violet solution (Merck) for 24 h. After washing the Transwells with PBS, cells that migrated to the lower side werecounted using a phase-contrast microscope.
Immunostaining
Cells (1 ⫻ 105) were plated on glass coverslips (MatTek),
precoated with or without ECM for 24 h, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100
for 10 min, and blocked with PBS containing 3% BSA and 0.01%
Tween 20 (PBSBT) for 1 h. Cells were then incubated with theindicated primary antibodies (GM130, BD Biosciences, 610822;
␣3, Millipore, P1B5) at 4 °C overnight, followed by incubation
with Alexa Fluor 647– conjugated secondary antibodies (Molecular Probes) for 1 h. For lectin staining, cells on coverslips wereincubated with either biotinylated SNA lectin (Vector Laboratories, B-1305) or wheat germ agglutinin (WGA) lectin (J-Oil
Mills, J220) at 4 °C for 30 min in DMEM, fixed and blocked withPBSBT for 1 h, followed by incubation with streptavidin-conjugated Alexa Fluor 647 at 4 °C for 1 h in the dark. All samples
were mounted using Prolong Diamond Antifade mounting
medium (Molecular Probes). Images were acquired by sequential excitation using an Olympus FV1000 laser-scanning confo-

Downloaded from http://www.jbc.org/ at Tohoku Medical and Pharmaceutical University on April 22, 2019lation of adhesive molecules such as integrin ␤1, which, in turn,contributes to tumorigenesis (73). In this study, ␣3 deficiency
significantly inhibited sialylation, indicating a novel link between sialylation and this integrin, which demonstrated theimportance for both forms of functional expression. Although
the MS analysis clearly showed that the complex between
PI4KII␣ and integrin ␣3 regulates the biosynthesis of sialylation
on N-glycans but not O-glycans, we could not completely
exclude other possibilities for the regulation of N-glycan structures other than sialylation or gangliosides. In addition, it has
been reported that GOLPH3 containing a PI4P binding domain
could control Golgi localization of core 2 N-acetylglucosaminyltransferase 1 for the biosynthesis of O-glycans (74). Thus,
further studies are required to explain the underlying mechanism for different influences, such as localization of glycosyltransferase, pH environment (75), and cholesterol homeostasis(76) through this system. Taken together, this study may provide a new concept for the regulation of glycosylation and could
suggest insights for the development of cancer treatment.

Importance of the PI4K-integrin ␣3␤1 complex for sialylation
PA oligosaccharide preparation of N-glycosylation andquantitative analysis of sialylated N-glycans by anion
exchange HPLC

Cell spreading and adhesion experiment

To release N-glycans from glycoproteins, the lyophilized cellpellets (1–2 mg) were heated with 200 ␮l of anhydrous hydrazine at 100 °C for 10 h using an oil bath. Removal of hydrazine
and acetylation of N-glycans were carried out with a graphite
carbon column (81). The released N-glycans were then 2-pyridylaminated using the Pyridylamination Manual Kit (Takara
Bio Inc.), and then the excess 2-aminopyridine was removed via
phenol– chloroform extraction (81) with a gel filtration column(82). The prepared PA oligosaccharides treated with or without
neuraminidases were subjected to a HPLC system (JASCO)
equipped with a TSKgel DEAE-5PW column (7.5 ⫻ 75 mm,
Tosoh) and analyzed as described previously (19). HPLC chromatogram data were analyzed using chromNAV software(JASCO). The amounts of total and neutral N-glycans were calculated based on the peak area of neutral fraction with or without neuraminidase treatment. Relative ratios of sialylated
N-glycans versus total N-glycans were calculated by subtracting
the neutral portions from the total and then dividing by the
total. The data were normalized to the control as 1.

Coverslips were coated with 1 ␮g/ml laminin-332 or 2 mg/ml
gelatin in PBS containing 0.1% BSA at 4 °C for 24 h and thenblocked with 5% BSA in DMEM at 37% for 1 h. Cells were
replated on the coverslips in DMEM with 0.1% BSA. After incubation for 10 min, the areas of adherent cells were measured
using ImageJ. We defined cells spread at more than 0.025 ␮m2
as adherent cells. After incubation for 30 min, the cells were
fixed with 4% paraformaldehyde and stained with phalloidin–
Alexa Fluor 647 (Molecular Probes).
Immunoprecipitation and Western blotting
Immunoprecipitation was performed as described previously with minor modifications (11, 80). Briefly, cells were
gently rinsed three times with PBS at room temperature andsolubilized in cold lysis buffer A (20 mM Tris-HCl (pH 7.4), 150
mM NaCl, and 1% Brij98), which included protease inhibitors.
Protein concentrations of lysates were determined via BCA
assay (Pierce). The lysates were immunoprecipitated using
either anti-GFP-agarose (MBL), anti-integrin ␣3 antibody(Millipore, P1B5), or anti-integrin ␤1 (P5D2, DSHB) with AbCapcher Protein A-R28 (Protenova, Tokushima, Japan) for 1 h
at 4 °C and gentle rotation. After washing with lysis buffer, theimmunoprecipitates were subjected to SDS-PAGE. The proteins were probed with anti-integrin ␣3 (Santa Cruz Biotechnology, sc-6592) or anti-GFP antibody (Rockland, 600-101215) and then detected using anti-goat IgG-conjugatedhorseradish peroxidase (Santa Cruz Biotechnology) with an
Immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore). For analysis of the lectin precipitates, we used lysis buffer B (20 mM Tris-HCl (pH 7.4), 150 mM
NaCl, and 1% Triton X-100). Cell lysates were precipitated witheither SSA-agarose (J-Oil Mills, J318), MAM (also called MAL1)–agarose (J-Oil Mills, J310) or concanavalin A (ConA)–
agarose (J-Oil Mills, J303), which specifically recognize either
␣2,6- and ␣2,3-sialylation or total N-glycans, respectively. The
precipitants were detected using either anti-integrin ␤1 (BD
Biosciences, 610468) or anti-EGFR (Cell Signaling Technology,4267). Antibody agents against Akt (9272) and p-Akt (4060)
were purchased from Cell Signaling Technology. ␣-Tubulin(Sigma, T6199) was used as a loading control.
Flow cytometric analysis
Flow cytometric analysis was performed as described previously with minor modifications (11, 79, 80). Briefly, semiconfluent cells were detached from the culture dishes using trypsin
containing 1 mM EDTA. The cells were subsequently stainedwith or without primary mouse anti-␣3 (P1B5), anti-␤1 (P5D2),
and anti-␣5 integrin (Millipore, HA5) in PBS, followed by incubation with Alexa Fluor 647– conjugated secondary antibodies.
For staining cells with biotin-conjugated lectins (MAM and
SNA), we used PBS buffer containing 0.5 mM CaCl2 and MgCl2.
Flow cytometric analysis was performed using a FACSCalibur
flow cytometer and Cell Quest Pro software (BD Biosciences).

Generation of CRISPR/Cas9-based integrin ␣3 KO cells
CRISPR/Cas9-based integrin ␣3 cells were established as
described previously (83, 84). Briefly, the single guide RNAspecifying oligo sequences spanning human integrin ␣3 (forward, 5⬘-CACCGCATCGGGCACAGCGAGCTCC-3⬘; reverse,
5⬘-AAACGGAGCTCGCTGTGCCCGATGC-3) were chosenfrom the human KO library (85). Then the oligos were cloned
into pSpCas9 (BB)-2A-GFP, which was a kind gift from Dr.
Feng Zhang (Addgene plasmid ID 48138). The plasmid wastransfected into cells according to the manufacturer’s instructions (Amaxa Cell Line Nucleofector kitV). After 4 days oftransfection, GFP-positive cells were sorted using a FACSAria
II (BD Bioscience). Following 10-day culture, ␣3-negative and
GFP-negative cells were sorted, and the procedure wasrepeated twice. The KO cells were defined by flow cytometry
and Western blot analysis as described above.
Mass spectrometry of glycans
Total plasma membrane proteins of cells were preparedusing a plasma membrane protein extraction kit (101Bio). For
N-glycan analysis, a previous report (86) suggests that the pellets are dissolved and denatured and then digested by glycopeptidase F (Takara Bio Inc.). The released N-glycans were captured on hydrazide beads (BlotGlyco, 5 mg, Sumitomo BakeliteCo., Ltd.) using a process recommended by the manufacturer.
Sialic acids of the N-glycans on the beads were then differentially amidated with methylamine for ␣2,3-sialic acids and isopropylamine for ␣2,6-sialic acids using the sialic acid linkage–
specific alkylamidation (SALSA) method (87). The derivatizedN-glycans were liberated, labeled with aoWR (a component of
BlotGlyco, Sumitomo Bakelite Co., Ltd.) (86), and purified
according to the manufacturer’s instructions. The obtained glycan solutions were mixed with a 2,5-dihydroxybenzoic acidsolution (10 mg/ml in 30% acetonitrile) at a ratio of 1:10. Aliquots (0.5 ␮l) of the mixed solutions were deposited onto a
J. Biol. Chem. (2019) 294(12) 4425–4436

4433

Downloaded from http://www.jbc.org/ at Tohoku Medical and Pharmaceutical University on April 22, 2019

cal microscope with an UPlanSApo ⫻60/1.35 oil objective
operated with F10-ASW version 4.02 software.

Importance of the PI4K-integrin ␣3␤1 complex for sialylation

Author contributions—T. I., T. F., and J. G. conceptualization; T. I.,
S. I., A. K., Y. W., and J. G. data curation; T. I., S. I., A. K., Y. W., andJ. G. formal analysis; T. I. and J. G. supervision; T. I., S. I., A. K.,
Y. W., T. F., and J. G. validation; T. I., S. I., A. K., and Y. W. investigation; T. I., S. I., A. K., Y. W., T. F., and J. G. methodology; T. I. and
S. I. writing-original draft; T. I., S. I., A. K., Y. W., T. F., and J. G. writing-review and editing; S. I. visualization; A. K. and J. G. funding
acquisition.
Acknowledgments—We thank Dr. Takashi Nishikaze for informative
suggestions regarding the SALSA method and Dr. Wai Wai Thet Tin
for technical assistance with glycan analysis.

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MALDI target plate and dried. MS spectra were acquired witha MALDI quadrupole ion trap TOF mass spectrometer(AXIMA-QIT, Shimadzu Corp.). For O-glycan analysis, pelletsof the membrane fractions were dissolved in a 1% SDS solution.
Aliquots (10 ␮l) of the solution were transferred into 90 ␮l of
0.6 M sodium borohydride containing 60 mM NaOH and incubated at 45 °C for 16 h. Then the mixtures were neutralized,
diluted to 1 ml, and applied onto a solid-phase extraction cartridge (Sep-Pak C18 Vac 1cc, 50 mg, Waters Corp.), and thenthe cartridge was washed with distilled water (1 ml). The eluents and washings were combined and lyophilized. The
obtained residues were dissolved in 1% acetic acid in methanol(100 ␮l) and evaporated using a centrifuge evaporator. The procedure of dissolution and evaporation was repeated. The
obtained residues were permethylated according to previous
reports (88). The permethylated glycans were dissolved in a
2,5-dihydroxybenzoic acid solution (20 ␮l, 10 mg/ml in 30%
acetonitrile), and aliquots (0.5 ␮l) of the mixed solutions were
deposited onto a MALDI target plate and dried. MS spectra
were acquired with a MALDI-TOF mass spectrometer (Ultraflex, Bruker Daltonik).

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THE

JOURNAL• RESEARCH •www.fasebj.org

N-acetylglucosaminyltransferase-I as a novel regulatorof epithelial-mesenchymal transition
Guowei Zhang, Tomoya Isaji, Zhiwei Xu, Xu Lu, Tomohiko Fukuda, and Jianguo Gu1
Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University,
Sendai, Japan

ABSTRACT: N-Glycans are involved in numerous biologic processes, such as cell adhesion, migration, and invasion.

To distinguish the functions of complex high-mannose types of N-glycans, we used the clustered, regularly interspaced, short palindromic repeats/Cas9 system to establish N-acetylglucosaminyltransferase (GnT)-I–knockout(KO) cells. Loss of GnT-I greatly induced cell-cell adhesion and decreased cell migration. In addition, the expression
levels of epithelial-mesenchymal transition (EMT) markers such as a-SMA, vimentin, and N-cadherin were suppressed, whereas the expression of claudin-1 was promoted, suggesting a mesenchymal-epithelial transition–like
phenotype, an opposite process to the EMT, was occurred in the KO cells. The phosphorylation levels of Smad-2,
epidermal growth factor receptor, and integrin-mediated focal adhesion kinase (FAK) were consistently suppressed.
Furthermore, the restoration of GnT-I in the KO cells suppressed the cell-cell adhesion and augmented the expression of EMT markers as well as that of FAK activation. The expression levels of integrins were upregulated in
the KO cells, although their functions were decreased, whereas their expression levels were downregulated in the
rescued cells, which suggests a negative feedback loop between function and expression. Finally, we also found thatthe expression of GnT-I was important for cell survival, resistance to cancer drugs, and increased colony formation.
The results of the present study demonstrate that GnT-I works as a switch to turn on/off EMT, which further
supports the notion that on most surface receptors, the N-glycans differentially play essential roles in biologic
functions.—Zhang, G., Isaji, T., Xu, Z., Lu, X., Fukuda, T., Gu, J. N-acetylglucosaminyltransferase-I as a novel
regulator of epithelial-mesenchymal transition. FASEB J. 33, 000–000 (2019). www.fasebj.org

KEY WORDS: cell adhesion • N-glycosylation • integrins

Epithelial-to-mesenchymal transition (EMT) is a cellulartrans-differentiation process that allows polarized epithelial cells to undergo multiple biochemical changes andenables them to acquire a mesenchymal identity that results in a loss of cell-cell adhesion. EMT is integral in celladhesion, migration, and wound healing and pathologically contributes to cancer metastasis and resistance totherapy (1). This switch in cell behavior is executed mainly
ABBREVIATIONS: AAL, Aleuria aurantia lectin; BSA, bovine serum albumin; ConA, concanavalin A; CRISPR/Cas-9, clustered regularly interspaced short palindromic repeats/caspase-9; DSA, Datura stramonium
agglutinin; ECM, extracellular matrix; EGFR, epidermal growth factor
receptor; EMT, epithelial-mesenchymal transition; E-PHA, Phaseolus vulgaris erythroagglutinin; FAK, focal adhesion kinase; FBS, fetal bovine serum;
FN, fibronectin; GlcNAc, N-acetylglucosamine; GnT, N-acetylglucosaminyltransferase; KO, knockout; L-PHA, Phaseolus vulgaris leucoagglutinin;
MAA, Maackia amurensis agglutinin; MET, mesenchymal-epithelial transition; PHA, phytohemagglutinin; PNGase, peptide-N-glycosidase; Res, rescued; SW, swainsonine; TbRII, TGF-b receptor type II; WT, wild type; ZEB,
zinc-finger E-box–binding homeobox
1

Correspondence: Division of Regulatory Glycobiology, Institute of
Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsusima, Aobaku, Sendai, Miyagi 981-8558,
Japan. E-mail: jgu@tohoku-mpu.ac.jp

doi: 10.1096/fj.201801478R

0892-6638/19/0033-0001 © FASEB

by specific transcription factors [e.g., the Snail, Twistrelated protein, and zinc-finger E-box-binding homeobox(ZEB) families] at transcriptional levels (2). Mesenchymalepithelial transition (MET) is a reversible biologic processthat involves the transition from motile, spindle-shaped
mesenchymal cells to planar arrays of polarized cells that
acquire cell-cell adhesion. Recent studies have shown that
MET participates in the establishment and stabilization of
distant metastases by allowing cancerous cells to regain
epithelial properties and integrate into distant organs (3).
Several studies have focused on the sequential EMT-MET
process that underlies the fate of cells (4, 5), and the flexible
transitions are related to different stages of metastasis (6).
Details of the mechanism involved in this process, however, await elucidation.
An important hallmark of EMT is the attenuation of theepithelial marker E-cadherin and the enhancement ofmesenchymal markers such as N-cadherin (7). E-cadherin
is a central component of cell-cell adhesion junctions, theloss of which facilitates cell migration and alters integrinmediated cell adhesion (8). Integrins control cell interactions with the extracellular matrix (ECM), which is criticalto EMT progression (9, 10). In fact, TGF-b-induced EMT
enhances the expressions of aVb6 and aVb3 integrins,1

asebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on December 20, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNu

which in turn alters cell-matrix adhesion to promote the
EMT process (11, 12). Ectopic expression of b4 integrin
upregulates cell proliferation and induces EMT (13). BothE-cadherin and integrin are glycoproteins. Aberrant expression of N-glycans has been observed in several EMT
models (14). Studies by our group, as well as those ofothers, have found that alteration of N-glycans bymanipulation of the expression of several glycosyltransferases can change the functions of glycoproteins,which include cell adhesion molecules and surface receptors, such as E-cadherin, integrins and epithelial growth
factor receptors (EGFR). For example, overexpression of
N-acetylglucosaminyltransferase (GnT)-V promotes Ecadherin destabilization, whereas expression of GnT-III
stabilizes it (15). TGF-b–induced EMT increases the a1,6fucosylation expression catalyzed by a1,6 fucosyltransferase and overexpression of a1,6-fucosyltransferase
promotes EMT progression (16, 17). In addition, overexpression of GnT-III inhibits the integrin a3b1–mediatedcell adhesion and migration (18, 19), as well as the bindingof a5b1 to fibronectin (FN) (20). Several studies have envisaged the possibility of using glycan-based therapies forcancer treatment (21–23).
Thus far, most studies have focused on the complex Nglycans, and little is known about the functions of highmannose types of N-glycans. In fact, those N-glycans havebeen detected at relatively low levels in glycoproteins
expressed either on the cell surface or in secretions (24).
The expression levels of high-mannose N-glycans are
known to increase in breast cancer (25), but they are decreased in gastric cancers (26). Swainsonine (SW), an inhibitor of Golgi a-mannosidase II, is known to block theprocessing of high-mannose N-glycans into complex
N-glycans, but not into hybrid types of N-glycans (27, 28).
Treatment with SW inhibits tumor growth and metastasis(29, 30), but the underlying mechanism for the function of
SW remains unclear. The blocking of complex N-glycans
has shown promise in the development of anticancer
drugs. Phase II clinical trials of the hydrochloride salts of
SW in patients with renal carcinoma have been discouraging (31). Thus, the discovery of new therapeutics basedon high-mannose N-glycans is urgent.
GnT-I is a key enzyme in the cis-Golgi cisternaethat transfers N-acetylglucosamine (GlcNAc) onto theN-glycan core (containing 5 mannose) after the mannosebranch is trimmed by a-mannosidase-I. The additionof GlcNAc by GnT-I is necessary for the action ofa-mannosidase-II and GnT-II and then GnT-III, -IV, or -V
in a catalytically ordered pathway (32, 33). Deficiency of
GnT-I leads to loss of the downstream N-glycosylation
pathway and results in the presence of high-mannose
types of N-glycans only (34–36). GnT-I is known to be
crucial for embryo development (37), and necessary incytokine signaling (34). The knockdown of GnT-I using
short hairpin RNA suppresses cell migration, invasion,and tumor growth (38). However, the molecular mechanisms of the functions of GnT-I largely remain unclear.
In the present study, we generated GnT-I–knockout(KO) cells by using the clustered, regularly interspaced,short palindromic repeats/caspase-9 (CRISPR/Cas-9) system and found that GnT-I worked as a switch to activate
2Vol. 33

February 2019

EMT and up-regulated integrin-mediated functions. The
mesenchymal markers were decreased in the KO cells, butthey were increased in the restoration of GnT-I genes in KO[rescued (Res)] cells. Furthermore, we found that the expression of GnT-I was closely related to drug resistance andcolony formation. Thus, targeting GnT-I could be a potential
therapeutic strategy for cancer treatment.
MATERIALS AND METHODS
Antibodies and reagentsIn the present study, experiments were performed with the following antibodies: the antibody against the human b1 integrin
subunit (P5D2) was obtained from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA); rabbit
mAb against GnT-I (ab180578) from Abcam (Cambridge, MA,USA); mouse mAb against Smad-2 (3103), rabbit mAbs against
vimentin (5741), ZEB-1 (3396), claudin-1 (13255), Snail (3879),
p-EGFR (4267), EGFR (3777), and p-Smad-2 (3108) from Cell
Signaling Technology (Danvers, MA, USA); mouse mAbs
against N-cadherin (610920), b1 integrin (610468), focal adhesion kinase (FAK; 610088) and p-FAK (611807) from BD Biosciences (Franklin Lakes, NJ, USA); mouse mAbs against a5 integrin(328002), aV integrin (327902), b3 integrin (336402), and b5 integrin(345202) from BioLegend (San Diego, CA, USA); mouse pAb
against a3 integrin (Sc-6592) from Santa Cruz Biotechnology(Dallas, TX, USA); mouse mAb against b4 integrin (MAB2059)
from MilliporeSigma (Burlington, MA, USA); and mouse mAbagainst a-tubulin (T6199) and a-SMA (A2547) from MilliporeSigma. Alexa Fluor 647 goat anti-mouse IgG and streptavidin
were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
The peroxidase-conjugated goat antibodies against mouse andrabbit IgG were obtained from Cell Signaling Technology; sulfoNHS-SS-biotin from Thermo Fisher Scientific; and biotinylated
Datura stramonium agglutinin (DSA; B-1185), Phaseolus vulgaris
leucoagglutinin (L-PHA; B-1115), Phaseolus vulgaris erythroagglutinin (E-PHA; B-1385), concanavalin A (ConA; B-1005), Maackia
amurensis agglutinin (MMA; B-1265), and Aleuria aurantia lectin(AAL; B-1395) from Seikagaku Corporation (Tokyo, Japan).
Cell culture
The HeLa (Riken, Wako, Japan), 293T (Riken), and MDA-MB-231(ATCC, Manassas, VA, USA) cell lines were cultured in DMEM
with 10% fetal bovine serum (FBS), in a humidified atmosphere
containing 5% CO2 at 37°C.
Genomic deletion of GnT-I–KO in HeLa cells
The pSpCas9(BB)-2A-GFP (PX458) plasmid was a gift from Dr.
Feng Zhang (plasmid 48138; Addgene, Cambridge, MA, USA)(39). The target site on human GnT-I exon 1 (CGAAAGTACTCGAAGAAGTC), which was chosen from the genomescale CRISPR-Cas9 KO library (40), was cloned into PX458.
GnT-I–KO cell lines were created by electroporating cells with
vector containing the target sequence according to the manufacturer’s recommendation (Amaxacell line Nucleofectorkit R;
Lonza, Basel, Switzerland). After 3 d of transfection, green fluorescent protein–positive cells were sorted on a FACSAria II (BD
Biosciences). Approximately 10 d thereafter, FACS sorting was
performed with L-PHA, which preferentially binds to the
Galb4GlcNAcb6 Mana3 (GlcNAcb2Mana3) structure to detect
GnT-I deletion (KO). The process was repeated 3 times to obtain
the GnT-I–KO cell line.

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Establishment of GnT-I–Res HeLa cells
The full open reading frame of human GnT-I was cloned into thepENTR/D-TOPO vector and then transferred into the CSII-EFRfa destination vector by LR recombination, according to the
manufacturer’s instructions (Thermo Fisher Scientific). To prepare lentivirus, 293T cells were cotransfected with CSII-EF-RfaGnT-I and lentiviral packaging vectors (pCAG-HIVgp and
pCMV-VSV-G-RSV-Rev). The culture supernatants were collected 48 h after infection, and the virus was introduced into GnTI–KO HeLa cells via addition of the supernatant to the culture
medium. The cells that highly reacted with L-PHA were selectedby using FACSAria II for GnT-I-Res cells, and the stable GnT-IRes cell line was used for subsequent studies.

under a light microscope. Three independent experiments were
performed for each assay.
Soft agar assay
Cells were suspended in DMEM containing 0.3% agarose andlayered onto solidified 0.6% agarose containing DMEM at adensity of 1 3 104 per dish. After incubating for 20 d, the plates
were stained with 0.005% crystal violet, and the colonies wereidentified randomly by stereo microscope (Stemi 2000-CS; Zeiss,
Oberkochen, Germany) and counted. Three independent experiments were performed for each assay.
Cell growth assay

Western blot analysis
Total cell lysates were prepared with lysis buffer containing
20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% Triton X-100, protease inhibitors, and phosphatase inhibitors (Nacalai Tesque,
Kyoto, Japan) and normalized by protein concentrations with abicinchoninic acid protein assay kit (Thermo Fisher Scientific). An
equal amount of protein from each sample was subjected to SDSPAGE and transferred to PVDF membranes (MilliporeSigma).
The membranes were detected with primary and secondary antibodies and visualized by chemiluminescence (MilliporeSigma).
For peptide-N-glycosidase F (PNGase F) treatment, the indicated cell lysates were treated with PNGase F at 37°C for 1 haccording to the manufacturer’s instruction and then subjected toWestern blot analysis.
Flow cytometric analysis
After detachment, cells were washed with ice-cold PBS andstained with primary antibodies or lectins in PBS containing
1 mM EDTA and 0.1% bovine serum albumin (BSA) (m/v) at 4°C
for 20 min, followed by incubation with Alexa Fluor 647–
conjugated secondary antibodies according to the primary antibodies or lectins (Thermo Fisher Scientific) for 20 min at 4°C.
Nonstained cells were included as controls, and dead cells anddebris were excluded from the analysis, based on scatter characteristics. Data for at least 10,000 live events per sample were acquired via FACSCalibur flow cytometry (BD Biosciences). Afterincubation with doxorubicin for 1 d, the identified cells were detached and washed with ice-cold PBS and then subjected to flow
cytometry. The fluorescence was measured with an FL2 (phycoerythrin) band-pass filter to monitor doxorubicin cellular uptake.
Untreated cells were used as controls, and dead cells and debris
were excluded from the analysis, based on scatter characteristics.
Cell migration assay
For the in vitro wound-healing assay, cells were seeded in 6-wellplates precoated with 10 mg/ml FN (MilliporeSigma) and growninto a confluent monolayer. Then the cells were scratched via a
pipette tip. Images of the wound area were photographed after
8 h, and the distance of cell migration was measured by QCapture
Pro 7 software (QImaging, Surrey, BC, Canada). Another cell
migration test was performed with a 24-well Transwell chambersystem (8-mm pore control inserts; BD BioCoat; BD Biosciences).
The bottom side of the chamber was coated with 10 mg/ml FN,
and blocked with 1% BSA. Cells were seeded in the upper FNpretreated chamber at 4 3 104 cells in 0.2 ml serum-free DMEM.
Then, 0.6 ml complete medium containing 10% FBS was placed in
the bottom well. After incubation for 8 h, the migrated cells on thelower surface were fixed, stained with crystal violet, and counted
SWITCHING OF EMT BY GNT-I

Cells (5 3 104) were seeded into 6-well culture dishes overnight.
Cells in the same area were photographed by phase contrast on 4
consecutive days and counted, and the fold change in number ofcells was normalized to the number on d 0.
Cell aggregation assay
Cells were harvested and resuspended in DMEM containing 1%
BSA at a concentration of 2 3 105 cells/ml. For each condition, 1 mlof the cell suspension was added to a 15-ml tube, with or without
2 mM EDTA. The cells were incubated on a rotator (MACSmix Tube
Rotator; Miltenyi Biotec, Bergisch-Gladbach, Germany) at 12 rpm
for 6 h at 37°C, and images of random fields were obtained with amicroscope. Cell aggregation assays were performed in triplicate.
Cell-spreading assay
A cell-spreading assay was performed as previously describedwith minor modifications (41). In brief, cells were harvested andsuspended in DMEM containing 0.1% BSA at 2 3 104 cells/ml.
After seeding on the FN (10 mg/ml)-pretreated dishes for 30 min,the cells were washed with PBS to remove the nonadherent ones,
and the attached cells were fixed with 4% paraformaldehyde in
PBS. The representative images were photographed by phasecontrast microscopy (Olympus, Tokyo, Japan).
Cell surface biotinylation
Cells cultured on dishes were gently washed with PBS and incubated with ice-cold PBS that contained 0.2 mg/ml Sulfo-NHSSS Biotin for 1 h at 4°C. After incubation, the cells were washed 3times with ice-cold PBS and harvested with lysis buffer. Biotinylated proteins were immunoprecipitated with anti-b1 integrinmAb (P5D2) and Ab-Capcher Mag (ProteNova, Higashikagawa,Japan) and subjected to SDS-PAGE. The biotinylated proteins
were visualized with a Vectastain ABC Kit (Vector Laboratories,
Burlingame, CA, USA) and an ECL kit.
Cell viability assays
Cells were cultured at 5 3 103 cells per well in 96-well plates andexposed to the indicated concentrations of paclitaxel for 3 d or
doxorubicin for 2 d. Cell viability was detected by adding 10 ml of
5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT; Dojindo, Kumamoto, Japan) to 100 ml culture
medium and incubating the mixture for an additional 3 h at 37°C.
The medium was then removed, and DMSO (MilliporeSigma)
was added to dissolve the resultant formazan crystals within thecells. The optical absorbance was assessed at 570 nm with amicroplate reader (BioTek Instruments, Winooski, VT, USA).
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Statistical analysis
All data are expressed as means 6 SEM. Prism, v.5.0, was used toperform statistical analyses (GraphPad Software, La Jolla, CA,
USA). Significant differences were analyzed with Welch’s t test.
Values of P , 0.05 were considered statistically significant.

RESULTS
Knockout of the GnT-I gene blocked complex
types of N-glycans
To distinguish the functions of only high-mannose
N-glycans and mixed N-glycans including high-mannose,
hybrid, and complex types, we established GnT-I–KO cells
using CRISPR/Cas9–based technology. As expected, theexpression of GnT-I was not at detectable levels (Fig. 1A).
Flow cytometric analysis and lectin blots using different
kinds of lectins were performed to confirm the efficiency of
GnT-I–KO in HeLa cells (Fig. 1B, C) and in MDA-MB-231

A

B

cells (data not shown). The reactive abilities of L-PHA,
E-PHA, and MAA lectins preferentially recognized
branched GlcNAc N-glycans, bisected N-glycans, and a2,3
sialylated N-glycans, respectively. Those reactive abilities,
however, were greatly suppressed in the KO cells comparedwith those in wild-type (WT) cells (Fig. 1B). Lectin blots using
DSA and AAL lectins preferentially recognized branched
GlcNAc N-glycans and fucosylated N-glycans, respectively,
which showed that the reactive abilities were almost totally
blocked. The binding ability with ConA lectin, however,preferentially recognized high-mannose types of N-glycans
that were augmented in the KO cells (Fig. 1C). These results
show that the GnT-I gene knockout was successful.

Deletion of GnT-I-induced cell aggregation
and a MET-like phenotype
Next, we used these cell lines to compare the cell biologic
functions of GnT-I. First, during cell passages we noticed

E-PHA

L-PHA

MAA

KO

WT
kDa
50

WT
Cell counts

GnT-I

α-Tubulin

KO50

Fluorescence intensity

C
kDa

WT

KO

kDa

WT

KO

kDa

WT

KO

250

150

150

*

*
100

150

100

75
75

*

100

*
75
50
50

DSA

AAL

ConA

Figure 1. Deﬁciency of complex types of N-glycans in GnT-I–KO cells. A) Equal amounts of cell lysates from WT and KO HeLa
cells were analyzed by immunoblot with an anti-GnT-I antibody; a-tubulin served as the loading control. B) The WT and KO cells
were collected and incubated with (bold line) or without (gray shadow) the lectins LL-PHA, E-PHA, and MAA, which
preferentially recognize branched GlcNAc N-glycans, bisected N-glycans, and a2,3 sialylated N-glycans, respectively, followed byincubation with Alexa Fluor 647 streptavidin and ﬂow cytometric analysis. The vertical dashed lines indicate the peaks of lectins
expressions in WT cells. C ) The same amounts of cell lysates from WT and KO cells were analyzed by immunoblot with the lectins
DSA, AAL, and ConA, which preferentially recognize branched GlcNAc N-glycans, fucosylated N-glycans, and high-mannose
N-glycans, respectively. Asterisks denote no speciﬁc bands.
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February 2019

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that the KO cells tended to form islands, which was notobserved in the WT cells (Fig. 2A), suggesting that knockoutof GnT-I may induce cell-cell adhesion. We performed a celladhesion assay in the suspension. Both the WT and KO cells
were incubated with rotation for 6 h. The KO cells tended toform many aggregates that resulted in larger sizes, whereas
the WT cells remained as either single cells or smaller aggregates (Fig. 2B). The cell aggregates disappeared in thepresence of EDTA, suggesting that the cell-cell adhesion had
proceeded in a calcium-dependent manner. A similar
phenomenon was observed in the MDA-MB-231 cells(data not shown). Of particular interest, the expression
levels of mesenchymal markers such as a-SMA,
vimentin, ZEB-1, N-cadherin, and Snail were greatly
suppressed in the KO cells, whereas epithelial markers
such as claudin-1, which is a tight junction protein, wereincreased, compared with those in WT cells (Fig. 2C). It isnoteworthy that the expression levels of E-cadherin in
both WT and KO cells were undetectable. These results
suggest that the deletion of GnT-I induces cell aggregation and MET-like phenotypes.
Deletion of GnT-I inhibited cell spreading andmigration, but did not affect cell proliferation
As previously described, the expression of GnT-I wasessential for cell-cell adhesion, and, therefore, we next

A

B

explored whether GnT-I is also necessary for integrinmediated cell-ECM adhesion. By using a cell-spreading
assay, we found that the WT cells had already begun tospread after the cells were replated on FN-coateddishes for 30 min, whereas the KO cells continued to
exhibit a rounded shape with white dots under phasecontrast photography (Fig. 3A, B). Unexpectedly, there
were no significant changes in the proliferation of these2 types of cells (Fig. 3C). However, we investigatedsome related cellular signaling pathways and foundthat p-Smad-2, p-FAK, and p-EGFR, which correspondswith TGF-b receptor-, integrin- and EGFR-mediatedsignaling, respectively, were decreased in the KO cells
compared with those in the WT cells (Fig. 3D), which
confirmed the notion that a complex type of N-glycans
on these signal receptors plays an important role in cellular signaling.
Cell migration is considered a crucial step in themetastatic cascade (42), and we examined the effectsof GnT-I on cell migration by using wound healing
and Transwell assays. Wound closure capability wassignificantly suppressed in the KO cells comparedwith that in the WT cells (Fig. 4A, B). The integrinmediated Transwell migration was also greatly
inhibited (Fig. 4C, D). These data clearly show that theexpression of GnT-I is essential for integrin-mediatedfunctions.

C
WT

KO

50

α-SMA
WT

WT

kDa
50

Vimentin

250

ZEB-1
150

KO

KO

N-cadherin
100

Snail
25
20

Claudin-1KO+

α-Tubulin
50

EDTA

Figure 2. Comparison of the cell-cell adhesion and expression levels of EMT markers between WT and KO cells. A) The WT and
KO cells were cultured in normal culture dishes for 1 d and were photographed with a phase-contrast microscope. B) The WT
and KO cells were detached from culture dishes and separated into single cells in 10% FBS culture medium at 2 3 105 cells/ml,
with or without EDTA, followed by a constant rotation (12 rpm) at 37°C for 6 h. An aliquot of each of these cell suspensions was
photographed with a phase-contrast microscope. C ) The same amounts of cell lysates from the WT and KO cells were analyzed byimmunoblot with the anti-a-SMA, vimentin, ZEB-1, N-cadherin, snail, and claudin-1 antibodies, and a-tubulin was used as theloading control. Arrows: cell aggregation. Scale bars, 50 mm.
SWITCHING OF EMT BY GNT-I

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B
Percentage of cell spreading

A

D

120

kDa

WT

KO

80

p-FAK
100
40

FAK

***

100

0

WT

p-Smad2

KO
50

C 4

WT

Smad2

KO

Fold cell growth

50
3
100

p-EGFR

2

1

100

EGFR

0
0

1

2

α-Tubulin

3Time (days)

50

Figure 3. Effects of GnT-I deﬁciency on cell spreading, cell proliferation, and cellular signaling. A) The WT and KO cells weredetached and then replated onto FN-coated plates. After incubation for 30 min, cells were ﬁxed, and representative images weretaken via a phase-contrast microscope. Insert: a magniﬁed view of the area indicated by the box. Scale bar, 50 mm. B) The
percentages of spread cells were statistically analyzed. C) The WT and KO cells were starved with serum-free DMEM for 24 h andthen cultured in DMEM containing 10% FBS. The number of cells was counted on the indicated days, and the counts werenormalized to that of each group at 0 h. Data are means 6 SEM (n = 3). D) The same amounts of cell lysates from the WT and KO
cells were analyzed by immunoblot with anti-p-FAK, FAK, p-Smad-2, Smad-2, p-EGFR, and EGFR antibodies. a-Tubulin was usedas the internal control. ***P , 0.001.

The expression levels of integrins were
up-regulated in the KO cells
Because integrin-mediated cell adhesion was inhibited in the
KO cells, we next sought to determine whether the expression of integrin was also suppressed. Flow cytometricanalysis and Western blot were performed to evaluate theexpression levels of different integrins. The expression levels
on the cell surface of integrins, such as a3, a5, aV, b1, b3, b4,
and b5, were increased in the KO cells, compared with theirexpression levels in the WT cells (Fig. 5A). Because of thelack of complex N-glycans on integrin a3 and b1 in the KO
cells, the bands of those integrins shifted on SDS-PAGE
much faster than in the WT cells (Fig. 5B). In the WT cells, theso-called immature band of b1was similar to that in the KO
cells, confirming that immature b1 carries a high-mannose
type of N-glycan. These differences dissipated after the removal of N-glycans by PNGase F. Treatment with PNGase F
showed enhanced expression levels of a3 and b1 in the KO
cells. These results suggest a negative feedback loop from
integrin function to expression.
Restoration of GnT-I in KO cells restored cell
behavior and induced an EMT-like phenotype
Given the observation that the KO cells exhibited aberrant
behavior, we restored GnT-I expression in the KO cells
6Vol. 33

February 2019(Res) to determine whether it would rescue these phenotypes. The efficiencies of Res cells were confirmed byWestern blot with the anti-GnT-I antibody (Fig. 6A) andflow cytometric analysis of L-PHA lectin (Fig. 6B).
Integrin-mediated FAK phosphorylation (Fig. 6A) increased, whereas the expression levels of a5 and b1 (Fig.
6B) decreased in the Res cells, further supporting the notion of a negative feedback loop from function to expression, as previously described. The expression levels of
mesenchymal markers, such as vimentin, ZEB-1, andN-cadherin, were increased in the Res cells, compared withthose in the KO cells (Fig. 6C). Furthermore, the cell islands
caused by cell-cell adhesion, which could be observed in
the KO cells, disappeared in the Res cells (Fig. 6D). Integrinmediated cell migration increased significantly in the Res
cells (Fig. 6E, F). These results showed the importance of
GnT-I switching by the EMT process.
Influences of integrin and GnT-I oncell-cell adhesion
As previously described, deletion of GnT-I induced
integrin expression, which indicated that complex
N-glycans are not essential for integrin expression. We usedbiotinylation to further compare the expression levels of b1
among the WT, KO, and Res cells. The expression levels ofb1 and the associated a-subunits on the cell surface were

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A

0h

B

8h

Migration Rate
Relative to WT (%)

150

WT

100

50

***
0

WT

KO

KO

D

C

WT

Migration ability
Relative to WT (%)

150

KO

100

50

***

0

WT

KO

Figure 4. Comparison of cell migration between the WT and KO cells. A) Cells were grown to more than 90% conﬂuence, andthe monolayers were incised with a pipette in each well. Photographs were taken by phase-contrast microscopy at 0 and 8 h. B)
The quantiﬁcation of cell migration rate relative to the WT (100%) was expressed as means 6 SEM of results in 3 independent
experiments. C ) A cell migration chamber was used to examine the migration capacity of WT and KO cells. Migrated cells were
stained and photographed. D) Relative cell migration was expressed as the percentage of migrating cells compared with thatof WT cells (100%). Results are representative of at least 3 independent experiments. Scale bars: 50 mm (A); 100 mm (C ).
***P , 0.001, WT vs. KO.

enhanced in KO cells, but restored to the levels of WT in the
Res cells (Fig. 7A). The deletion of GnT-I enhanced theexpression levels of integrins, but the integrin-mediatedcell-ECM adhesion including FAK phosphorylation andcell adhesion and migration, all were down-regulated. We
speculated as to whether the suppression of cell-ECM adhesion via blocking the process for complex-type N-glycans
on integrins could induce cell-cell adhesion, as observed in
the KO cells. To test the hypothesis, we added a functional
blocking b1 antibody (P5D2) (43), to attenuate cell-matrix
adhesion in the cell culture. All 3 types of cells clearly
exhibited cell aggregation in the presence of anti-b1 integrin
antibody (Fig. 7B). These data may partly explain why cellaggregation occurred in the KO cells and could further
suggest that the modification of complex types of N-glycans
on b1 integrin is very important for its ability to function.

cancer drugs paclitaxel for 3 d (Fig. 8A) and doxorubicinfor 2 d (Fig. 8B) at the indicated concentrations. The KO
cells were more sensitive to both drugs, compared with theWT and Res cells. When we used fluorescence-activatedcell sorting to monitor the cellular uptake of doxorubicin,
we consistently observed higher fluorescence intensities in
KO cells (Fig. 8C).
The ability of tumor cells to form colonies from a single
cell is a well-known feature of metastatic potential (46).
Cells that undergo EMT are also known to form morecolonies (47), and, therefore, we evaluated the effects of
GnT-I on colony formation in HeLa cells. As shown in Fig.
8D, the colony formation in soft agar was significantly
suppressed in the KO cells, compared with the WT and the
Res cells. These data suggest that the expression of GnT-I
greatly contributes to tumor survival and formation.

Deletion of GnT-I increased drug sensitivity

DISCUSSION

Because recent studies revealed the involvement of EMT
on the efficacy of cancer drug responses (44, 45), we evaluated whether GnT-I deficiency affects drug sensitivities
in HeLa cells. The 3 cell groups were incubated with the

In the present study, we found that GnT-I works as aswitch to activate EMT, based on the following observations: 1) deletion of GnT-I suppressed the expression ofmesenchymal markers and cell-ECM adhesion and induced

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A
α3

α5

αV

β1

β3

β4

β5

Cell counts

WT

KO

Fluorescence intensity

B

PNGase F (-)
WTPNGase F (+)

KO

WT

KO

150

PNGase F (-)

PNGase F (+)

WT

WT

KO

KO

150

MatureImmature
100

100

kDa
kDa
75
75

IB:β1

IB:α3

Figure 5. Effects of GnT-I deﬁciency on the expression levels of integrins and the effects of N-glycosylation on a3b1 integrin. A)
The WT and KO cells were collected and incubated with (bold line) or without (gray shadow) the indicated integrins, followed byincubation with Alexa Fluor 647 conjugated with goat anti-mouse antibody, and then subjected to ﬂow cytometry. The verticaldashed lines indicate the peaks of integrins expressions in WT cells. B) The same amounts of cell lysates (20 mg) obtained fromthe WT and KO cells were treated with (+) or without (2) peptide: N-glycosidase F (PNGase F), and then immunoblot analysis
was performed with anti-b1 and -a3 antibodies.

cell-cell adhesion; 2) loss of GnT-I decreased integrinmediated cell migration; and 3) restoration of GnT-I efficiently rescued the expression of mesenchymal markers andcell migration, and decreased cell-cell adhesion. In addition,
the expression of GnT-I was important for cell survival
against anticancer drugs.
One study has shown that mice lacking GnT-I die at
midgestation (37). Biochemical and morphologic analysesof embryos have revealed GnT-I–null embryos that aredevelopmentally retarded, particularly in neural tissue.
The neural crest is known to be a transient embryonic cell
population that migrates long distances through the vertebrate embryo during development, wherein the integrinfamily of cell adhesion receptors are necessary for proper
cell migration, proliferation, survival, and differentiation.
In fact, both major ECM molecules, laminin and FN, havereceptors that include the a3b1 and a5b1 integrins and areabundantly expressed in neural crest migration pathways(48, 49). Furthermore, a loss of function of the murine a5
integrin by gene targeting posed a recessive embryoniclethal threat at midgestation that was related to defects inthe development of the cardiac neural crest, mesoderm
formation, and cell movement and function (50, 51).
In our study, GnT-I deficiency greatly suppressed
8Vol. 33

February 2019

integrin-mediated cellular signaling (FAK phosphorylation), cell-ECM adhesion, and migration, which may
partly explain some of the important phenotypes shownin the GnT-I–null embryos. We speculate that the defectin integrin functions is one of the main causes of theembryonic lethality at midgestation of GnT-I–KO mice.
In fact, there is an increasing body of evidence implicating N-glycans in integrin function (52). For example,
alterations in the oligosaccharide portion of integrinsthat are modulated by glycosyltransferases, such as
GnT-III, GnT-V, and a2,6 sialyltransferase, differentiate
in the regulation of cell malignant phenotypes, such as inintegrin-mediated cell migration and cell spreading. The
expression of GnT-V stimulates a3b1 integrin-mediatedcell migration, whereas the overexpression of GnT-III
inhibits GnT-V–induced cell migration (19). Therefore,
the lack of GnT-I may result in the loss of the dynamic
and precise post-translational regulation that plays acrucial role in embryo development. It is intriguing that
GnT-I deficiency increased the expression levels of many
types of integrins, although integrin functions were
suppressed, whereas the restoration of GnT-I decreased
integrin expression and enhanced integrin-mediated celladhesion and cellular signaling. These findings suggest

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A

KO

B

RES

L-PHA

α5

C

β1

KO

RES

150

50

GnT-I

p-FAK

100

KO

Cell counts

kDa

250

ZEB-1
kDa

FAK

100

α -Tubulin

50

D KO

N-cadherin
100

RES
50

Vimentin50

α -Tubulin

Fluorescence intensity

E KO

RES

Migration Index
Relative to KO (%)

F

400

***

300

200

100

0

KO

RES

Figure 6. Forced expression of the GnT-I in KO cells Res cell behavior and induced EMT-like phenotypes. A) Equal amounts ofcell lysates from the KO and restoration of GnT-I in the KO cells (Res cells) were analyzed by immunoblot with anti-GnT-I, -pFAK, and -FAK antibodies. a-Tubulin was used as an internal control. B) The KO and Res cells were collected and incubated with(bold line) or without (gray shadow) anti-GnT-I antibody, followed by incubation with Alexa Fluor 647 conjugated with goat antimouse antibody and counting by ﬂow cytometry. The vertical dashed lines indicate the peaks of the indicated expressions in KO
cells. C ) The expression levels of EMT markers such as N-cadherin, ZEB-1, and vimentin were examined by Western blot analysis.
D) Images were taken with a phase-contrast microscope to show representative cell morphologies of the KO and Res cells. Arrows:
cell aggregations. Scale bar, 50 mm. E, F) Photographs (E ) and quantitative analysis (F ) of the cell migration assays were used todetermine migration capabilities of the KO and Res cells. Scale bar, 100 mm. ***P , 0.001, KO vs. Res.

the existence of a negative feedback loop from thefunction to the expression of integrins. Furthermore, theresults support the notion that N-glycans closely regulate integrin function.
EMT is an important phenotype characterized by a lossof the cell-cell junction and the acquisition of cell migration, which plays crucial roles, not only in physiologic
processes, such as embryo implantation, embryogenesis
and organ development, but also in tissue repair andpathologic processes such as tissue fibrosis, tumor invasiveness, and metastasis (53). Different signaling
pathways—for example, receptor tyrosine kinase, Notch,
Wnt, and TGF-b—are known to provide the necessary
stimuli that modulate gene expression and trigger EMT
and cell migration (54). The regulation of EMT by GnT-I
may have crosstalk with those signaling pathways. In fact,glycosylation and signaling mutually regulate and affectcell behavior. Inhibition of GnT-V–mediated N-glycosylation suppresses PI3K and MAPK signaling (55, 56), but
the inverse is true of the PI3K/Akt (55) and ras/raf/ets2(57) signaling pathways, which regulate GnT-V expression. In addition, core fucosylation is critical to the activation of the TGF-b receptor type II (TbRII) and Smad-2
signaling pathways (58), and high-mannose type TbRIISWITCHING OF EMT BY GNT-I

decreases TGF-b–induced Smad-2 activation (59),whereas TGF-b receptor–mediated signaling promotes
GnT-V expression (60). Our data show that GnT-I deficiency exhibited a MET-like phenotype and that restoration of GnT-I efficiently reversed MET to an EMT-likephenotype, which may also partly explain why the formation of neural tissue was delayed in the GnT-I–nullembryos. The inhibition of complex types of N-glycans by
SW efficiently and consistently inhibit cell invasion and
EMT processes in esophageal carcinoma cells (30). In addition, the deletion of GnT-I suppressed the phosphorylation of both EGFR and Smad-2, which further confirmed
the switching function of GnT-I toward EMT. Thus, highmannose types of N-glycans may maintain MET-likephenotypes, whereas GnT-I initiates the EMT process.
We also demonstrated how the deletion of GnT-I augmented cell-cell adhesion, which may have 2 explanations.
The first concerns a weakening of the cell-ECM adhesion in
KO cells, as previously described. It is reasonable tospeculate that the cell-cell adhesion and cell-ECM adhesion regulate each other in an opposing manner. Indeed,treatment with a blocking b1 integrin antibody (P5D2)
greatly induced cell-cell adhesion in the WT and Res HeLa
cells, which was observed in the KO cells without this9

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A

B

Biotinylation
KO

WT

RES

kDa

150

100

IP:β1

Figure 7. Effects of GnT-I on integrin b1 maturation and the cell-cell adhesion induced by a blocking anti-b1 integrin antibody.
A) The indicated cells were incubated with Sulfo-NHS-SS biotin for 1 h, and then the cell lysates were immunoprecipitated with
anti-b1 integrin mAb (P5D2) and the biotinylated proteins were detected. B) The indicated cells were replated in 6-well culture
dishes in normal culture medium, with or without a blocking anti-b1 integrin antibody (P5D2, 10 mg/ml). After incubation for
16 h, phase-contrast photographs were taken. Arrows: cell aggregation. Scale bar, 75 mm.

treatment. Consistently, the KO of b1 integrin resulted
in cell-cell adhesion and in the suppression of FAK
phosphorylation and cell migration (61). The secondA

explanation involves the possibility that high-mannose
types of N-glycans may enhance cell-cell adhesion.
E-cadherin is the core transmembrane glycoprotein of

B
120

KO

WT

C
120

RES

WT

KO

Negative

RES

80

40

***
**
0
0

0.005

0.05

80

Cell counts

Cell viability (%)

Cell viability (%)

WT

40

***

KO

***

***

0

0.5

5

Paclitaxel (μM)

0

0.5

2.5

5

15

RES

30

Doxorubicin (μM)
Fluorescenceintensity

WT

KO

RES

E
Colony formation (%)

D

150

100

50

0

***
WT

KO

RES

Figure 8. Loss of GnT-I chemosensitized the cells to cancer drugs. A–B) The cells were cultured in normal culture medium
containing paclitaxel (A) and doxorubicin (B) at the indicated concentrations. Percentages of cell viability were measured via
MTT assay. **P , 0.01, ***P , 0.001 vs. paclitaxel- or doxorubicin-treated WT cells, which was set as 100%. C ) After incubation
with (bold line) or without (gray shadow) doxorubicin for 1 d, the indicated cells were collected and then subjected to ﬂow
cytometry via the FL2 (phycoerythrin) channel. D) The indicated cells (1 3 104/per well) were cultured in soft-agar plates for 20 d,the colonies were stained with crystal violet, and images were taken by light microscope. E) The stained colonies were counted, andthe quantitative data are presented as means 6 SEM of results in 3 independent experiments. Scale bar, 5 mm. ***P , 0.001
compared to the WT group, which was set as 100%.
10Vol. 33 February 2019

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ZHANG ET AL.

asebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on December 20, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNuthe adherens-junction, although the expression level ofE-cadherin in HeLa cells is marginal. The modification ofE-cadherin with complex N-glycans has been associatedwith the formation of dynamic and weak adherens
junctions, whereas E-cadherin, modified by highmannose types or fewer N-glycans, is known to promote the establishment of stable adherens junctions (62).
E-cadherin was modified by N-glycans containing b1,6
branched and sialylated structures during acquisition of
the malignant phenotype (63), whereas overexpressionof GnT-III prolonged the turnover of E-cadherin on thecell surface by suppression of extensive branched complex N-glycan formation by the action of GnT-V and thenenhanced homophilic adhesion (15). Up-regulation of
GnT-V expression was observed in TGF-b–induced EMT(60), and overexpression of GnT-V promoted EMT and
keratinocyte migration in GnT-V–transgenic mice (64).
Therefore, smaller N-glycans, such as the high-mannoseor bisected types, may promote cell-cell adhesion.
It is also worth noting why GnT-I deficiency did notaffect cell proliferation (Fig. 3B). Studies have shown thatlost or reduced branched-complex N-glycans catalyzed by
GnT-IV and -V lead to a reduction in the retention time ofglycoprotein receptors on the cell surface related to a
weakening of their interactions with a galectin lattice (65,66). Thus, it could be more plausible to speculate that deletion of GnT-I inhibits cell proliferation. However, ourresults showed there was no significant difference in cell
growth between WT and KO cells, although the level ofphosphorylated EGFR was decreased in the KO cells
compared with that in the WT cells (Fig. 3). In fact, a study
showed that the proliferation of CHO cells is not affectedby the genetic disruption of multiple a1,2-mannosidases
and induced to generate high-mannose types of N-glycans
in HEK293 cells (67). Knockdown of GnT-I by short hairpin RNA consistently did not alter cell proliferation rates ina human PC-3 prostate tumor cell line (38). These results
indicate that complex types of N-glycans remodeled byglycosyltransferases on glycoprotein receptors may positively or negatively regulate cellular signaling for cellproliferation. For instance, the lack of a1,6-fucosylation in
TbRII reduced its binding with ligands, and subsequently
suppressed intracellular signaling by phospho-Smad-2,
which suggests that a1,6-fucosylation positively regulates
TGF-b downstream signaling (58). In contrast to TGF-b–
mediated signaling, a1,6-fucosylation on an activin receptor, a member of the TGF-b superfamily, negatively
regulates phospho-Smad-2 signaling (68). In addition, cells
expressing high levels of GnT-V increase migration (69)and EMT behavior by up-regulating EGFR signaling (64).
However, lower expression of GnT-V is found in lungcancer, and GnT-V inversely regulates EMT behavior bysuppressing Smad activation in human lung cancer cells(70). Therefore, even the same types of N-glycans on different receptors may play distinctive roles in their cellularsignaling.
Inhibition of N-linked glycosylation has generated interest in its potential use as an anticancer agent (71, 72),
because aberrant N-glycosylation is implicated in cancer
growth and metastasis. In our study, we found that the KO
cells were more sensitive to cancer drugs than WT and Res
SWITCHING OF EMT BY GNT-I

cells, and more drugs were detected in the KO cells. The
treatment with SW promoted cell apoptosis induced bypaclitaxel (73). Inhibition of N-glycosylation treated withtunicamycin also greatly increased the sensitivity todoxorubicin (74). These results indicate that the functionsof some membrane glycoproteins such as ABC transporters and P-glycoprotein (75), which are involved in
the export and import of drugs, may be regulated by
N-glycans. N-glycosylation of ABC transporters has been
associated with functional activity in rat hepatocytes (76),and the inhibition of protein glycosylation has enhanced
the effect of chemotherapy by interfering with the localization and function of transporter proteins in several cancer cell lines (77). In addition, the decrease in the functionsof integrin in KO cells suggests an increase in the sensitivity
to drugs, given that b1 deficiency increases the inhibitory
effects of AG1478, an EGFR tyrosine kinase inhibitor (61).
The underlying mechanisms for drug sensitivity must be
studied further.
In summary, our findings indicate that GnT-I plays apivotal role in the regulation of key processes in cancer
cells, such as EMT, cell adhesion, migration, and chemoresistance. Thus, we believe that GnT-I is a promisingtherapeutic target for cancer.
ACKNOWLEDGMENTS
This work was supported, in part, by Grant-in-Aid for
Scientiﬁc Research 15H04354 (to J.G.) from the JapaneseSociety for the Promotion of Science, and by Grant-in-Aid for
Scientiﬁc Research on Innovative Areas 18H04868 (to J.G.)
from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan. The authors declare no conﬂicts ofinterest.

AUTHOR CONTRIBUTIONS
G. Zhang, T. Isaji, and J. Gu designed the research;
G. Zhang performed all the experiments; Z. Xu, X. Lu,
and T. Fukuda assisted with the experiments; G. Zhang,
T. Isaji, T. Fukuda, and J. Gu analyzed and interpreted thedata; G. Zhang, T. Isaji, and J. Gu wrote and revised themanuscript; and all authors approved the ﬁnal version of
the manuscript.
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Accepted for publication September 17, 2018.

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cro

ARTICLEO-GlcNAcylation regulates integrin-mediated cell adhesion
and migration via formation of focal adhesion complexes
Received for publication, September 19, 2018, and in revised form, December 2, 2018 Published, Papers in Press, December 26, 2018, DOI 10.1074/jbc.RA118.005923

Zhiwei Xu‡1, Tomoya Isaji‡1, Tomohiko Fukuda‡, Yuqin Wang§, and Jianguo Gu‡2
From the ‡Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai Miyagi 981-8558, Japan and the §Department ofPharmacology, Pharmacy College, Nantong University, Nantong, Jiangsu Province 226001, China
Edited by Qi-Qun Tang

O-GlcNAcylation is controlled by OGT3 and is a specific typeof post-translational modification that consists of the covalentattachment of single GlcNAc to the nucleus and cytoplasm of
the serine or threonine residue of an extremely large family oftarget proteins (1, 2). This post-translational modification isessential for cell survival and division (3), and aberrant
This work was supported in part by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research 15H04354; National Natural Science Foundation of China Grant 31670807; and Grant-in-Aid for Scientific
Research on Innovative Areas 18H04868 from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan. The authors declarethat they have no conflicts of interest with the contents of this article.
1
Both authors contributed equally to this work.
2
To whom correspondence should be addressed. Tel.: 81-22-727-0216; Fax:
81-22-727-007; E-mail: jgu@tohoku-mpu.ac.jp.
3
The abbreviations used are: OGT, O-GlcNAc transferase; KD, knockdown;
DOX, doxycycline; shRNA, short hairpin RNA; FAK, focal adhesion kinase;FA, focal adhesion; ECM, extracellular matrix; VSV, vesicular stomatitis virus
glycoprotein; DMEM, Dulbecco’s modified Eagle’s medium.

O-GlcNAcylation provokes tumorigenesis, diabetes, and Alzheimer’s disease by regulating cell signaling, transcription,
metabolism, and cytoskeletal formation (4 –7). The increased
O-GlcNAcylation seems to be a general characteristic of cancer
cells. For example, higher levels of O-GlcNAcylation expression
have been observed in cancers of the liver (8), lung, colon (9), andbreast (10). Furthermore, numerous breast cancer cell lines haveshown higher levels of O-GlcNAcylation, and the levels of OGT
expression in aggressive breast cancer cell lines are much higher
than those seen in less aggressive breast cancer cell lines (11).
O-GlcNAc modifications have also been observed in important
target proteins, such as p53 (12), HIF-1␣ (13), ␤-catenin (10), andG6PD (14), which are involved in the regulation of malignant cancer characteristics by controlling cellular metabolism and proliferation. On the other hand, the suppression of OGT expression inbreast or liver cancer cell lines decreases cell motility, which suggests that O-GlcNAcylation could be involved in cell migration(10, 15).
Cell migration is a highly integrated multistep process that
includes the development of cytoplasmic protrusions, attachment, and spreading (16). The migratory capacity of cancer
cells is initially mediated by alterations in the expression of cellsurface molecules known as integrins (17). It is becoming
increasingly clear that integrins are crucial for cell migration in
the tumor microenvironment (18). Following ligand binding,integrins cluster into focal contacts that contain different focal
adhesion (FA)-associated proteins, such as ␣-actinin, vinculin,talin, FAK, and paxillin, which link the integrins to the actin cytoskeleton (19). The processes of adhesion formation and disassembly drive the migration cycle through ligand binding, which in turnregulates integrin activity and cytoskeletal complex formation as
well as adhesion dynamics (20). O-GlcNAcylation appears tooccur in actin cytoskeletal regulatory proteins, such as paxillin (21)and talin (22), as well as in microtubule assembly proteins, such as
tubulin (23), and in microtubule-associated proteins (24). However, whether and how O-GlcNAcylation impacts cell migration
remains unclear.
In the present study, we used the doxycycline shRNAinducible system to knock down the OGT gene to identify
the biological functions of O-GlcNAcylation and its regulatory mechanisms in cell adhesion and migration. We foundthat the knockdown of OGT aberrantly increased cell adhesion, FA formation, and integrin ␤1 activation, which in turndecreased cell migration. Thus, our findings may provide
J. Biol. Chem. (2019) 294(9) 3117–3124© 2019 Xu et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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O-GlcNAcylation is a post-translational modification of a protein serine or threonine residue catalyzed by O-GlcNAc transferase(OGT) in the nucleus and cytoplasm. O-GlcNAcylation plays
important roles in the cellular signaling that affect the different
biological functions of cells, depending upon cell type. However,whether or not O-GlcNAcylation regulates cell adhesion andmigration remains unclear. Here, we used the doxycycline-inducible short hairpin RNA (shRNA) system to establish an OGT knockdown (KD) HeLa cell line and found that O-GlcNAcylation is a
key regulator for cell adhesion, migration, and focal adhesion(FA) complex formation. The expression levels of OGT and
O-GlcNAcylation were remarkably suppressed 24 h after inductionof doxycycline. Knockdown of OGT significantly promoted celladhesion, but it suppressed the cell migration on fibronectin. The
immunostaining with paxillin, a marker for FA plaque, clearly
showed that the number of FAs was increased in the KD cells compared with that in the control cells. The O-GlcNAcylation levels of
paxillin, talin, and focal adhesion kinase were down-regulated in
KD cells. Interestingly, the complex formation between integrin
␤1, focal adhesion kinase, paxillin, and talin was greatly increasedin KD cells. Consistently, levels of active integrin ␤1 were significantly enhanced in KD cells, whereas they were decreased in cells
overexpressing OGT. The data suggest a novel regulatory mechanism for O-GlcNAcylation during FA complex formation, which
thereby affects integrin activation and integrin-mediated functions
such as cell adhesion and migration.

FAK was modified by O-GlcNAc

new insight into integrin-mediated cell migration and explain why
O-GlcNAcylation is usually highly expressed in some malignant cancers.

Results
Established OGT knockdown (KD) cells
A growing number of studies have shown that OGlcNAcylation plays a critical role in the regulation of tumor
cell growth (11) and cancer metastasis (25, 26). To investigate
the effects of O-GlcNAc expression on cell adhesion and migration, we used the DOX-dependent inducible shRNA KD system
to establish OGT KD HeLa cells. In this cellular system, OGT
and O-GlcNAc were expressed at normal levels in the absenceof DOX, whereas both expressions were drastically suppressedin the presence of DOX in the culture medium at the indicatedconcentrations, as shown in Fig. 1A. Furthermore, similar suppression levels were observed even following incubation at thelowest concentration of 0.1 ␮g/ml after 24 h (Fig. 1B), suggesting an effective KD of OGT and a rapid turnover of O-GlcNAc
levels in HeLa cells. After culture for 48 h, elongated cell shapes
were converted to a more-rounded morphology, and the KD
cells showed significantly increased cell spreading areas compared with those in the control cells (Fig. 1C). These observations suggest the impact that O-GlcNAcylation exerts on cellmorphology.

3118 J. Biol. Chem. (2019) 294(9) 3117–3124

Knockdown of O-GlcNAcylation enhanced cell adhesion andFA formation and suppressed cell motility
Next, we used a fibronectin (FN)-coated dish to investigate
the effects of OGT KD on cell adhesion, FA formation, and cell
motility. To verify the initial stage of cell adhesion, we performed a 20-min cell adhesion assay on FN. Interestingly, thenumber of adhered cells was drastically increased in the KD
cells compared with that in the control cells (Fig. 2A). During
cell adhesion, integrins and cytoplasmic proteins such as paxillin, talin, and FAK become clustered in the plane of the cell
membrane and in well-developed aggregates, the so-called FA
plaque, which can be detected by immunofluorescence microscopy (27). Consistent with their enhancement of cell adhesion,in the present study, OGT KD cells also promoted an increasein FA formation, by comparison with the activity in control cells(Fig. 2B). By contrast, the KD cells showed a significant reduction in cell motility, as observed by video microscopy (Fig. 2C).
These data indicate that a loss of O-GlcNAcylation promotes
cell adhesion and focal contact formation while suppressing cell
migration.
Talin, FAK, and paxillin were O-GlcNAc–modified proteins
Previous studies have revealed that some forms of protein FA
plaque, such as paxillin and talin, are modified by O-GlcNAc(21, 22). In the present study, we investigated whether
O-GlcNAc modification of those target proteins also occurredin HeLa cells in this system. Consistent with previous studies,

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Figure 1. Knockdown of OGT suppressed O-GlcNAcylation and enhanced
cell spreading in HeLa cells. A and B, the expression levels of OGT and
O-GlcNAcylation from cell lysates of DOX-controlled OGT KD HeLa cells wereverified with concentrations of DOX at 0, 0. 1, 0.5, 1.0, and 5.0 ␮g/ml for 72 h(A) or at the indicated time with 0.1 ␮g/ml DOX (B). The control (Ctrl) indicatesthe cells treated without DOX. Cell lysates from the indicated cells were subjected to Western blotting with the O-GlcNAc (CTD110.6), OGT, and ␣-tubulin
antibodies. C, representative images of cell spreading are shown after incubation for 48 h. Cells were incubated with (KD) or without (Ctrl) 0.1 ␮g/ml DOX
for 24 or 48 h on a normal culture dish, after which the cell areas were measured. Values represent the mean ⫾ S.E. (error bars) (n ⫽ 50). **, p ⬍ 0.01
(Welch’s correction t test). Scale bars, 15 ␮m. Experiments were independently repeated at least two times.

Figure 2. Reduced O-GlcNAcylation promoted cell adhesion and FA formation but decreased cell migration. HeLa cells were cultured in the presence (KD) or absence (Ctrl) of DOX for 24 h. A, 20 min after replating cells onto
FN-coated 96-well plates, the attached cells were fixed, and then the nuclei
were stained and counted. Representative fields were photographed via fluorescent microscopy. Scale bars, 30 ␮m. Values represent the mean ⫾ S.E.
(error bars) (n ⫽ 11). **, p ⬍ 0.01 (Welch’s correction t test). B, cells wereallowed to spread on FN-coated coverslips for 1 h. Cells were then stainedwith anti-paxillin antibody (green) and TO-PRO-3 (blue). The numbers of focal
adhesions were quantified by ImageJ software. Scale bars, 5 ␮m. Values represent the mean ⫾ S.E. (n ⫽ 11). **, p ⬍ 0.01 (Welch’s correction t test). C, cell
motility was observed by time-lapse video microscopy. Values represent themean ⫾ S.E. (n ⫽ 30). **, p ⬍ 0.01 (Welch’s correction t test). Experiments wereindependently repeated at least two times.

FAK was modified by O-GlcNAc

Figure 3. Decreased O-GlcNAcylation levels of paxillin, talin, and FAK in OGT-KD cells. HeLa cells (A) or cells transfected with talin (B) or FAK (C) wereincubated without (Ctrl) or with (KD) DOX. The cell extracts were immunoprecipitated (IP) with the indicated antibodies and Western blotted with
anti-O-GlcNAc or the indicated antibodies, respectively. Experiments were independently repeated at least three times.

Reduction of O-GlcNAcylation promoted complex formation
FAK is a key component of the signal transduction pathways
triggered by integrins. When cells bind to the extracellularmatrix (ECM), FAK is usually recruited to integrin-mediatednascent FA, because it interacts directly through the cytoskeletal proteins talin and paxillin, with the cytoplasmic tail of
integrin ␤1 (28). Therefore, we compared the ability of control
and KD cells to form FA complexes. As shown in Fig. 5A, thecomplexes immunoprecipitated with anti-FAK antibody showed
higher levels of paxillin in KD cells than in control cells. Consistently, KD cells demonstrated a greater number of complex formations composed of both ␤1 integrin and talin (Fig. 5B) and talin and
FAK (Fig. 5C). A similar phenomenon was also confirmed in
OGT-KD 293T cells (data not shown).
Knockdown of O-GlcNAcylation activated integrin ␤1
Given the increase in FA complex formation in KD cells, it isreasonable to speculate that OGT-KD may affect integrin activation. Integrin-mediated adhesion can recruit FA proteins toform FA plaque and then trigger conformational activation,so-called inside-out signaling, of integrin ␤1 in the ectodomain,which then can be recognized by a specific antibody (29, 30)
that we used to examine the expression levels of active integrin
␤1 in both control and KD cells. The expression levels of active
␤1 in immunostaining (Fig. 6A) or cell lysates (Fig. 6B) wereclearly up-regulated in the KD cells compared with control
cells. In contrast to KD cells, the expression levels of active ␤1were suppressed in the OGT-overexpressing HeLa cells, whichfurther suggested that O-GlcNAcylation negatively regulates
integrin-mediated inside-out signaling. Thus, we were convinced that OGT could be a novel regulator for FA complex
formation and integrin activation by dynamically regulatingcell adhesion and migration.

Discussion
In the present study, we clearly showed that O-GlcNAcylation
negatively regulates integrin-mediated cell adhesion and FA
complex formation as well as integrin activation, which resultsin the control of cell migration on the ECM (Fig. 7). Our findings are the first to demonstrate that OGT may function as a key
regulator of FA complex formation during cell–ECM adhesion.
These results provide clues to understanding the roles of
O-GlcNAcylation in cell migration.
Cell migration is a central process in the development andmaintenance of multicellular organisms (16). Although thedetailed mechanisms underlying cell migration remain unclear,
it is reasonable to postulate that integrin-mediated cell adhesion could regulate migration, which would allow communication between cell–ECM contact and the actin cytoskeleton
through focal adhesions (31). The dynamic balance between
adhesion receptors and the binding of ECM ligands provides
FA turnover that regulates adhesion formation and disassembly(32). In the framework of this model, an imbalance in the processes of attachment and detachment leads to conformationalchanges that mediate abnormal adhesion (19). In the present
study, we clearly demonstrated that the suppression of
O-GlcNAcylation inhibited HeLa cell migration, whereas itenhanced cell–ECM adhesion (Fig. 2), which indicated that
O-GlcNAcylation is involved in the regulation of integrin-mediated cell adhesion. Consistently, FAK serves as a key regulatorof FA assembly and disassembly processes that are fundamental
for efficient cell migration (33). Indeed, there were more stressfibers and focal adhesions in FAK-deficient cells, whereas cell
motility was inhibited (34). Aberrant cell–ECM adhesiveness islikely to suppress cell migration, and proper cell adhesion is an
important determinant for cell migration (35). Thus, our data
are reasonable in that the knockdown of O-GlcNAcylation
aberrantly increased cell adhesion, as well as spreading and FA
complex formation, which in turn decreased cell migration.
Consistently, a loss of paxillin phosphorylation at Ser-250
markedly inhibits focal adhesion turnover and cell migration(36).
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O-GlcNAc modifications were detected on both paxillin andtalin, whereas O-GlcNAcylation levels for both proteins weresignificantly decreased in KD cells, compared with that seen incontrol cells (Fig. 3, A and B). Importantly, we also found that
FAK, a key molecule for integrin-mediated signaling, was also atarget protein for O-GlcNAcylation, which was decreased in
the KD cells (Fig. 3C). The suppression of O-GlcNAcylation on
FAK and talin was also confirmed in DOX-induced OGT KD
293T cells (data not shown). To further establish the occurrence of O-GlcNAcylation in these proteins, we conducted achemoenzymatic labeling assay using an azido-GalNAc sugar,
as described under “Experimental procedures.” Clearly, talin,
FAK, and paxillin were labeled, which proved that they are
O-GlcNAcylated proteins (Fig. 4, A–C). These results suggestthat O-GlcNAcylation may affect both integrin ␤1–mediatedcomplex formation and FA formation, which confirms this process as a regulator of cell adhesion and migration.

FAK was modified by O-GlcNAc

Figure 4. Confirmation of O-GlcNAcylation on talin, FAK, and paxillin. Cell lysates of 293T cells transfected with talin (A), FAK (B), or WT HeLa cells (C) were
immunoprecipitated (IP) with anti-GFP, anti-VSV, or anti-paxillin antibodies, respectively, followed by click chemistry labeling of O-GlcNAc residues with (⫹) orwithout (⫺) GalT and UDP-GalNAz, and were detected using an ABC kit, as described under “Experimental procedures.” Experiments were independently
repeated at least two times.

We were intrigued as to why a knockdown of OGT would
enhance integrin activation. Integrins are the major cell surface
receptors used to assemble and recognize a functional ECM
and to facilitate cell signaling and migration (37). The organization of cell adhesions is complex and includes a number ofcytoplasmic proteins, such as paxillin, talin, FAK, vinculin, and␣-actinin (29). Integrin activation is associated with an array of
biological and pathological conditions involving both outside-in and inside-out signaling (38). Accumulating data haveindicated that the cytoplasmic domain of the integrin ␤1 subunit cooperatively promotes integrin activation through thebinding of talin (39). Consequently, our results clearly showed
the interaction of integrin ␤1 with talin, and the association of
FAK, paxillin, and/or talin both were greatly increased in the
KD cells, which suggests that the KD of OGT promotes insideout signaling (Fig. 5). A reciprocal relationship between
O-GlcNAcylation and O-phosphorylation has been observed in
the specific serine or threonine residue of particular proteins

3120 J. Biol. Chem. (2019) 294(9) 3117–3124(40,41),and,therefore,howO-GlcNAcylationaffectstheO-phosphorylation of FA complex proteins is worthy of clarification.
The O-GlcNAcylation of FAK is noteworthy. Integrins do
not possess enzymatic activity; rather, they associate with anumber of cytoplasmic protein kinases, such as FAK and Src.
Tyrosine-phosphorylated FAK is well-known to be a promoter
of interactions with various Src homology 2– and 3– containing
proteins and to initiate enzymatic cascades via these associatedkinases that ultimately lead to changes in cell behavior (42). By
contrast, serine or threonine phosphorylation on FAK is not
well-understood. FAK phosphorylation at either Ser-732 or
Ser-722 has recently been recognized as important for
microtubule organization, nuclear movement, and neuronalmigration during cell adhesion (43, 44). Interestingly, phosphorylation of both Ser-843 and Ser-910 on FAK exhibited synchronized phosphorylation during cell mitosis (45), which may
be related to O-GlcNAcylation because expression levels of
OGT change during mitosis (46). Furthermore, a cluster of ser-

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Figure 5. Increased focal adhesion complex formation in OGT KD cells. Cell lysates from the control (Ctrl) and KD HeLa cells that were transfected withexpression plasmids of FAK (A), talin (B), or both FAK and talin (C) were immunoprecipitated (IP) by the indicated antibodies and then subjected to Western
blotting as described under “Experimental procedures.” The relative ratios are shown at the bottom (n ⫽ 3 individual experiments). Values represent themean ⫾ S.E. (error bars). *, p ⬍ 0.05 (Welch’s correction t test). Cell lysates were used as input. Experiments were independently repeated at least three times.

FAK was modified by O-GlcNAc

cell migration. Emerging data have already established that
O-GlcNAc modification has a critical role in the progress ofhuman diseases, and particularly diseases such as cancer, diabetes, and Alzheimer’s (7). Intriguingly, FAK has been associated with insulin resistance in adipocytes in the early stages oftype II diabetes (48, 49) and has also been implicated in thedeposition of ␤-amyloid plaque (50, 51). It would be reasonable
to assume that dynamic regulation of FAK O-GlcNAcylation
with phosphorylation may partially serve as a possible explanation for a number of diseases.

Experimental procedures
Antibodies and reagents
Figure 7. Proposed molecular mechanism for the regulation of cell adhesion and migration by O-GlcNAcylation. During cell adhesion, integrin may
form a complex with focal adhesion proteins, such as FAK, talin, and paxillin,to connect with an actin cytoskeleton, which mediates appropriate cell adhesion to promote cell migration. It is well-known that focal adhesion assemblyand disassembly processes are fundamental for efficient cell migration. Most
focal adhesion proteins can be modified by O-GlcNAc and O-phosphate on
serine or threonine residues. In the present study, suppression of the
O-GlcNAcylation of paxillin, talin, and FAK aberrantly enhanced integrin activation, integrin-mediated cell adhesion, and complex formation, which inturn led to an inhibition of cell migration, which strongly suggests that OGT
functions as a key regulator for cell adhesion.

ine phosphorylation sites was recently identified at the initiation of the FA-targeting domain in FAK (47), which may
suggest that some of those sites could be modified by
O-GlcNAcylation. Thus, to elucidate the roles of OGT in cellbiology, it is necessary to identify the specific sites and functions of O-GlcNAcylation in FAK.
Our results indicate that O-GlcNAcylation plays importantroles in regulating cell adhesion, FA complex formation, andExperiments were performed using the following antibodies:mAb against O-GlcNAc (CTD110.6, 9875S) and peroxidaseconjugated secondary antibody against rabbit (7074S) from Cell
Signaling Technology; the rabbit polyclonal antibody against
OGT (O0164) and mAb against ␣-tubulin (T6199) and VSV(V5507) from Sigma; mAb against integrin ␤1 (610468) andpaxillin (610052) from BD Biosciences; mAb against active
integrin ␤1 (HUTS-4; 2079Z) and peroxidase-conjugated secondary antibodies against mouse (AP124P) and goat (AB324P)
from Millipore; Alexa Fluor 488 – conjugated anti-mouse(A11029) from Invitrogen; TO-PRO-3 (T3605) from Molecular
Probes, Inc.; and GFP-agarose (MBL, D153-8) and goat antibody against GFP (Rockland, 600-101-215). The mAb against
human ␤1 (P5D2) was obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Human FN and doxycycline hyclate (D9891) were from Sigma-Aldrich. An ABC kit
was acquired from Vector Laboratories, and Ab-Capcher Mag
was from ProteNova (Takamatsu, Japan).
J. Biol. Chem. (2019) 294(9) 3117–3124

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Figure 6. Comparison of the expression levels of active integrin ␤1 among the control (Ctrl), KD, and OGT-overexpressing cells. A, a representative
immunostaining pattern with anti-active ␤1 or anti-␤1 antibodies in the control, KD, and OGT-overexpressing (OGT) HeLa cells. Cells were cultured on
FN-coated coverslips for 1 h and then subjected to immunostaining analyses. The relative fluorescence intensities of KD and OGT-overexpressing cells werecompared with the control, and relative fluorescence intensity was 1.0 for the control cells. Scale bar, 5 ␮m. B, the expression levels of active and total integrin
␤1 were verified by immunoblotting with the indicated antibodies in control and KD HeLa cells or parent (WT) or transiently OGT-overexpressing HeLa cells. The
relative ratios are shown at the bottom (n ⫽ 10 random fields of view). Values represent the mean ⫾ S.E. (error bars). *, p ⬍ 0.05 (Welch’s correction t test); n.s,
not significant (p ⬎ 0.05). Experiments were independently repeated at least two times.

FAK was modified by O-GlcNAc
Cell culture and expression plasmids

Establishment of doxycycline-inducible OGT knockdown cells
We used CS-RfA-ETBsd DOX-dependent inducible RNAi
mediated by a single lentivirus vector (RIKEN) for the knockdown experiment (54). The following oligonucleotides wereinserted into pENTR/H1/TO (sense, CACCGCTGAGCAGTATTCCGAGAAACTCGAGTTTCTCGGAATACTGCTCAGCC; antisense, AAAAGGCTGAGCAGTATTCCGAGAAACTCGAGTTTCTCGGAATACTGCTCAGC) with minor
modification from a procedure established in a previous report(13). Using LR clonase, inserted oligonucleotide was then transferred to CS-RfA-ETBsd, which encodes tetracycline-dependent transactivators for shRNA expression. To prepare theviruses, PEI MAX was used to transfect the resultant vector into
293T cells with packaging plasmids. HeLa and 293T cells werethen infected by the obtained viruses and selected for stable
integration with 10 ␮g/ml blasticidin. The shRNA-mediatedsilencing of OGT was induced by the addition of DOX in theestablished cell line, and the cells cultured by DOX-free
medium were used as the control in the present study.
Immunoprecipitation and Western blotting
The cells were washed with PBS, and lysed in lysis buffer (20mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) withprotease and phosphatase inhibitors (Nacalai Tesque, Kyoto,
Japan). The supernatants were collected, and the protein concentrations were determined using a bicinchoninic acid protein
assay kit (Pierce). Equal amounts of proteins were subjected to
SDS-PAGE and then transferred to polyvinylidene difluoride
membranes. To detect active integrin ␤1, we prepared samples
under nonreducing conditions. The membranes were blocked
either with 5% nonfat milk in TBST or with 3% BSA for 2 h atroom temperature, and then the proteins were probed with
antibodies against O-GlcNAc, OGT, ␣-tubulin, active integrin␤1 (HUTS-4) (55, 56), integrin ␤1 (Millipore), paxillin, VSV,and GFP. After being washed, the membranes were incubated

3122 J. Biol. Chem. (2019) 294(9) 3117–3124

Cell adhesion assay
Cell adhesion assays were performed in a 96-well CellCarrier(PerkinElmer Life Sciences) coated with FN (5 ␮g/ml) overnight. HeLa cells were pretreated with or without DOX (0.1
␮g/ml) for 24 h. Cells were replated at a density of 104 cells/wellin plates using serum-free DMEM with 0.1% BSA, followed
by incubation at 37 °C for 20 min. Nonadherent cells were
removed by washing three times with PBS. Cells were fixed with
4% formaldehyde and stained 4⬘,6-diamidino-2-phenylindole(Invitrogen) and were then imaged by fluorescent microscopyusing an Operetta CLS (PerkinElmer Life Sciences). To countthe number of nuclei in the each well, images were analyzed
using Harmony software (PerkinElmer Life Sciences).
Immunofluorescence
Cells were plated onto FN-coated glass coverslips (MatTek
Corp., Ashland, MA) for 1 h, washed with PBS, and fixed with
4% paraformaldehyde. For permeabilization, the cells weretreated with 0.1% Triton X-100 in PBS. The cells were blockedwith 0.1% Tween 20 and 3% BSA in PBS and then stained withpaxillin, active ␤1 (HUTS-4), total ␤1 (P5D2), and OGT antibodies overnight at 4 °C. The samples were followed by incubation with anti-mouse Alexa Fluor 488 – conjugated secondary
antibody and were then incubated with TO-PRO-3. Images wereacquired by sequential excitation using an Olympus FV1000 laserscanning confocal microscope with an UPlanSApo ⫻60/1.35 oilobjective and high-sensitivity gallium arsenide phosphide detector
units operated by F10-ASW version 4.02 software. To count the
number of FAs, we followed a protocol previously described using
ImageJ (57), excluding focal adhesions that were less than 0.2 ␮m2,
because these disappeared quickly (58). OGT-overexpressing cells
were identified via co-immunostaining with OGT. The relative
fluorescence intensities of active integrin ␤1 and total integrin ␤1
were quantified using ImageJ software.
Video microscopy
Glass-bottom dishes (Asahi Glass, Shizuoka, Japan) wereprecoated with FN (10 ␮g/ml) in PBS, let stand at 4 °C overnight, and were then blocked with 1% BSA. Ten thousand cells
were suspended in 2 ml of DMEM containing 3% fetal bovine
serum medium, which was then added to each FN-coated glassbottom dish and monitored for 12 h using AxioVision equipment (Carl Zeiss, Oberkochen, Germany). Images wereacquired using an inverted microscope (Axio Observer.D1,
Carl Zeiss) every 10 min with 5% CO2 at 37 °C in a heatedchamber equipped with temperature and CO2 controllers

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HeLa and 293T cell lines (RIKEN, Japan) were maintained at
37 °C in DMEM high-glucose (Invitrogen) supplemented with
10% fetal bovine serum under a humidified atmosphere that
contained 5% CO2. To express GFP-tagged talin (52) and 2⫻
VSV-tagged FAK, expression vector pEGFP-N1-talin-GFP(Addgene 26724) and pRKVSV-FAK were kindly provided by
Dr. Anna Huttenlocher (52) and Dr. Kenneth Yamada (53),respectively. The pcDNA3.1/myc-his expression vector containing human OGT was kindly provided by Dr. Yuanyuan
Ruan (School of Basic Medical Sciences, Fudan University,
Shanghai, China). Transfection was performed using PEI MAX(molecular mass, 40 kDa; Polysciences Inc.) and following
the dictates of the United States patent application (number
US20110020927A1) with minor modifications. Briefly, 24 hprior to transfections, cells were seeded on a 10-cm dish, andexpression vectors with PEI MAX (1 mg/ml in 0.2 M hydrochloric acid) were preincubated for 15 min at a 1:3 ratio in 2,000 ␮lof a solution that contained 20 mM CH3COONa buffer, pH 4.0,
and 150 mM NaCl. Cells and DNA complexes were further
incubated for 24 h with 10 ml of normal culture medium topromote expression.

with horseradish peroxidase– conjugated secondary antibodies. Detection was accomplished using a horseradish peroxidase substrate (Millipore) according to the manufacturer’s
instructions. For immunoprecipitation, the supernatant (500␮g of protein) was incubated with an anti-VSV or an anti-paxillin with an Ab-Capcher Mag. GFP-talin was immunoprecipitated with GFP-conjugated beads. The immunoprecipitates
were washed with lysis buffer and subjected to SDS-PAGE. The
immunocomplexes then were detected using the indicatedantibodies. An mAb against ␣-tubulin was used as the loading
control.

FAK was modified by O-GlcNAc(Onpu-4 and CO2; AR BROWN, Tokyo, Japan) during timelapse imaging. Cell motility was evaluated using an AxioVision
Tracking module (Carl Zeiss).
Chemoenzymatic labeling assay

Statistics
All results shown are the results of at least two independent
experiments and are shown as representative data. The values
represent the mean ⫾ S.E. p values were calculated using
Welch’s correction t test using GraphPad Prism version 5(*, p ⬍ 0.05; **, p ⬍ 0.01).
Author contributions—T. I. and J. G. designed the research; Z. X.
and T. I. performed all experiments; T. F. and Y. W. assisted withexperiments; T. I., T. F., Y. W., and J. G. analyzed and interpreted thedata; Z. X., T. I., Y. W., and J. G. wrote and revised the manuscript;
and all authors approved the final version of the manuscript.
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Contents lists available at ScienceDirect

BBA - General Subjects
journal homepage: www.elsevier.com/locate/bbagen

Deﬁciency of α1,6-fucosyltransferase promotes neuroinﬂammation byincreasing the sensitivity of glial cells to inﬂammatory mediators
Xu Lua,b, Dongmei Zhanga,c, Hayato Shojia, Chengwei Duana, Guowei Zhanga, Tomoya Isajia,⁎⁎
Yuqin Wangb, Tomohiko Fukudaa, , Jianguo Gua,a
Division of Regulatory Glycobiology, Tohoku Medical and Pharmaceutical University, Institute of Molecular Biomembrane and Glycobiology, 4-4-1 Komatsushima, Aobaku, Sendai, Miyagi 981-8558, Japanb
Department of Pharmacology, Pharmacy College, Nantong University, Nantong, Jiangsu Province 226001, Chinac
Clinical Medical Research Center, The Second Aﬃliated Hospital of Nantong University, Nantong, Jiangsu Province 226001, China

A R T I C L E I N FO

A B S T R A C T

Keywords:
Core fucosylation
Cytokines
Fut8
Glial cell
Neuroinﬂammation

Background: α1,6-Fucosyltransferase-deﬁcient (Fut8−/−) mice displayed increased locomotion and schizophrenia-like behaviors. Since neuroinﬂammation is a common pathological change in most brain diseases, thisstudy was focused on investigating the eﬀects of Fut8 in microglia and astrocytes.
Methods: Brain tissues were analyzed using immunohistochemical staining. Core fucosylation and protein expression were analyzed using lectin blot and western blot, respectively. Fut8-knockout (KO) cells were established by the CRISPR/Cas9 system.
Results: The number of Iba-1 positive cells and GFAP positive cells were signiﬁcantly increased in both untreated
and lipopolysaccharide stimulated inﬂammatory conditional Fut8−/− mice by comparison with both wild-type(Fut8+/+) and hetero (Fut8+/−) mice. Stimulation with pro-inﬂammatory factors, such as IFN-γ and IL-6, induced expression levels of fucosylation in primary microglia and astrocytes, as well as in glial cell lines. Cell
motility and iNOS expression were easily induced by IFN-γ in Fut8-KO BV-2 cells compared with wild-type (WT)
cells. In a similar manner, both Fut8-KO C6 cells and primary astrocytes treated with 2-ﬂuoro-L-fucose, a speciﬁc
inhibitor for fucosylation, showed a higher response to IL-6-stimulated phospho-STAT3 signaling, comparedwith WT cells.
Conclusions: Core fucosylation negatively regulates the states of neuroinﬂammation by modulating the sensitivity of microglia and astrocytes to inﬂammatory mediators. The disorders of Fut8−/− mice are caused not only
by neurons but also by glial cell dysfunction.
General signiﬁcance: Core fucose is a novel regulator for neuroinﬂammation in the central nervous system.

1. Introduction

processes, such as tumor formation [4,5], inﬂammation and immune
response [6–9], and central nervous system (CNS) diseases [10,11].
Fut8-deﬁcient (Fut8−/−) mice exhibit a schizophrenia-like phenotype with a decrease in working memory [10] and long-term potentiation [12]. Very recently, a complete loss of core fucosylation inpatients was reported [13]. Those patients showed growth retardation,
severe developmental and growth delays and also including neurological impairment, which was quite similar to that seen in the phenotypes of Fut8−/− mice [10,14]. These data suggested core fucosylation

α1,6-Fucosyltransferase (Fut8) transfers a fucose residue from GDPfucose to the innermost N-acetylglucosamine (GlcNAc) residue via α1,6linkage to form α1,6-fucosylation [1], which is referred to as core fucosylation in mammals [2,3]. In fact, N-glycans with core fucosylation
are widely distributed in a variety of glycoproteins, and diﬀerently
regulate their functions. Accumulating data suggests that Fut8 and its
products play important roles in various physiological and pathologicalAbbreviations: Fut8, α1,6-Fucosyltransferase; CNS, central nervous system; IL-6, interleukin-6; IFN-γ, interferon gamma; LPS, lipopolysaccharide; 2FF, 2-ﬂuoro-Lfucose; Iba-1, ionized calcium binding adaptor molecule 1; GFAP, glial ﬁbrillary acidic protein; AAL, Aleuria Aurantia Lectin; PhoSL, Pholiota Squarrosa Lectin;
ConA, concanavalin A; SNA, Sambucus nigra lectin; MAA, Maackia amurensis lectin; iNOS, inducible nitric oxide synthase; NO, nitric oxide; TGF-β, transforming
growth factor β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-Diamidino-2-phenylindole Dihydrochloride n-Hydrate; STAT1, signal transducer
and activator of transcription 1; STAT3, signal transducer and activator of transcription 3; gp130, glycoprotein 130⁎
Corresponding authors.
E-mail addresses: tfukuda@tohoku-mpu.ac.jp (T. Fukuda), jgu@tohoku-mpu.ac.jp (J. Gu).
https://doi.org/10.1016/j.bbagen.2018.12.008
Received 14 September 2018; Received in revised form 5 December 2018; Accepted 13 December 2018
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plays important roles in the CNS.
The pathophysiology of schizophrenia has not yet been fully elucidated. Studies involving in vivo imaging suggest that neuroinﬂammation may contribute to the pathogenesis of schizophrenia, which could
be due to a dysfunction in glial cells [15,16]. In the CNS, glial cells are
made up mostly of microglia, astrocytes, and oligodendrocytes. For
several decades, many researchers believed that glial cells outnumberedneurons at a ratio of 10:1 [17], but recently researchers used isotropic
fractionators to demonstrate that the ratio of glial cells to neurons is
actually about 1:1 [18]. Anyway, glial cells are known to play crucial
roles in neuronal functions [19,20]. As the only resident macrophagelike cells in the CNS, microglia are the pre-eminent form of active immune defense [21], even though they only account for 10–15% of all
glial cells [22]. In addition to their surveillance role, microglia participates in maintaining synapses and homeostasis in the CNS [23,24].
Astrocytes are the most abundant glial type, and make up ~20–40%[25]. They provide trophic support for neurons [26], and also participate in the maintenance of synapses and in the process of neuroinﬂammation [27,28].
Microglial cells play a key role in neuroinﬂammation. After activation, microglia quickly release several pro-inﬂammatory mediatorssuch as interleukin-1β (IL-1β), interleukin-6 (IL-6), and interferon
gamma (IFN-γ), and induce reactive astrocytes, which leads to thefurther injury of neurons [28]. In fact, postmortem studies have discovered a higher level of activation and increased microglia density inschizophrenia [29]. Gene-set analyses have also shown that genetic
alterations of astrocytes could increase the risk for schizophrenia [30].
These results suggest the potential role of microglia and astrocytes inthis disorder.
Recently, several studies have also demonstrated that N-glycosylation might play important roles in schizophrenia. Postmortem examinations of the cortex of schizophrenia patients have shown alterations in the N-glycosylation of α-amino-3-hydroxy-5-methyl-4isoxazolepropionate receptor (AMPAR), N-methyl-D-aspartate receptor(NMDAR) and γ-aminobutyric acid type A (GABAA) receptors [31–33].
Furthermore, in the superior temporal gyrus of elderly patients withschizophrenia, the expression levels of Fut8 were decreased [34].
The present study was focused on glial cells to explore the underlying mechanisms of the disorders found in the brains of Fut8−/− mice.
We found that the initial status of microglia and astrocytes activationand the neuroinﬂammation model induced by lipopolysaccharide (LPS)
both were signiﬁcantly enhanced in Fut8−/− mice, compared withwild-type (Fut8+/+) mice. In accordance with the in vivo data, thesensitivities to inﬂammatory stimulators such as IFN-γ or IL-6 weregreatly increased in glial cell lines lacking Fut8 (Fut8 KO cells) or primary astrocyte cells treated with 2-ﬂuoro-L-fucose (2FF) [35–37], aﬂuorinated analog of fucose, which could dramatically inhibit fucosylation in cells, compared with those in wild-type (WT) cells. Along withprevious results [10–12], these data clearly demonstrate the importance of Fut8 in microglia and astrocytes, and the disorders of
Fut8−/− mice are caused not only by neurons but also by glial cell
dysfunction.

Smad2 Ser465/467 (3108S), rabbit mAb against signal transducer andactivator of transcription 1 (STAT1) (14994S), and rabbit mAb against
p-STAT1 Tyr701 (9167S) were purchased from Cell Signaling
Technology; mouse mAb against gp130 (MAB5029) was from R&D
Systems; mouse mAb against α-tubulin (T6199) was from Sigma andrabbit pAb against glyceraldehyde-3-phosphate dehydrogenase(GAPDH) (sc-25,778) was from Santa Cruz.
Biotinylated Aleuria Aurantia Lectin (AAL), concanavalin A (ConA),
sambucus nigra lectin (SNA) and maackia amurensis lectin (MAA) wereobtained from J-oil Mills (Tokyo, Japan). Biotinylated Pholiota
Squarrosa Lectin (PhoSL), which speciﬁcally recognizes core fucosylated
N-glycans, was a generous gift from Dr. Yuka Kobayashi (J-oil Mills,
Tokyo, Japan).
The peroxidase-conjugated goat against mouse and rabbit IgG antibodies, and donkey against sheep/goat IgG antibody were obtained
from Promega, Cell Signaling Technology and Millipore Corporation,
respectively. Goat anti-mouse IgG Alexa Fluor 568, goat anti-rabbit IgG
Alexa Fluor 488, streptavidin-conjugate Alexa Fluor 647 were purchasedfrom
Invitrogen.
4′,6-Diamidino-2-phenylindole
Dihydrochloride n-Hydrate (DAPI) was obtained from Wako, Japan.
LPS puriﬁed from Escherichia coli 0111:B4 was the product of Sigma,
recombinant mouse IFN-γ was purchased from Prospec Bio, recombinant rat IL-6 was purchased from PEPROTECH, and 2FF waspurchased from Synchem, Inc., IL, USA.
2.2. Animals
The generation of Fut8−/− mice by a gene-targeting technique has
been described previously [10]. ICR genetic background F1 heterozygous mice were mated with JF1/Ms. (Japanese fancy mouse 1, M. m.
molossinus–derived inbred strains) [38] mice to produce F2 generation
mice, and the F2 generation mice were paired with other F2 generation
mice to generate the Fut8−/− mice. All experiments were conductedwith male and female mice 5–6 weeks old. Mice were housed in groups
in each cage under conditions of constant temperature (22 ± 2 °C) andhumidity (55 ± 5%) on a 12-h light-dark cycle (lights on:
07:00–19:00) with free access to food and water. All animal experiments were performed in accordance with protocols approved by the
Animal Care and Use Committee of the Graduate School of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University.
2.3. Cell culture
Mouse microglia cell line BV-2 was kindly provided by Professor
Elisabetta Blasi (University of Modena and Reggio Emilia, Modena,
Italy). Rat glioma cell line C6 was purchased from American Type
Culture Collection (Rockville, MD, USA). Cells were cultured in
Dulbecco's modiﬁed Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidiﬁed atmospherewith 5% CO2. The medium was replaced every 3 days.
Mouse primary cells from brain tissues were prepared as previously
reported with some modiﬁcations [39]. Brieﬂy, newborn (day 0–1) ICR
Fut8+/+ mice were euthanized by decapitation using scissors, thecortex was removed, and cut into pieces on ice, and then digested with
0.125% trypsin for 20 min at 37 °C. A 100 μm Nylon (Falcon) cellstrainer was used to ﬁlter the undigested tissue. This procedure wasfollowed by centrifugation at 300g for 5 min, and then the cells were resuspended and plated on poly-D-lysine (PDL, 50 μg/mL)-coated culture
ﬂasks. Single-cell suspensions were cultured in DMEM/F12 supplemented with 10% FBS and 1% penicillin-streptomycin (100 U/mL). The
medium was replaced with fresh medium after 24 h and changed every
3 days. In order to isolate the primary microglia, after 14 days, themixed cells were shaken gently at 180 rpm for 2 h at 37 °C, and then the
supernatants containing microglia were collected and plated on the new
PDL-coated culture dishes. The remaining cells were washed withphosphate buﬀered saline (PBS), and then detached with 0.25% trypsin

2. Materials and methods
2.1. Materials
The experiments were performed using the following antibodies:mouse mAb against inducible nitric oxide synthase (iNOS) (ab49999)
and goat pAb against ionized calcium binding adaptor molecule 1 (Iba1) (ab5076) were purchased from Abcam; rabbit pAb against Iba-1(019–19,741) was from Wako, Japan; mouse mAb against glial ﬁbrillary acidic protein (GFAP) (MAB360) was from Millipore
Corporation; mouse mAb against signal transducer and activator oftranscription 3 (STAT3) (9139S), rabbit mAb against p-STAT3 Tyr705(9145S), mouse mAb against Smad2 (3103S), rabbit mAb against p

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analyzed via FACSCalibur ﬂow cytometer (BD Biosciences).

to obtain the primary astrocytes. After centrifuge, the primary astrocytes were also cultured in PDL-coated culture dishes. All cells weremaintained in a 37 °C incubator containing 95% air and 5% CO2. Immunoﬂuorescent staining with Iba-1 and GFAP antibodies was used to
diﬀerentiate the microglia and astrocytes. The purity of both the microglia and astrocytes was > 90%.

2.7. Western blot and lectin blot analyses
The mice were euthanized by decapitation after 24 h of LPS (1 mg/kg) administration. Brain hippocampi were rapidly removed, placed onice, and then homogenized in 4 volumes of TBS (20 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 1% protease) with φ2.0 Zirconia Beads by Micro
Smash MS-100 (Digital Biology), according to the manufacturer's instructions. After centrifugation at 8000 rpm for 15 min, the supernatants were collected and used for analysis.
Cells cultured under diﬀerent conditions were washed with PBS andlysed with lysis buﬀer that contained 20 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 1% Triton-X100, and 1% protease and phosphatase inhibitor
cocktail (Nacalai Tesque, Japan). Protein concentrations were measured using a bicinchoninic acid protein assay kit (Thermo Fisher
Scientiﬁc, Wilmington, DE, USA).
Equal amounts of proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF (Millipore, Billerica, MA, USA) membranes for future
detection. For western blot, after blocking with 5% skim milk or 3%
bovine serum albumin (BSA) for 2 h at room temperature, the membranes were incubated with speciﬁc primary antibodies at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase(HRP)-conjugated secondary antibody. For lectin blot, the membranes
were blocked in 3% BSA overnight at 4 °C, followed by incubation withbiotinylated AAL, ConA, SNA or MAA lectin for 2 h at room temperature, and then the immunoreactive bands were probed with a
Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Finally,
speciﬁc proteins were visualized using an ECL select™ reagent(Amersham, Piscataway, NJ, USA).

2.4. Immunoﬂuorescence
After intraperitoneal injection with LPS (1 mg/kg) for 24 h, animals
were deeply anesthetized with sodium pentobarbital and intracardiallyperfused with 50 ml of PBS, followed by 50 ml of 4% paraformaldehyde(PFA; Sigma-Aldrich) in 0.1 M PBS. Brains were post-ﬁxed in 4% PFA0.1 M PBS at 4 °C overnight, followed by immersion in 20% sucrose0.1 M PBS for 48 h. The brains were cut into 40 μm sections that included the dorsal dentate gyrus from bregma −1.60 mm to −2.60 mm
using a cryostat (MICROM HM560, Microm International GmbH,
Walldorf, Germany). Frozen sections were mounted on glass slides(Matsunami Glass, Japan). The sections were incubated with PBS containing 1% normal goat serum and 0.3% Triton X-100 (PBSGT) at room
temperature for 2 h. The sections were incubated overnight at 4 °C withrabbit anti-Iba1 (1:200) and mouse anti-GFAP (1:200) antibodies.
Sections were washed 3 times with PBS every 10 min, then incubatedwith goat anti-mouse IgG Alexa Fluor 568 (1:500) and goat anti-rabbit
IgG Alexa Fluor 488 (1:500) in PBSGT for 2 h at room temperature.
Finally, sections were washed 3 times with PBS every 10 min andcoverslipped with ﬂuorescent mounting medium (Dako, Carpinteria,
CA, USA). Immunoﬂuorescent images were analyzed using a confocal
laser-scanning microscope (A1Rsi: Nikon, Tokyo, Japan). The numberof Iba-1 positive cells in the images (0.04 cm2) and the length of eachsoma radius were measured using NIS-Elements AR Analysis (Nikon,
Tokyo, Japan), the area of superﬁcial somas was then calculated byfollowing the equation (4πR2).
2.5. Generation of CRISPR/Cas9-based Fut8- KO Cells

2.8. Video microscope

The CRISPR/Cas9-based Fut8-KO cells were established, as described previously [40]. Brieﬂy, the sgRNA-specifying oligo sequences(sequences one: 5′-CACCGCAGAATTGGCGCTATGCTAC-3′ and 5′-AAA
CGTAGCATAGCGCCAATTCTGC-3′; sequences two: 5′-CACCGATTCGTCCACAACCTTGGC-3′ and 5′-AAACGCCAAGGTTGTGGACGAATC-3′)
spanning Mus musculus fucosyltransferase 8 (NM_001252614) and thesgRNA-specifying oligo sequences (sequences one: 5′-CACCGGAGATAAGTTATTCTCCGC-3′ and 5′-AAACGCGGAGAATAACTTATCTCC-3′; sequences two: 5′-CACCGATTTGATTCGTCCACAACCT-3′ and 5′-AAACA
GGTTGTGGACGAATCAAATC-3′) spanning Rattus norvegicus fucosyltransferase 8 (NM_001002289.1) were cloned into the pSpCas9 (BB)-2AGFP (Addgene plasmid ID: 48138) vector [40], which was a kind giftfrom Dr. Feng Zhang. The plasmid was electroincorporated into the BV2 and C6 cells according to the manufacturer's instructions (Amaxa®
cell line Nucleofector R kit V). After 72 h of transfection, GFP-positive
cells were sorted using the FACS Aria II (BD Bioscience). Cells that were
Fut8 positive and GFP negative were sorted approximately three times
using PhoSL during the following 3-week culture. The Fut8-KO cells
were conﬁrmed by ﬂow cytometric and lectin blotting analyses, as
described in the following section.

BV-2 Cells were plated at a density of 1 × 104 in a glass-bottom dish(Asahi Glass, Shizuoka, Japan). After the cells attached over night, theculture media were replaced with fresh media without phenol red butwith IFN-γ (20 ng/ml), and then cell motility was monitored for 12 husing Axio Vision (Carl Zeiss, Jena, Germany). Images were acquired
using an inverted microscope (AxioObserver.D1; Carl Zeiss) every
10 min with 5% CO2 at 37 °C in a heated chamber equipped withtemperature and CO2 controllers (Onpu-4 and CO2; AR Brown, Tokyo,
Japan) during time-lapse imaging. Cell migration was evaluated using
an AxioVision Tracking module (Carl Zeiss).

2.9. Immunoprecipitation
After being washed with PBS, cells were treated with lysis buﬀer, as
previously described. Anti-gp130 antibody and Ab-Capcher Protein AR28 agarose (Protenova, Tokushima, Japan) were ﬁrst mixed together
for 1 h on ice, and then gently mixed again every 10 min. The cell lysates were then immunoprecipitated with the antibody-agarose solution
for 1 h at 4 °C with rotation. The immunoprecipitates were then washedtwice with TBS and subjected to SDS-PAGE.

2.6. Flow cytometric analysis
2.10. Statistical analysis

Cells were grown to ~80% conﬂuency, then detached from culture
dishes, washed with ice-cold PBS, and subsequently stained with biotinylated PhoSL for 1 h on ice, followed by incubation with streptavidin-conjugate Alexa Fluor 647 for 1 h on ice in the dark. A negative
control was prepared only with streptavidin-conjugate Alexa Fluor 647.
During incubation, the cells were gently mixed every 10 min by
ﬂicking. Finally, cells were washed 3 times with ice-cold PBS and

Results are reported as the means ± S.E.M. Statistical analyses were
performed using an unpaired Student's t-test with Welch's correction ora one-way analysis of variance (ANOVA) with Tukey's post hoc test by
GraphPad Prism version 5. Statistical signiﬁcance was deﬁned as
p < .05.
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Fig. 1. Eﬀects of core fucosylation on the activation of glial cells in mice brain tissues. Brain sections of mice were prepared and analyzed, as described in“Experimental procedures”. (A) The representative microscopy images immunostained with anti-Iba1 (green) antibodies, anti-GFAP (red) antibodies, and DAPI (blue)in the hippocampus of brain tissues of mice under normal conditions; (B) The representative microscopy images after the administration of LPS by intraperitoneal
injection (i.p. 1 mg/kg, 24 h). (C) Quantiﬁcation of Iba-1 positive cells using representative tissue sections stained with anti-Iba1 antibody. Data represent themean ± S.E.M.; p values were calculated using one-way ANOVA with Tukey's multiple comparison test. *p < .05 vs. Fut8+/+ mice (n = 3, n: number of slices;
N = 3, N: number of the mice per group). (D) Quantiﬁcation of microglia soma size using representative tissue sections stained with anti-Iba1 antibody. Data
represent the mean ± S.E.M.; p values were calculated using one-way ANOVA with Tukey's multiple comparison test. **p < .01 vs. Fut8+/+ mice (n = 96, n:
number of recordings; N = 3, N: number of the mice per group).

3. Results

the basal level of microglia activation in vivo.

3.1. The basal status of microglia activation was increased in Fut8−/− mice

3.2. Microglia and astrocytes of Fut8−/− showed a greater level ofsensitivity to LPSFirst, we checked the basal status of glial cell activation by detecting
Iba-1 and GFAP, which are markers for microglia and astrocytes, respectively, in the hippocampus regions in vivo. The immunochemical
staining with anti-Iba-1 antibody clearly showed that the Iba-1-positive
cells were signiﬁcantly increased in Fut8−/− mice under normal conditions without treatment, compared with that of the Fut8+/+ mice
(Fig. 1A and C). Interestingly, the number of Iba-1-positive cells in the
Fut8+/− mice fell between those of both the Fut8+/+ and Fut8−/−
mice, which may have been due to the lower enzyme activity of Fut8.
Furthermore, the sizes of the microglia, evaluated by the superﬁcial
area of the central soma, in Fut8−/− mice were larger than those in the
Fut8+/+ mice, while the sizes in the Fut8+/− mice fell between thoseof both the Fut8+/+ and Fut8−/− mice (Fig. 1A and D). These results
suggest that the deﬁciency of Fut8 could have spontaneously increased

It is well known that neuroinﬂammation is a common pathologicalchange in many disorders of the CNS such as Alzheimer's disease [41]and Parkinson's disease [42], and that it also could participate in thepathology of schizophrenia [43]. Here, we established a systemic inﬂammatory model induced by a low dose of LPS at 1 mg/kg via intraperitoneal injection, as described in “Experimental procedures”. The
immunochemical staining of brain tissues with anti-Iba-1 antibody
showed greater increase in microglia activation, which included both
numbers and sizes in Fut8−/− mice, compared with those in Fut8+/−
and Fut8+/+ mice (Fig. 1B, C and D). Consistent with the immunohistochemical analysis, western blot analysis has shown thatwithout LPS treatment the basal expression levels of Iba-1 are increased
in both Fut8−/− and Fut8+/− mice, compared with that in Fut8+/+

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Fig. 2. Eﬀects of LPS on fucosylation, and expression of Iba-1 and GFAP in mice brain tissues.
Tissue lysates from mice hippocampi were prepared
as described in “Experimental procedures”. (A) The
expression levels of Iba-1 and GFAP were examinedby western blotting with anti-Iba-1 and anti-GFAP
antibodies. GAPDH was used as a loading control.
(B) Quantitative analysis of Iba-1 protein expression
in normal and LPS-stimulated Fut8+/+ and Fut8−/−
mice. Data represent the mean ± S.E.M; p values
were calculated using unpaired t-test with Welch's
correction. **p < .01 vs. Fut8+/+ mice (n = 3). (C)Quantitative analysis of GFAP protein expression innormal and LPS-stimulated Fut8+/+ and Fut8−/−
mice. Data represent the mean ± S.E.M; p values
were calculated using unpaired t-test with Welch's
correction. **p < .01 vs. Fut8+/+ mice (n = 3). (D)The expression levels of fucosylation were detectedby AAL lectin blot. Asterisks indicate nonspeciﬁcbands.

mice (Fig. 2A, B), and the injection of LPS greatly increased the expression levels of Iba-1 in the Fut8−/− and Fut8+/− mice, but only
modestly increased the levels in Fut8+/+ mice (Fig. 2A, B). Western
blot analysis also showed a signiﬁcantly increased expression of GFAP
in both untreated and LPS-treated Fut8−/− and Fut8+/− mice, compared with Fut8+/+ mice (Fig. 2A, C). These results indicated that Fut8
may negatively regulate microglia and astrocytes responses to extrinsic
stimuli during the process of neuroinﬂammation.
It is worth mentioning that the inﬂammation induced by LPS increased the expression of fucosylation. The lectin blotting with AAL,
which preferentially recognizes core fucose [44], showed an increase inboth Fut8+/− and Fut8+/+ mice brain tissues for 24 h following an
injection of LPS. It is reasonable to speculate that speciﬁc reactive
bands simply could not be detected in the Fut8−/− mice (Fig. 2D).

expression of fucosylation in microglia.
3.4. Eﬀects of Fut8 on neuroinﬂammation in BV-2 cells
To explore the roles of core fucosylation in microglia-mediatedneuroinﬂammation, we established a core fucosylation-deﬁcient BV-2
cell line using the CRISPR/Cas9 system described in “Experimental
procedures”. The eﬃciency of Fut8 KO was conﬁrmed by AAL blot
analysis (Fig. 4A) and ﬂow cytometric analysis stained with PhoSL(Fig. 4E). These results clearly showed that core fucosylation wascompletely abolished in Fut8 KO cells. The results of ConA, SNA and
MAA lectin blotting showed no signiﬁcant diﬀerences between WT and
KO cells (Fig. 4B, C and D), which suggested that only core fucosylation
was speciﬁcally blocked. Here, we chose KO2 cells for the following
experiments.
Western blot assay revealed that IFN-γ induced iNOS expression
with a dose-dependent manner in both the WT and KO cells. However,
the induction of iNOS expression by IFN-γ at doses of both 10 ng/ml
and 20 ng/ml was much higher in the KO cells, compared with that in
WT cells (Fig. 5A, B). The time-course eﬀects of IFN-γ at 20 ng/ml also
clearly showed that the iNOS expression levels were higher in the KO
cells, compared with those in WT cells (Fig. 5C, D). In addition, we used
time-lapse microscopy to detect cell motility, which is known to indicate the activation of microglia after inﬂammatory stimulation. We
found that those cells did not move under normal culture condition,
while after IFN-γ stimulation, the cell motility was signiﬁcantly increased in the KO cells (Fig. 5E).
Consistently, the IFN-γ-induced phosphorylation levels of STAT1 in
the KO cells were slightly higher than those in the WT cells (Fig. 5F, G).
While examining the involvement of crosstalk between IFN-γ- andtransforming growth factor β (TGF-β)-mediated signaling, a common
anti-inﬂammatory cytokine [48,49], we detected phosphorylation

3.3. Neuroinﬂammation upregulated fucosylation expression in microglia
We isolated primary microglial cells from Fut8+/+ mice brain tissues and stimulated them with a commonly used stimuli for inﬂammation, IFN-γ [45], which also shows an increased expression inschizophrenia patients [46]. The IFN-γ stimulation consistently increased the expression of fucosylation detected by lectin blotting withAAL (Fig. 3A), and induced the expression of iNOS, which is an enzyme
responsible for the inﬂammation-induced production of nitric oxide(NO) [47]. To further conﬁrm the eﬀects of neuroinﬂammation on fucosylation expression in microglia, we used BV-2, a microglial cell line,
as a cell model. The reactivity with AAL was also enhanced in a dose(Fig. 3B) and time-dependent manner (Fig. 3C). The expression of iNOS
induced by IFN-γ was observed at a ﬁnal concentration at 10 ng/ml,
and remarkably appeared at 20 ng/ml (Fig. 3B). In addition, iNOS expression reached a peak at 24 h, after which it diminished (Fig. 3C).
These data showed that neuroinﬂammation might upregulate the%%$*HQHUDO6XEMHFWV  ²

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Fig. 3. Changes in fucosylation and iNOS expression in primary microglia cells and in BV-2
cells treated with IFN-γ. (A) Primary microglia cells
were prepared as described in “Experimental procedures”, and were then treated with IFN-γ (20 ng/ml)
at the indicated times. Equal amounts of cell lysates
were detected via AAL lectin blot (upper panel) andanti-iNOS antibody (middle panel). (B) BV-2 cells
were treated with IFN-γ for 24 h at the indicatedconcentrations, and then harvested. The cell lysates
were detected via AAL lectin blot (upper panel) andanti-iNOS antibody (middle panel). (C) BV-2 cells
were treated with IFN-γ (20 ng/ml) at the indicatedtimes, and then harvested for AAL lectin blot (upper
panel) and anti-iNOS antibody (middle panel)testing. α-tubulin was used as a loading control.

levels of Smad2, which is a speciﬁc TGF-β downstream signaling molecule. As shown in Fig. 5H and I, the expression of p-Smad2 was detected under stimulation with IFN-γ at 12 and 24 h in the WT cells,
while it was undetectable in the KO cells. These data indicated that a
deﬁciency in Fut8 could increase the sensitivity of microglia to inﬂammatory stimulators and increase the inﬂammatory reaction while
decrease the anti-inﬂammatory reaction.

neuroinﬂammation, we used glioma C6 cells as an astrocyte cell model.
We established the Fut8-KO cells via the CRISPR/Cas9 system, which
was conﬁrmed by AAL lectin blotting (Fig. 7A) and ﬂow cytometricanalysis (Fig. 7B). We chose the KO2 cells for further experiments.
Consistent with data obtained from primary astrocytes (Fig. 6), theresponses stimulated by IL-6 in KO cells were sharper and stronger thanthose in the WT (Fig. 7C, D). When considering the core fucosylationinﬂuence on the functions of glycoproteins expressed on the cell surface, we attempted to detect whether these receptors were modiﬁed bycore fucosylation. We found that glycoprotein 130 (gp130), one subunitof IL-6 receptor, contained core fucosylation as detected by AAL lectinin the WT cells, and the reactivity with AAL was abolished in the KO
cells (Fig. 7E). Unfortunately, at the present time, we could not conclude whether IFN-γ and IL-6 receptors are also modiﬁed by core fucosylation, since the appropriate antibodies for immunoprecipitation
were unavailable.

3.5. Eﬀects of Fut8 expression on cellular signaling in astrocytes
It is well known that astrocytes also participate in neuroinﬂammation [50]. Thus, we isolated the primary astrocytes from the Fut8+/+
mice brain tissues, and treated them with or without 2FF. After treatment for 3 days, the inhibitory eﬀects of 2FF were observed at a ﬁnal
concentration at 30 μM, and remarkably appeared at 100 μM (Fig. 6A).
IL-6 is an important inﬂammatory cytokine in schizophrenia [46], andthe responses which it stimulates were compared between the cells. As
shown in Fig. 6B and C, IL-6-mediated phosphorylation levels of STAT3
were higher in the 2FF-pretreated cells, compared with those in thecontrol cells.
To further explore the eﬀect of Fut8 in astrocytes during

4. Discussion
In the present study, we investigated the potential roles of corefucosylation involved in neuroinﬂammation, and found that a higher

Fig. 4. Established the Fut8 KO BV-2 cell line. The Fut8-deﬁcient BV-2 cells were established using the CRISPR/Cas9 system, as described in “Experimental
procedures”. (A) Equal amounts of cell lysates were detected by AAL lectin blot with α-tubulin used as the loading control. Asterisks indicate nonspeciﬁc bands. Equal
amounts of cell lysates were detected by ConA lectin blot (B), SNA lectin blot (C), and MAA lectin blot (D). (E) The expression level of core fucosylation on the cellsurface recognized by PhoSL, was analyzed via ﬂow cytometry.
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Fig. 5. Eﬀects of core fucosylation on cellular signaling and cell motility in BV-2 cells treated with IFN-γ. (A) After cells were stimulated with IFN-γ at theindicated doses for 24 h, the expression levels of iNOS in both WT and KO cells were examined by western blotting with anti-iNOS antibody. (B) Quantitative analysis
of iNOS protein expression in IFN-γ-stimulated WT and KO cells. Data represent the mean ± S.E.M; p values were calculated using unpaired t-test with Welch's
correction. *p < .05 vs. WT cells (n = 3). (C) After cells were stimulated with IFN-γ at 20 ng/ml for the indicated time, the expression levels of iNOS in both WT and
KO cells were examined by western blotting with anti-iNOS antibody. (D) Quantitative analysis of iNOS protein expression in IFN-γ-stimulated WT and KO cells. Data
represent the mean ± S.E.M; p values were calculated using unpaired t-test with Welch's correction. *p < .05 vs. WT cells (n = 3). (E) Cell motility of BV-2 cells was
examined via video microscope, as described in “Experimental procedures”. The cell moving distances of both WT and KO cells were recorded during the stimulation
with IFN-γ (20 ng/ml) for 12 h. Data represent the mean ± S.E.M; p values were calculated using unpaired t-test with Welch's correction. **p < .01 vs. WT cells(n = 33, n: numbers of recording). (F) After cells were stimulated with IFN-γ (20 ng/ml) at the indicated times, the expression levels of phosphorylation for STAT1, a
downstream signaling of IFN-γ, in both WT and KO cells were examined by western blotting with anti-phospho-STAT1 antibody. Total STAT1 was used as a loading
control. (G) Quantitative analysis of p-STAT1 protein expression in IFN-γ-stimulated WT and KO cells. Data represent the mean ± S.E.M; p values were calculatedusing unpaired t-test with Welch's correction. *p < .05 vs. WT cells (n = 3). (H) After cells were stimulated with IFN-γ (20 ng/ml) at indicated times, the expression
levels of the phosphorylation of Smad2, a speciﬁc signaling of TGF-β, were examined by western blotting in both WT and KO cells with anti-phospho-Smad2
antibody. Total Smad2 was used as a loading control. (I) Quantitative analysis of p-Smad2 protein expression in IFN-γ-stimulated WT and KO cells. Data represent themean ± S.E.M; p values were calculated using unpaired t-test with Welch's correction. **p < .01 vs. WT cells(n = 3).

activation of microglia and astrocytes was observed in Fut8−/− mice,compared with that in Fut8+/+ mice. The experiments using primarycells and cell models of microglia and astrocytes suggested that suppression of core fucosylation resulted in an increase in sensitivity for
pro-inﬂammatory cytokines such as IFN-γ and IL-6. Furthermore, theresults of mouse inﬂammatory models induced by LPS also showed thatthe response for LPS was greatly increased in the Fut8−/− mice, compared with that in Fut8+/+ mice, although the responses were diﬀerent
from results obtained from mouse embryonic ﬁbroblasts [51], which
may be due to diﬀerent cell types and the existence of blood-brain
barrier. It is also worth noting that those responses in the Fut8+/− micealways occurred on a level that fell between that of Fut8−/− andFut8+/+ mice. These results clearly suggest that core fucosylation negatively regulates the functions of microglia and astrocytes in neuroinﬂammation.
Neuroinﬂammation could protect the CNS from harmful stimuli
arising from both endogenous and exogenous substances in physiological conditions. However, uncontrolled or persistent neuroinﬂammation is potentially harmful and can result in cellular damage, which isparticularly relevant to neurodegenerative diseases [52]. Our dataclearly showed that the microglia cells in Fut8−/− mice exist in aspontaneously activated state, while those in Fut8+/+ mice are in a

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Fig. 6. Eﬀects of 2FF on fucosylation and cellular signaling in primary astrocytes. (A) Primary astrocytes were prepared as described in “Experimental
procedures”, and were cultured with 2FF, an inhibitor of fucosylation, for 3 days at the indicated concentrations. Equal amounts of cell lysates were detected by AAL
lectin blot. α-Tubulin was used as a loading control. Asterisks indicate the nonspeciﬁc bands. (B) The primary astrocytes were pretreated with or without 2FF at
30 μM for 3 days, and then were further stimulated with or without IL-6 at the indicated times. The expression levels of phosphorylated STAT3 in those cells were
examined by western blotting with anti-phospho-STAT3 antibody. Total STAT3 was used as a loading control. (C) Quantitative analysis of the eﬀects of 2FF on pSTAT3 protein expression in IL-6-stimulated primary cells. Data represent the mean ± S.E.M; p values were calculated using unpaired t-test with Welch's correction.
**p < .01 vs. control cells (n = 3).

resting state under normal conditions (Fig. 1), which suggests the occurrence of persistent neuroinﬂammation in the Fut8−/− mice. Further
in vitro experiments have shown that a lack of core fucosylation could
increase the response of the microglia cell line BV-2 to IFN-γ (Fig. 5),
and the IL-6-mediated cellular signaling was greatly promoted by primary astrocytes treated with 2FF, an inhibitor of fucosylation, which
was also conﬁrmed by the deletion of Fut8 in glioma C6 cells (Figs. 6,7).
All these data indicated that deﬁciency of core fucosylation might increase the sensitivity of microglia and astrocytes to stimuli. It is also
worth mentioning that both in vivo and in vitro experiments showed theincreased expression of AAL after long-time inﬂammatory stimulation.
This interesting phenotype may be due to a negative feedback of inﬂammation.
We wondered why the sensitivity of microglia and astrocytes increases with a lack of core fucosylation. Although the underlying molecular mechanism remains unclear, the following scenario is plausible.
First, a lack of core fucosylation on these cytokine receptors mightenhance the interaction between a receptor and its ligand, and could
promote several pro-inﬂammatory signaling (Fig. 8). Previous studies
by our group and by those of other groups have indicated that many
receptors expressed on the cell surface contain core fucose: TGF-β1
receptors [14,53], epidermal growth factor (EGF) receptors [54,55], T
cell receptors (TCR) [6,9], and integrin α3β1 [56]. The present study
found that gp130, an important transmembrane protein of IL-6 signaling complexes, also contain core fucose (Fig. 7). Loss of core fucoseon activin receptors [11] and AMPARs, one type of ionotropic glutamate receptor, enhanced the formation of receptor complexes [12],which constitutively activated intracellular signaling. The results of thepresent study showed an enhancement of the IL-6-induced p-STAT3
signal pathway in Fut8 KO C6 cells (Fig. 7) as well as in the Fut8 KO BV2 cells (data not shown), and the IFN-γ-induced p-STAT1 signaling inFut8 KO BV-2 cells (Fig. 5) as well as in the Fut8 KO C6 cells (data notshown), all of which could share similar mechanisms. The detailed
molecular mechanisms require further studies.
Second, core fucosylation deﬁciency could down-regulate anti-inﬂammatory signaling (Fig. 8). It is known that microglia can exist in
two diﬀerent states: one is an activated state (M1), which is typiﬁed by
the expression of inﬂammatory cytokines and reactive oxygen species
such as NO produced by iNOS; the other is a state of alternative activation (M2), which exhibits the properties of an anti-inﬂammatory
phenotype involved in the production of interleukin 4 (IL-4), interleukin 10 (IL-10), and TGF-β etc., that are implicated in inhibiting inﬂammation and restoring homeostasis in the CNS [57]. As describedabove, the TGF-β-induced Smad2/3 signaling pathway is also one of theimportant anti-inﬂammatory signaling pathways. Core fucosylation
diﬀerentially regulates the biological functions of receptors. Our previous study showed that a lack of core fucose led to a marked reductionin the ligand-binding ability and the downstream signaling of several
receptors [8,14,53]. A lack of core fucose in TGF-β receptors suppressed
its binding with TGF-β and subsequently inhibited its downstream
signaling such as in the phosphorylation of Smad2/3, which resulted in
the development of an emphysema-like phenotype in Fut8−/− mice
[14,53]. Furthermore, in a cigarette smoke-induced emphysema model,
the Fut8+/− mice showed a higher inﬂammatory response than that of
Fut8+/+ mice [8]. In this study, coincidentally, we observed a modest
expression of phosphorylated Smad2 following stimulation with IFN-γ
in WT BV-2 cells, but this expression was below the detection level inthe KO cells (Fig. 5). These data suggested that the anti-inﬂammatory
signaling pathways, at least TGF-β signaling pathway, could be downregulated in Fut8 deﬁcient cells.
In summary, results from the present study clearly showed that corefucosylation exerts dual eﬀects in microglia and astrocytes. Deﬁciencyof Fut8 enhanced the pro-inﬂammatory signaling pathways, while inhibited the anti-inﬂammatory signaling pathways (Fig. 8). It should be

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Fig. 7. Eﬀects of core fucosylation on cellular signaling in C6 cells. Establishment of the Fut8 KO C6 cells was performed via a CRISPR/Cas9 system, as describedin “Experimental procedures”. (A) Equal amounts of cell lysates were detected by AAL lectin blot. (B) The expression level of core fucosylation recognized by PhoSL
on the cell surfaces was subjected to ﬂow cytometric analysis. (C) The expression levels of phosphorylated STAT3 in both WT and KO cells treated with or without IL6 stimulation at indicated times, were examined by western blotting with anti-phospho-STAT3 antibody. Total STAT3 was used as a loading control. (D) Quantitative
analysis of p-STAT3 protein expression in IL-6-stimulated C6 WT and KO cells. Data represent the mean ± S.E.M; p values were calculated using unpaired t-test with
Welch's correction. *p < .05 vs. WT cells (n = 3). (E) Equal amounts of cell lysates were immunoprecipitated (IP) with anti-gp130 antibody, which was followed by
AAL lectin blot (upper panel). Whole cell lysates were directly blotted with anti-gp130 (as an input, middle panel) and α-tubulin (as a loading control, lower panel)
antibodies.

noted that only modest changes in the pro-inﬂammatory and anti-inﬂammatory signaling could be detected between WT and Fut8 KO cells
in this study, but in concerted responses these small eﬀects may resultin a big impact in vivo, as shown in Fig. 1. Considering the importantroles of microglia during brain development [58] and the participationof glial cells in schizophrenia [16], and the schizophrenia-like phenotype [10] as well as the high rate of mortality [14] found in Fut8−/−
mice, we concluded that the disorders in CNS with deﬁcient core fucosylation may be caused by not only neurons but also by glial cells.

Fig. 8. A simpliﬁed model for core fucosylation
on a neuroinﬂammation model. Based on our observations in the present study, loss of core fucosylation could cause diﬀerences in the regulation of thesensitivities of microglia and astrocytes to stimuli.
Core fucose deﬁciency seemed to enhance the proinﬂammatory signaling, such as IFN-γ/p-STAT1 andIL-6/p-STAT3 signaling pathways, while decrease
the anti-inﬂammatory signaling, such as in the TGFβ/p-Smad2 signaling pathway, which has been also
observed in lung tissues as well as in ﬁbroblast cells[14]. It should be noted that only modest changes
could be detected in the pro-inﬂammatory and antiinﬂammatory signaling levels between WT and Fut8
KO glial cells. Considering most cytokine receptors,
such as TGF-β receptor and gp130, could be corefucosylated, we believe that the concerted responsesof these small eﬀects could result in a signiﬁcant
impact in vivo, as observed in Fut8 KO mice as well as
in Fut8-deﬁcient patients [10,12,13].

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X. Lu, T. Fukuda and J. Gu designed the research; X. Lu performed
the mice in vivo experiments, primary microglia and BV-2 related experiments; H. Shoji performed the primary astrocytes experiments; D.
Zhang performed the C6 cell-related experiments; C. Duan, G. Zhang
and Y. Wang assisted with experiments; X. Lu, T. Isaji, T. Fukuda, and J.
Gu analyzed and interpreted the data; X. Lu, D. Zhang, T. Isaji, T.
Fukuda, and J. Gu wrote and revised the manuscript; and, all authors
approved the ﬁnal version of the manuscript.
Funding sources
This work was supported in part by a Grant-in-Aid for ScientiﬁcResearch (15H04354 to J.G.; 17 K08284 to T.F.) from the Japan Society
for the Promotion of Science; grant from National Natural Science
Foundation of China (No. 31670807); and by a Grant-in-Aid for
Scientiﬁc Research on Innovative Areas (18H04868 to J.G.) from the
Ministry of Education, Culture, Sports, Science, and Technology ofJapan.
Acknowledgments
We thank Dr. Kohei Takahashi, Dr. Osamu Nakagawasai and Dr.
Koichi Tan-No (Department of Pharmacology, Tohoku Medical andPharmaceutical University) for technical support.
Conﬂicts of interest
The authors declare that they have no conﬂicts of interest with thecontents of this article.
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ORIGINAL RESEARCH
published: 29 January 2018
doi: 10.3389/fimmu.2018.00078

Core Fucosylation of the T Cell
Receptor Is Required for T Cell
Activation
Wei Liang1†, Shanshan Mao1†, Shijie Sun1†, Ming Li1, Zhi Li 2, Rui Yu1, Tonghui Ma1,
Jianguo Gu 3, Jianing Zhang 4, Naoyuki Taniguchi 5 and Wenzhe Li1*
1
College of Basic Medical Sciences, Dalian Medical University, Dalian, China, 2 Clinical Laboratory, Dalian Municipal Central
Hospital, Dalian, China, 3 Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology,
Tohoku Medical and Pharmaceutical University, Sendai, Japan, 4 School of Life Science and Medicine, Dalian University ofTechnology, Panjin, China, 5 Systems Glycobiology Research Group, Advanced Science Institute, RIKEN, Saitama, Japan

Edited by:Michael Loran Dustin,
Harvard University, United States
Reviewed by:
Balbino Alarcon,
Consejo Superior de Investigaciones
Científicas (CSIC), Spain
Christoph Wülfing,
University of Bristol, United Kingdom*Correspondence:
Wenzhe Liliwenzhe@dlmedu.edu.cn†

These authors have contributed
equally to this work.
Specialty section:
This article was submitted
to T Cell Biology,
a section of the journal
Frontiers in Immunology
Received: 30 September 2017
Accepted: 11 January 2018
Published: 29 January 2018

Citation:
Liang W, Mao S, Sun S, Li M, Li Z,
Yu R, Ma T, Gu J, Zhang J,
Taniguchi N and Li W (2018) Core
Fucosylation of the T Cell Receptor
Is Required for T Cell Activation.
Front. Immunol. 9:78.
doi: 10.3389/fimmu.2018.00078

CD4+ T cell activation promotes the pathogenic process of systemic lupus erythematosus (SLE). T cell receptor (TCR) complex are highly core fucosylated glycoproteins,which play important roles in T cell activation. In this study, we found that the corefucosylation of CD4+ T cells was significantly increased in SLE patients. Loss of corefucosyltransferase (Fut8), the sole enzyme for catalyzing the core fucosylation of
N-glycan, significantly reduced CD4+ T cell activation and ameliorated the experimental autoimmune encephalomyelitis-induced syndrome in Fut8−/− mice. T cell activation
with OVA323–339 loaded major histocompatibility complex II (pMHC-II) on B cell was
dramatically attenuated in Fut8−/−OT-II CD4+ T cells compared with Fut8+/+OT-II
CD4+ T cells. Moreover, the phosphorylation of ZAP-70 was significantly reduced in
Fut8+/+OT-II CD4+ T cells by the treatment of fucosidase. Our results suggest that corefucosylation is required for efficient TCR–pMHC-II contacts in CD4+ T cell activation,and hyper core fucosylation may serve as a potential novel biomarker in the sera from
SLE patients.
Keywords: core fucosylation, T cell receptor, T cell activation, systemic lupus erythematosus, T–B cell interaction

INTRODUCTION
Systemic lupus erythematosus (SLE) is a severe autoimmune disease that characterized by the
production of autoantibodies and the subsequent inflammatory disorders (1). Although the pathogenesis is not completely understood, the activation of CD4+ T cells seems plays an essential rolein the onset and development of SLE (2). Appropriate CD4+ T cell activation is crucially important
for adaptive immune responses and autoimmunity, but hyper-activation of these cells results in
autoimmune diseases. T cell recognition of peptide-loaded major histocompatibility complex II(pMHC-II) on the antigen-presenting cells (APCs) by T cell receptors (TCRs) is the most important
checkpoint for CD4+ T cell activation (3). During antigen recognition, the CD4 coreceptor binds
to the non-polymorphic surfaces of the membrane-proximal domains of the same pMHCs, which
results in a marked increase in the sensitivity of T cells to pMHCs on APC. When adequate agonistic
TCR signaling creates a favorable microenvironment for binding, CD28-B7 molecules provide
costimulatory lower the thresholds for TCRs triggering and activation. The signaling throughTCR induces a conformational change in leukocyte function-associated antigen-1, which greatly

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Fut8+/− mice (C57BL/6). OT-II (Jackson Laboratory) is a
C57BL/6 TCR transgenic strain, expressing a receptor specificfor peptide OVA323–339. Fut8+/+OT-II mice and Fut8−/−OT-II mice
were generated by crossing heterozygous Fut8+/−OT-II mice.
Mice were maintained in the specific pathogen-free laboratory
animal facility of Dalian Medical University. All animal work
was approved by the Ethics Committee at the Dalian Medical
University.

increases its affinity for intercellular adhesion molecule 1 andcontributes to immune synapse formation T cells and APC (4, 5).
Glycosylation plays a regulatory and often pivotal role in
T cell activation (6–10). Several studies have reported thatglycosylation could contribute to higher activation thresholds
of T cells. For instance, β1,6N-acetylglucosaminyltransferase
V (Mgat5) deficiency mediates lower T lymphocyte activationthresholds, and subsequently improves T cell activity in vitro
and results in autoimmune disease in vivo (11, 12). Deletion of
sialyltransferase ST3Gal-1 increase the sensitivity of TCRs to
low-affinity ligands in CD8+ T cells (13). Fucosyltransferase 1transgenic mice show increased TCR signaling and apoptosisthat results in thymocyte maturation arrest (14). Notably,
reduced N-glycosylation of TCR chains can improve functional
avidity and recognition by T cells (15) suggesting that the glycosylation of TCR has a unique role in the regulation of T cell
activation.
T cell receptors are heavily core-fucosylated glycoproteins.
The core fucosylation of protein is catalyzed by core fucosyltransferase (Fut8), which transfers fucose residue from GDPfucose to the innermost N-acetylglucosamine (GlcNAc) residueof N-linked glycans via an α1,6 linkage in the Golgi apparatus
of mammalians (Figure S1 in Supplementary Material). Fut8mediated core fucosylation is an important post-translational
process (16), which regulates protein conformation, stability, andfunctional expression. Studies have shown that the N-glycans
at Asn70 [GlcNAc(α1,6Fuc)-β1,4GlcNAc: (A2G2F)], Asn185(A2G2F), and Asn203 in the α chain (Cα) and Asn236 in the β chain(Cβ) extend from the surface of TCR on Drosophila melanogaster
cells (6, 17). Interestingly, they found that the Cα and Cβ of TCR
were connected by the hydrogen bonds of the core fucose residue
from Asn185α (A2G2F) to side chains of Glu181β and Ser182β (6, 17),
suggesting a crucial role of core fucosylation on the conformation of TCR. However, to the best of our knowledge, none of theprevious studies had addressed the regulatory role of TCR corefucosylation on CD4+ T cell activation.
B cells play a role in evoking T cell responses by functioning as APCs, and the presentation of peptide by MHC-II on the
B cells initiates T cell activation (18). Therefore, it is reasonable
to anticipate that the core fucosylation has significant functional
implications in T–B cell interaction, and thus affect the CD4+
T cell activation. In this study, we provide the first confirmationthat SLE patients exhibited hyper core fucosylation on CD4+
T cells, which significantly enhanced the activation of their CD4+
T cells. Knockout of Fut8 gene resulted in attenuated T–B cellinteraction via TCR–pMHC and the consequential reduced CD4+
T cell activation. Our data suggest that the core fucosylation may
serve as a potential novel biomarker with promising clinical andtherapeutic implications in SLE patients.

Patients
Serum samples were collected from a total of 17 SLE patients(14 women, 3 men; mean age, 49 years; range, 18–67 years) withand healthy controls (12 women, 12 men; mean age, 18–48 years)(Table S1 in Supplementary Material). The diagnosis of underlying disease was made based on clinical manifestation, serology, imaging, and/or histopathology. These participants were
Chinese, recruited at Dalian municipal central hospital. The antinuclear antibodies (ANA) titers of AD patients were detectedwith using Anti-nuclear Antibodies IgG Kit (EUROIMMUN,
Germany). The Ethics Committee at the hospital approved thestudy protocol.

Antibodies
Anti-CD16/32 (2.4G2), anti-CD3(145-2c11), anti-CD28 (37.51),
FITC-anti-MHC II (M5/114.15.2), FITC-anti-CD69 (H1.2F3),
PE-labeled anti-CD4 (GK1.5), APC-labeled anti-CD8 (53-6.7),
biotin-labeled anti-TCRβ (H57-597), and PE-Cy5-labeled antiTCRβ (H57-597) were obtained from e-Bioscience; anti-GAPDH,
horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG
and HRP-conjugated donkey anti-human IgG were obtained
from proteintech; additional biotin-conjugated lens culinaris
agglutinin (LCA) were purchased from Vector; anti-TCRαβ(ab25336), anti-pZAP70 (ab194800), anti-ZAP70 (ab32410),
Natural streptavidin protein (FITC) (ab136201), and streptavidin(HRP) (ab7403) were purchased from Abcam.

Histological Analysis
Formalin-fixed tissue samples were paraffin-embedded andsections were analyzed by hematoxylin–eosin (H&E) staining.
The sections were stained with biotin-conjugated LCA. Briefly,
sections were deparaffinized three times in xylene and hydrated
through a 100, 90, 80, and 70% ethanol to phosphate-bufferedsaline (PBS). To quench the endogenous peroxidase activity,
slides were incubated with 3% H2O2 for 30 min. Then, the slides
were incubated with biotin-conjugated LCA, and washed threetimes with PBS. The slides probed with HRP–streptavidin for
30 min, and visualized with 3,3′-diaminobenzidine. The intensityof LCA-positive staining in the spleen was analyzed by integrated
optical density using Image-Pro® Plus software (version 6.0;
Media Cybernetics, USA).

Cell Lysate

MATERIALS AND METHODS

Cells were solubilized in lysis buffer [Tris–HCl (50 mM), 1%
Triton X-100, 10% glycerol, phenylmethylsulfonylfluoride(100 μM), leupeptin (5 μg/mL), aprotinin (1 μg/mL), NaF(100 mM), 150 mM NaCl, 2 mM EDTA, and sodium orthovanadate (1 mM)] for 15 min at 4°C. Cell lysate was centrifuged at

MiceFut8−/− mice were generated as previously described (19),and homozygous wild-type (Fut8+/+) and Fut8−/− mice on the
C57BL/6 background were obtained by crossing heterozygous

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20,000 × g for 10 min at 4°C, and the supernatant was subjected
to immunoprecipitation or Western blot, as indicated below.

Western Blot and Lectin Blot Analysis
Protein samples were electrophoresed on 10% polyacrylamidegels. After electrophoresis at 240 mA for 30 min, proteins weretransferred to PVDF membranes. Membranes were blocked in
5% BSA in TBS-T (10 mM Tris–HCl, 150 mM NaCl, and 0.1%
Tween 20) at room temperature for 1 h, and then incubatedwith the biotin-labeled LCA, which preferentially recognizes
the core fucose, or primary Abs in 1% BSA in TBS-T overnightat 4°C. After washing, the membranes were covered with the
HRP-conjugated streptavidin or HRP-labeled secondary Abs atroom temperature for 1 h, and visualized with an ECL system(Amersham).

Fut8 Enzyme Activity Assay
The Fut8 enzyme activity was measured by using the previous
method (20). Five micrograms cell lysates as the enzyme source
were added to the assay buffer (200 mM MES, 1% Triton X-100)
supplemented with donor (500 μM GDP-L-fucose) and substrate[50 μM GnGn-Asn-4-(2-pyridylamine) butylamine (PABA)].
The mixture was incubated at 37°C for 8 h, and the reaction was
stopped by heating at 100°C for 5 min. The reactive solution
was then centrifuged at 12,000 × g for 10 min, and 10 μL of thereaction products were subjected to high-performance liquid
chromatography (HPLC) with a Fluorescent detector (Waters
Corporation, USA). The excitation and emission wavelengths are
320 and 400 nm, respectively.

Immunoprecipitation
Cell extracts (500 μg) were mixed with 20 μL of Protein
G-Sepharose (50%) and corresponding Abs, and then incubatedat 4°C overnight with continuous rotation. After washing threetimes in lysis buffer, the pull downed samples were boiled for 5 min
in Laemmli sample buffer with or without 2-mercaptoethanol.

PCR Array
Total RNAs were extracted from Fut8+/+ SPLs and Fut8−/− SPLs
with TRIzol reagent (Takara Bio). Mouse T Cell and B Cell
Activation PCR Array (SA Biosciences) was carried out according to the protocol of the manufacturer. The difference of gene
expression between Fut8+/+ SPLs and Fut8−/− SPLs was calculated.

T–B Cell Conjugate Formation

Mouse was immunized by subcutaneous injection with 200 μg
OVA mixed with an equal volume of complete Freund’s adjuvant(CFA) (Sigma). Two weeks later, mice were immunized with
200 μg OVA by subcutaneous injection. Mice sera were collected
at 0, 7, and 14 days post-immunization.

Conjugate formation between T cells and B cells were carried out
as described previously with slight modification (21). T cells and1 μg/mL OVA323–339 (NH2-ISQAVHAAHAEINEAGR-COOH)pulsed B cells were mixed at a 1:1 ratio and a quick centrifugation
to initiate cell–cell contact. To observe conjugate formation,
B cells and T cells were labeled with MHC-II-FITC and TCRβPE-Cy5 before mixing. T–B cell conjugates were then analyzed
by flow cytometry to determine the percentage of T–B cells that
had both TCRβ-PE-Cy5 and MHC-II-FITC positive staining.

Enzyme-Linked Immunosorbent
Assay (ELISA)

Remove of Core Fucose on Surface
of T Cells

The concentrations of IL-2 analyzed using mouse IL-2 ELISA
kits (Boster Biological Engineering, Wuhan, China), according
to the manufacturer’s instructions. The concentrations of IL-2
were calculated according to a standard curve prepared using
samples of known concentration. The absorbance was measured
at a test wavelength of 450 nm with a microplate reader.
The immunoglobulin isotypes were measured by mouse mAb
isotyping reagents (Sigma).

Purified CD4+ T cells (4 × 106) were treated with 100 mU Glyko®
α(1-2,3,4,6) Bovine Kidney Fucosidase (GKX-5006, Prozyme),
incubate 3 h at 37°C in the reaction buffer. The enzyme reaction
was terminated by centrifuging at 2,500 × g for 5 min and thecells were collected.

Animal Immunization

Confocal Microscopy
Conjugate formation between T cells and B cells were carriedout as described previously with slight modification (21). T cells
and 1 μg/mL OVA323–339 pulsed B cells were mixed at a 1:1 ratio
and a quick centrifugation to initiate cell–cell contact. Cell–cellconjugates were subsequently transferred to poly-d-lysine coatedcoverslips and incubated at 37°C for 30 min. Cells were fixedwith 4% PFA for 20 min, and then blocked with 5% BSA andanti-CD16/CD32 (2.4G2) mAb for 30 min. After washing, cells
were stained with anti-MHC-II Ab for 1 h. All images were takenusing a spinning disk confocal microscope (Leica).

Cell Proliferation Assay
The growth rate of cells was measured using MTT assay. CD4+
T cells (1 × 106) were cultured in 96-well culture plate with antiCD3ε (2 μg/mL) and anti-CD28 mAbs (1 μg/mL). After 48 h ofincubation, each well was added 10 μL of MTT solution, and thenthe absorbance was analyzed by a microplate reader (Thermo
Multiskan Ascent, Finland) at 570 nm.
In addition, T cell proliferation with the OVA323–339-loaded
B cells was analyzed by carboxyfluorescein diacetatesuccinimidyl
ester (CFSE, Sigma) dilution methods. CD4+ T cells (1 × 106)
were purified, and then labeled with 5 μM CFSE in PBS for 8 minat room temperature and coincubate with OVA323–339 loaded
Fut8+/+ OT-II B cells (1 × 106) for 48 h, and then analyzed by flow
cytometric analysis.

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MACS Magnetic Cell Sorting
Single splenic cell suspensions were prepared by first grinding
the tissues and then by passage through 30-μm nylon mesh.
Red blood cells were lysed by incubation with 0.14 M NH4Cl
and 20 mM Tris (pH7.4) for 3 min at room temperature.

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After the lysis of red blood cells, CD4+ T cells and B cells
were positively isolated with anti-CD4 Ab and anti-CD45R
Ab-conjugated magnetic beads (Miltenyi Biotec). The purified
cell populations were detected by fluorescence activated cellsorting analysis.

Lack of Core Fucosylation Ameliorated
EAE Symptoms with Reduced
CD4+ T Cell Activation
Wild-type (Fut8+/+) and Fut8−/− mice were generated previously(19). Histological analyses of the splenic architecture of Fut8+/+
and Fut8−/− mice were unremarkable in H&E staining. The Fut8
products, core-fucosylated N-glycans, are ubiquitously expressedin the Fut8+/+ spleen, as confirmed by LCA (Figure 2A), while
those were abolished in Fut8−/− spleens. LCA blot analysis also
confirmed the knockout of Fut8 expression in the whole celllysates of Fut8−/− SPLs (Figure 2B). The FUT8 enzymatic activity was not detected in the Fut8−/− SPLs using HPLC analysis(Figure 2C).
Experimental autoimmune encephalomyelitis is an activatedCD4+ T cell-mediated autoimmune disease model. Peptides(MOG35–55) and PTX could induce the migration of activatedT cells through the blood–brain barrier and caused several
neurologic symptoms. To determine the association between
core fucosylation and the activation of CD4+ T cells, EAE models
were established using Fut8+/+ and Fut8−/− mice. EAE is actively
induced but appear more quickly upon adoptive transfer ofactivated MOG35–55-specific T cells in Fut8+/+ mice (Figure 2D;
Video S1 in Supplementary Material), while Fut8−/− mice showedslight EAE symptoms (Figure 2D; Video S2 in Supplementary
Material). The body weights of Fut8+/+ mice were significantly
reduced, but no change was found in Fut8−/− mice during EAE
induction (Figure 2D). In addition, the proliferation of CD4+
T cells was significantly decreased in Fut8−/− EAE mice. Moreover,the proliferation of CD4+ T cells with MOG35–55-loaded B cells
was remarkably reduced by de-core fucosylation (Figure 2E).

Flow Cytometric Analysis (FACS)
Cells were isolated from tissue and incubated with an antiCD16/CD32 (2.4G2) mAb to block Fcγ receptors. The cells were
stained on ice for 15 min with several combinations of mAbs, as
indicated in the figure legends. Flow cytometry was performed
on a FACS-Calibur (Becton Dickinson, Mountain View, CA,
USA) and analyzed using FlowJo software (Tree Star).

Induction of Experimental Autoimmune
Encephalomyelitis (EAE)
For EAE induction, Fut8+/+ and Fut8−/− mice were subcutaneously injected with 100 μg of myelin oligodendrocyte glycoprotein peptides (MOG35–55) emulsified in CFA. Then, mice were
injected intraperitoneally with 200 μg pertussis toxin (PTX) (List
Biological Laboratories) on 0, 1, and 2 days. Clinical assessment
of EAE was performed according to the following scale: 0, nodisease; 1, limp tail; 2, hind-limb weakness; 3, partial hind-limb
paralysis; 4, complete paralysis of hind-limbs; and 5, moribund
state.

Statistical Analysis
Student’s t-test was used for statistical analysis. Data are presented
as mean values ± SEM, or as mean values ± SD. A probability
value of p < 0.05 was considered significant. *p < 0.05, **p < 0.01,***p < 0.001.

Lack of Core Fucosylation Suppressed
the IgG Class-Switching by Impaired
CD4+ T Cell Activation

RESULTS
Core Fucosylation Is Significantly
Upregulated in the Sera andCD4+ T Cells of SLE Patients

Flow cytometry analysis revealed that, although Fut8−/− micecontained normal proportions of CD4+ and CD8+ T cell
populations in the spleen (Figures 3A,B), they were markedly
reduced after OVA immunization contrast with the Fut8+/+ mice
(Figures 3A,B). Immunoglobulin class-switching is a biologicalmechanism that changes a mature B cell’s production of antibody
via its B cell receptor from one class to another. For example,
from an isotype called IgM to an isotype called IgGs. To illustrate
the effects of Fut8 in the class-switching of immunoglobulin, we
measured the class-switched (IgGs of different subclasses) andnon-switched (IgM) in the sera of Fut8+/+ and Fut8−/− mice using
mouse mAb isotyping reagents. In 4-week-old Fut8−/− mice, theamounts of IgG1, IgG2a, IgG2b, and IgG3 were significantly lower
than those in Fut8+/+ mice after OVA immunization, while thoseof IgM were relatively normal (Figure 3C). The cytokines, suchas IL-4, IL-5, IL-6, and IFNγ secreted by CD4+ T cells, contributeto the different IgG class-switching in the mice and human. It
is reasonable to consider that the reduced IgG class-switching
attributed to the low levels of cytokines secreted by CD4+ T cells
in Fut8−/− mice (Table 1). In addition, since the TGF receptor
signaling was attenuated in the Fut8−/− mice (19), the IgG3

Higher circulating levels of ANA were detected in sera from
SLE patients. In this study, we found that core fucosylation
was dramatically increased in the sera from patients with
SLE, as evidenced by LCA, which preferentially recognizes
the core fucose structure (22) (p < 0.001) (Figures 1A,B).
The expression of IgGs was also upregulated in sera of the SLE
patients (p < 0.05) (Figure 1B). These observations pinpoint
the contribution of hyper core fucosylation to SLE severity andpathogenesis.
The hyperactivity of B cells in SLE is T cell dependent, andCD4+ T cell activation plays a crucial role in SLE pathogenesis(23). We found that the percentage of CD4+ T cells in the peripheral blood of SLE patients is similar to those of healthy control(Figure 1C). However, the percentage of CD4+CD69+ T cells was
significantly increased in the SLE (n = 17) (Figure 1D). Moreover,the enzyme activity of Fut8 was dramatically increased in the
CD4+ T cells isolated from the SLE patients (Figures 1E,F), indicated that increased core fucosylation in SLE patients correlateswith CD4+ T cell activation.

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FIGURE 1 | Core fucosylation was significantly increased in the systemic lupus erythematosus (SLE) patients. (A) The sera of SLE patients were analyzed by lensculinaris agglutinin (LCA) blot and Western blot. Plates were incubated with biotin-conjugated LCA (1:20,000). Comparable results were obtained in three
independent experiments. (B) Densitometric analysis of the bands of IgG and LCA in sera in SLE patients. Data are shown as mean values ± SEM (*p < 0.05;***p < 0.001). (C) The percentage of CD4+ T cells in the peripheral blood of SLE patients. (D) The percentage of CD4+CD69+ T cells in the peripheral blood of SLE
patients (n = 17). (E,F) FUT8 activities in the CD4+ T cells of SLE patients by high-performance liquid chromatography (HPLC). CD4+ T cells were isolated with
anti-CD4 Ab-conjugated magnetic beads. The sorted cell populations were routinely more than 96% pure. Five micrograms cell lysates as the enzyme source weremixed with the assay buffer. After incubation at 37°C for 8 h, 10 μL of the supernatant was subjected to HPLC. Activity was expressed as pmol of GDP-fucose
transferred to the acceptor per hour per milligram of protein. Data are shown as mean values ± SD (n = 17; ns, not significant; *p < 0.05; **p < 0.01). S is the
peptide substrate and P is the product of fucosylation.

class-switching regulated by TGF signaling was also suppressedin the Fut8−/− mice.

FACS analysis showed that core fucosylation on the cellularsurfaces was abolished in Fut8−/− CD4+ T cells (Figure 4C). We
compared the phosphorylation levels of Fut8+/+CD4+ T cellswith Fut8−/−CD4+ T cells in response to costimulations with
anti-CD3/CD28 Abs. In those comparisons, the levels of pZAP70 in Fut8−/−CD4+ T cells were significantly lower than those in
Fut8+/+CD4+ T cells (Figure 4D). Moreover, the populations of
CD69+ cells (activated T cells) in Fut8−/−CD4+ T cells were lower
than those in Fut8+/+CD4+ T cells following OVA immunization,
while these were similar before immunization (Figure 4E).
Furthermore, cell proliferation of Fut8−/−CD4+ T cells was significantly reduced in response to the stimulation of anti-CD3/
CD28 Abs (Figure 4F).

Loss of Core Fucosylation Impaired the
Signal Transduction via TCR
T cell receptor signaling is very important for T cell activation.
As illustrated in Figure 4A, no different expression of TCR was
found in the Fut8+/+CD4+ T and Fut8−/−CD4+ T cells. Nonetheless,the core fucose of the N-glycans in the molecules was eliminatedby a disruption of the Fut8 (Figure 4B). Moreover, the MS spectra of N-glycans released from TCRs were analyzed. It is notablethat the high levels of signals were corresponded to the corefucosylated glycans bearing non-, mono-, or di-galactose in the
Fut8+/+CD4+ T cells, while those were completely disappearedin Fut8−/−CD4+ T cells (Figure S2 in Supplementary Material).
These results further confirmed in mice that TCRs are highly
core-fucosylated proteins and contributes to its activities.
In order to examine the role of core fucosylation in the activation of CD4+ T cells, we isolated CD4+ T cells from Fut8+/+
and Fut8−/− mice, and checked the level of core fucosylation.

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Core Fucosylation Is Essential for
TCR–pMHC Conjugates in CD4+ T Cell
Activation
B cells present antigenic peptide with MHC-II molecule as
APCs, and CD4+ T cells physiologically recognize a complex
of a peptide-loaded MHC-II in T–B cell interaction (18).

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FIGURE 2 | Continued

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FIGURE 2 | The experimental autoimmune encephalomyelitis (EAE) model was slightly induced in Fut8−/− mice. (A) Immunohistochemical analysis of Fut8+/+ spleen
and Fut8−/− spleen. The paraffin sections of spleens were deparaffinized and hydrated through a graded series of ethanol to phosphate-buffered saline. One section
was assessed by hematoxylin–eosin staining (magnification ×200). RP, red pulp; WP, white pulp. Another section was incubated with biotin-conjugated lens culinaris
agglutinin (LCA) (1:200) for 1 h. Finally, the slides were visualized with 3,3′-diaminobenzidine. The staining with LCA was showed by integrated optical density
analysis. Data are shown as mean values ± SD (***p < 0.001). (B) Lectin bolt of Fut8+/+ SPL and Fut8−/− SPL. The SPL lysates were run on 10% SDS-PAGE gel andstained with Coomassie blue staining and LCA (1:5,000). (C) High-performance liquid chromatography (HPLC) analysis of Fut8 activity. Fut8 activities were
examined using fluorescence-labeled sugar chain, GnGn-Asn-PABA, as an acceptor substrate, as described in Section “Materials and Methods.” The substrate (S)and Fut8 product (P) were eluted at 8 and 15 min, respectively. (D) Disease score of mice in Fut8+/+ and Fut8−/− mice EAE model. EAE induction of Fut8+/+ andFut8−/− mice (n = 7). Mice were immunized with 100 μg MOG35–55 peptide in complete Freund’s adjuvant and injected with 200 μg pertussis toxin, and detected thesigns of EAE daily for 30 days. Comparable results were obtained in four independent experiments. Body weights of mice were measured every 2 days after EAE
induction of Fut8+/+ and Fut8−/− mice (n = 7). (E) Cell proliferation from EAE mice. Purified splenic CD4+ T cells and/or MOG35–55 peptide-loaded B cells from EAE
models were incubated for 48 h at 37°C, and then the cell proliferation was detected by MTT assay. Comparable results were obtained in four independent
experiments. Data are shown as mean values ± SD (*p < 0.05; **p < 0.01).

FIGURE 3 | Lack of core fucosylation suppressed the IgG class-switching by impaired CD4+ T cell activation. (A,B) Flow cytometry analysis of the proportion of
CD4+ and CD8+ cells. SPLs were obtained from Fut8+/+ and Fut8−/− mice with or without OVA immunization. SPLs were filtered through nylon mesh, andresuspended. Numbers indicate the percentage of the total spleen cells within this quadrant, and 10,000 events were acquired for each analysis. Data arerepresentative of three independent experiments. Data are shown as mean values ± SD (ns, not significant; *p < 0.05; **p < 0.01). The number of Fut8+/+CD4+ T
and Fut8−/−CD4+ T cells were (1.29 ± 0.32) × 107 and (6.38 ± 0.89) × 106 of the SPLs, respectively (n = 5). (C) Comparison of the levels of serum immunoglobulin
isotypes in Fut8+/+ and Fut8−/− mice after OVA immunization. The immunoglobulin isotypes are compared by mouse mAb isotyping reagents (Sigma). Comparable
results were obtained in three independent experiments. Data are shown as mean values ± SD (*p < 0.05; **p < 0.01).

with pMHC-II ligands, we crossed Fut8+/− mice with OT-II TCR
transgenic mice (expressing CD4+ TCR specific for OVA323–339),
and obtained Fut8+/+OT-II and Fut8−/−OT-II mice. LCA blots

Therefore, we were interested in determining whether corefucosylation is involved in the TCR sensitivity to pMHC-II. To
explore the role of core fucosylation on the TCR interaction

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markedly increased at the site of T–B cell contact in Fut8+/+OT-II
T cells for 30 min and was ubiquitous on the Fut8−/−OT-II T cellsurface (Figures 5D,E). Next, T–B cell conjugates were quantitatively analyzed via flow cytometry analysis. Few conjugatesof T–B cells were observed in the absence of OVA323–339 peptide,whereas peptide-pulsed B cells effectively interacted with CD4+
T cells. The percentages of T–B cell conjugates in Fut8+/+OT-IIand Fut8−/−OT-II MHC-II+ TCRβ+ cells were 29.4 and 7.4%,
respectively (Figures 5F,G), indicated that core fucosylation
affected the T–B cell interaction. Moreover, the secretion of IL-2
was reduced in the culture media of Fut8−/−OT-II T–B cells compared with Fut8+/+OT-II T–B cells (Figure 5H). Furthermore, theT cell proliferation with OVA323–339-loaded B cells was analyzed
by CFSE dilution methods. Compared to the proliferation of
Fut8+/+OT-II CD4+ T cells, those of Fut8−/−OT-II CD4+ T cells
was significantly reduced with the cocultivation of OVA323–339loaded Fut8+/+OT-II B cells (Figure 5I). These results indicatedthat Fut8 deficiency contributes to attenuated T–B cell communication, and follows attenuated T cell activation.
To further elucidate the underlying mechanism of the
reduced T–B cell interaction caused by the disruption of Fut8,
T and B cell Activation PCR Array was used to compare mRNA
expression in Fut8+/+ SPLs with that in Fut8−/− SPLs following
OVA immunization. As illustrated in Table 1, the expression
levels of four genes (CD3e, CD4, CD8, and CD40L) associated with TCR complex formation, eight genes (IL-2Rα, IL-4,
IL-6, IL-10, IL-12, IFNγ, CXCL12, and CXCR4) involved in
T cell activation, and two genes (CD79a and CD81) associatedwith B cell activation, were downregulated in Fut8−/− SPLs.
Moreover, the gene expressions of signal molecules such as
MAPKKK, Vav1, PIK3, PKC, and Cyclin D3 were downregulated in Fut8−/− SPLs. Since core fucosylation of proteins is an
important post-translational process, it is not surprising thatmany molecules involved in T and B cell activation were downregulated in Fut8−/− SPLs.

TABLE 1 | Gene expression of Fut8−/− SPLs after OVA immunization.
Gene access number

Fold change (Fut8+/+/Fut8−/−)T-cell activation
CD3e
CD4
CD8
Cd40L
IL-2Rα
IL-4
IL-6
IL-10
IL-12
IFNγ
CXCR4
CXCL12

NM-007648
NM-013488
NM-009858
NM-011616
NM-008367
NM-021283
NM-031168
NM-010548
NM-008352
NM-008337
NM-009911
NM-001012477

2.15
3.16
2.51
2.17
3.04
2.18
2.14
2.25
3.89
3.08
2.04
2.07

B-cell activation
CD79a
CD81

NM-007655
NM-133655

3.35
2.82

Cell signaling
MAPKKK
Vav1
PIK3
PKC
Cyclin D3

NM_009316
NM-011691
NM-001077495
NM-008859
NM-001081636

2.41
3.28
2.36
2.00
3.84

Gene nameanalysis showed that the core fucosylation level of T and B cells
in Fut8−/−OT-II mice (Figure S3 in Supplementary Material).
Since MHC-II on the B cell surface can present peptides forrecognition and activation of T cells, in this study, B cells wereisolated from Fut8+/+OT-II and Fut8−/−OT-II spleen and incubated with monobiotin-labeled OVA323–339. Although electron
density was seen for the structures of I-Ad covalently linked toan OVA323–339, with a single N-glycan (24), the core fucosylation
did not affect the peptide presentation abilities of the MHCs
between Fut8+/+OT-II and Fut8−/−OT-II B cells (Figure S4 in
Supplementary Material). The CD4+ T cells were then stimulated with OVA323–339 loaded B cells and subsequent T cell priming was investigated. The freshly purified ex vivo CD4+ T cells
were stimulated with the OVA323–339-loaded B cells. As shownin Figure 5A, compared with Fut8+/+OT-II CD4+ T cells, thephosphorylation level of ZAP-70 was dramatically decreased in
Fut8−/−OT-II CD4+ T cells with stimulation of OVA323–339-loaded
B cells. Moreover, to remove the cell-surface fucosylation, theFut8+/+OT-II CD4+ T cells were treated with 100 mU Glyko®
α(1-2,3,4,6) Fucosidase, which can cleaves α1,6-linked fucose
more efficiently than other α-fucose linkages (25). The signaling via TCR was significantly suppressed in OT-II CD4+ T cells
treated with this fucosidase, when the T cells were stimulatedwith OVA323–339-loaded B cells (Figure 5B), indicated that the
Fut8 inactivation results in the less responsive for TCR stimulation with pMHC-II, despite similar TCR expression levels(Figure 4A). Moreover, the population of TCR+CD69+ cells was
significantly reduced in Fut8−/−OT-II CD4+ T/OVA323–339-loaded
B cells compared with Fut8+/+OT-II CD4+ T/OVA323–339-loaded
B cells (Figure 5C).
To further determine how Fut8 deficiency affects the T–B cellinteractions, the communication of CD4+ T cells and B cells wasobserved by confocal microscopy. OVA323–339-loaded MHC-II was

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DISCUSSION
The glycosylation and Golgi processing pathways have coevolved
with the larger regulatory network that controls T cell activation.
It is not surprising that changes of glycosylation are linked to
AD pathogenesis, such as galactosylation (26, 27) and sialylation (28, 29). The lower levels of sialylated IgG were foundin rheumatoid arthritis (RA) and Wegener’s granulomatosis
patients, and the sialylated IgG was increased in the sera ofpatients during remission (28, 29). Moreover, loss of galactose
residues on IgG1 is showed in the sera of RA patients (26, 27).
The significant differences of O-glycan on T helper cells weredetected in active SLE patients (30). Core-fucosylated glycans
that contain bisecting GlcNAc was increased on the IgG of SLE(31). In this study, core fucosylations were associated with SLEseverities, and significantly increased in the CD4+ T cells of
SLE patients. Coincidently, previous study by Fujii et al. foundthat the core fucosylation on T cells, required for activation of
TCR signaling with anti-CD3/CD28 Abs and induction in colitis,
is significantly increased in patients with inflammatory bowel
disease (32). Hence, one possible consequence of hyper core

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Core Fucosylation in T-Cell Activation

FIGURE 4 | Core fucosylation is required for the activation of CD4+ T cells. (A) Western blots analysis of T cell receptor (TCR). Whole cell lysates were resolved by
SDS-PAGE on a 8% gel, transferred to a PVDF membrane, and probed with anti-TCRβ Ab. Densitometric analysis of the bands of TCRβ normalized against
GAPDH. (B) Core fucose of N-glycan on TCRβ in Fut8−/−CD4+ T cells was detected by LCA blot. Whole cell lysates were immunoprecipitated with an anti-TCRαβantibody. The immunoprecipitates were resolved by SDS-PAGE on a 8% gel, and probed with the LCA and anti-TCRβ Ab. (C) Histograms of binding capacity with
LCA. Core fucosylation level on the surface proteins of Fut8+/+CD4+ T and Fut8−/−CD4+ T cells investigated by FACS analysis. (D) Downregulation of phosphorylated
Zap70 in Fut8−/−CD4+ T cells. Purified CD4+ T cells were serum-starved and were stimulated with anti-CD3/CD28 Abs for 5 min at 37°C. Cells were lysated in lysis
buffer for 15 min on ice. Whole cell lysates were subjected to 10% SDS-PAGE. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Densitometricanalysis of the bands of pZAP70 normalized against ZAP70. Data are reported as the mean ± SD from three independent experiments (**p < 0.01; ***p < 0.001).
(E) Loss of Fut8 reduced the CD4+CD69+ cells populations in the SPL after OVA immunization. SPLs were isolated from OVA-immunized and unimmunized mice(n = 5). Cells were stained with anti-CD69 and anti-CD4 Abs, and then detected by FACS. Data are reported as the mean ± SD from three independent experiments(*p < 0.05; ns, not significant). (F) Loss of Fut8 decreased the proliferation of CD4+ T cells. Purified CD4+ T cells were stimulated with anti-CD3ε Ab-coatedmicrobeads and anti-CD28 Ab for 0, 6, 24, and 48 h at 37°C. The growth rates of CD4+ T cell were detected by MTT assay. Data are reported as the mean ± SD
from three replicate cultures (*p < 0.05; **p < 0.01). The absorbance related to the formazan dye level was measured with a microplate reader at 570 nm.

fucosylation-induced T cell activation could be the developmentof SLE. However, the underlying mechanisms of how core fucosylation regulate T cell activation with TCR–pMHC interaction
remain unclear.
T cell recognition of pMHC-II ligands on B cells is thought
to be carefully coordinated in CD4+ T cells. Conformational
flexibility is likely responsible for the high degree of promiscuity or cross-reactivity that is evident in the TCR recognitionof pMHCs (33–35). There are two models to explain how
TCR–pMHC interactions result in T cell activation (36, 37). One

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model involves an activation threshold based on the occupancy
time of the TCR clustering with pMHCs. The alternative modelis that pMHC could induce a specific conformational change
of TCR complex and influence the quality of signal transductions via TCRs. Since the core fucose of N-glycan is located on
the cellular surface of T and B cells, and the core fucosylation
could affect the flexibility of N-glycan antenna (38) as well as
the conformational stability of proteins (39), it is reasonable toassume that core fucosylation of TCR would affect the geometry
and conformation of any TCR–pMHC clusters in the T–B cell

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Core Fucosylation in T-Cell Activationinteractions. Based on a previous study in D. melanogaster cells,
the closeness of the interaction of Cα with Cβ is shown by thehydrogen bonds of the core fucose residue (6, 17), suggested

core fucose possibly strengthens association of Cα with Cβ.
A water molecule also bridges the fucose exocyclic oxygen andthe side chain of Arg150β (17). In this scenario, core fucosylation

FIGURE 5 | Continued

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FIGURE 5 | Lack of core fucosulation reduced interaction ability between T cell receptor (TCR) and pMHC-II and impaired the signaling via TCR. (A) ZAP70
phosphorylation of Fut8+/+OT-II T–B cells. (T) B cell conjugate formation was initiated by centrifuging together CD4+ T cells with or without 1 μg/mL OVA323–339-loaded
B cells. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments. Densitometric analysis of the
bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01). (B) ZAP70 phosphorylation of Fut8+/+OT-II CD4+ T cells with orwithout fucosidase treatment. Purified CD4+ T cells were treated with or without 100 mU Bovine Kidney Fucosidase, and then coincubated with 1 μg/mL OVA323–339
loaded Fut8+/+OT-II B cells for 30 min. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments.
Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01; ***p < 0.001). (C) Compare of activationof Fut8+/+OT-II T + B cells and Fut8−/−OT-II T + B cells. CD4+ T cells were conjugated with OVA323–339 loaded B cell. After 30 min, the T–B cells were fixed and stainedwith anti-TCRβ and anti-CD69 Abs. Data are shown as mean values ± SD (**p < 0.01). (D,E) Distribution of MHC on the T and B cells. The T–B cells were fixed,
permeabilized, and stained with Abs to MHC-II (green). Data are from three separate experiments. The ratio of MHC II intensity at the T–B cell conjugate site relative
to non-conjugate areas. Data are shown as the mean values ± SD (**p < 0.01). (F) FACS analysis of the conjugates between B cells and CD4+ T cells. B cells(OVA323–339-loaded or not loaded) were labeled with anti-MHC-II (FITC) and CD4+ T cells were labeled with anti-TCRβ (PE-Cy5). T and B cells were quickly mixed andconjugated by a brief centrifugation step. They were then incubated at 37°C for 30 min. A representative staining profile with anti-MHC-II and anti-TCRβ mAb isshown. The conjugate cells are double positive (MHC-II+TCRβ+) cells. (G) Percentage of T–B cell conjugates (both MHC-II and TCRβ positive cells) was calculated.
Data are reported as the mean ± SD (**p < 0.01) in four independent experiments. (H) The secrition of IL-2 was downregulated in the culture media of Fut8−/−OT-II
T–B cells. OD values were measured at 492 nm using a microplate reader. Data are reported as the mean ± SD (**p < 0.01) in three independent experiments.
(I) Proliferation of Fut8+/+OT-II CD4+ T cells with OVA323–339 loaded Fut8+/+OT-II B cells. Fut8+/+OT-II CD4+ T cells were purified and labeled with carboxyfluoresceindiacetatesuccinimidyl ester (CFSE). Fut8+/+OT-II CD4+ T cells were cocultivated with 1 μg/mL OVA323–339 loaded Fut8+/+OT-II B cells for 48 h, the divided cells wereanalyzed by FACS. One representative experiment is shown. M1–M4 indicates daughter cell populations which have subsequently lost half of their CFSE signal.

is expected to provide exciting opportunities to control the
TCR function. In the present study, the percentage of T–B cell
conjugates (TCR+MHC-II+) was reduced by a factor of 3.97
(29.4/7.4%) in Fut8−/−OT-II cells, indicated that the core fucosylation has significant functional implications in TCR–pMHC
interaction.
Given the intimate relationship between pMHC recognition
and TCR signaling, the signaling via TCR is also be regulated byglycosylation. It has long been appreciated that Mgat5 deficiency
could enhance TCR recruitment to the synapse and results ingreater TCR internalization/endocytosis (11). Physiologically,

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TCR recognition of pMHC-II ligands on APCs such as dendritic
cells and B cells are the most important checkpoint for CD4+ T cell
activation (40). Engagement of the TCR complexes leads to a signaling cascade of protein tyrosine kinases, such as ZAP70. To explore
the role of core fucosylation on the T cell activation, we generated
Fut8+/+OT-II and Fut8−/−OT-II mice. Fut8 deficiency results inthe attenuated phosphorylation of ZAP70 in Fut8−/−OT-II CD4+
T cells with OVA323–339-loaded B cells. Also, the phosphorylationof ZAP-70 was significantly reduced in Fut8+/+OT-II CD4+ T cellsby the treatment of fucosidase. Moreover, in Fut8−/−OT-II cells,
the number of CD4+ T cells activation (CD69+) was decreased

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by a factor of 3.5. The proliferation of the Fut8−/−OT-II CD4+
T cells cocultivated with OVA323–339-loaded B cells was decreased
compared with the Fut8+/+OT-II CD4+ T cells. It is conceivablethat low T–B cell conjugates was proportional to the decreased
T cell activation and proliferation in Fut8−/− CD4+ T cells. Core
fucosylation is likely to be important in all three proposed stages
involved in the T cell activation. First, core fucosylation is essential for the TCR structural formation. Second, core fucosylationof TCR could regulate the recognition of pMHC and affect T cell
activation threshold. Third, fucose-specific lectins (41) might
participate the events in the T–B cell interaction.
Systemic lupus erythematosus is characterized by the
overproduction of auto antibodies, mainly IgG. However,B lymphocyte hyperactivity in SLE is T cell dependent. T cells
from SLE patients are activated with a decreased activationthreshold and regulated abnormally (32). Indeed, overactive CD4+ T cells had been implicated in the pathogenesis of
SLE (42). Although we know that CD4+ T cell deregulation
contributes to SLE pathogenesis, but the mechanism is stilllargely unknown. Studies in SLE patients and murine models
of lupus have shown enhanced level of IL-4 (43), IFNγ (44),and IL-6 (45, 46). Compared to Fut8+/+ mice, the IgG classswitching was significantly reduced in the sera of Fut8−/− mice
after OVA immunization due to low levels of IL-4, IL-5, IL-6,
IFNγ, and TGF secreted by Fut8−/−CD4+ T cells. Developmentof SLE is associated with genetic, hormonal, environmental,
and immunological factors, mainly those related to the helper
T cell activation. To investigate the mechanisms of the CD4+
T cell activation that is important for SLE pathogenesis, we
induced the EAE model using Fut8+/+ and Fut8−/− mice. Loss of
Fut8 reduced CD4+ T cell activation and ameliorated the EAE
in Fut8−/− mice. Fut8−/− mice are resistant to the induction of
EAE, whereas Mgat5−/− mice are hypersensitive to it (47). In
this regard, Fut8 and Mgat5 function as opposing regulators ofT cell activation thresholds and susceptibility to AD. The hyper
core fucosylations are associated with SLE severities, however,whether altered core fucosylation is a consequence or an underlying cause of the SLE cascade remains unclear.
FUT8 is able to modify multiple proteins, followed by thechange of their functions. Because the most of immune response
molecules are glycoproteins, FUT8 knockdown/knock out
affected their function associated with immune response. Okada
et al. (10) showed that loss of core fucosylation caused an inhibitory receptor PD-1 deprivation on the cellular surface and augmented T cell activation, when Fut8−/−CD4+ T cells transferred

into Rag2−/− mice. Although we did not measure the cell-surface
PD-1 expression in the T cell activation and T–B cell interaction,
the core fucosylation could regulate the PD-1 expression bymodulating TCR signaling strength. Physiologically, there may
be some equilibria of the core fucosylation on the TCR and PD-1
to regulate T cell activation. The complexity of in vivo condition
made us cannot conclude the inability for TCR signaling pathway
is the sole reason for the T cell activation, but our study firstrevealed that Fut8 is essential for TCR–pMHC contact and thefollowing CD4+ T cell activation. The balance between specificand degenerate T cell recognition of pMHC-II holds important
implications for protective immunity versus autoimmunity. Witha better understanding of how core fucosylation regulates of theadaptive immune system, its use in therapy for SLE may prove to
be a useful intervention.

ETHICS STATEMENT
All animal work was approved by the Ethics Committee at the
Dalian Medical University. The Ethics Committee at the hospital
approved the study protocol.

AUTHOR CONTRIBUTIONSWL, SM, and SS designed research and performed experiment;
ML, ZL, and RY analyzed the experimental data; TM, JG, JZ, andNT corrected paper; WL designed research and wrote paper. All
authors reviewed the results and approved the final version of themanuscript.

FUNDING
This work was supported by the National Nature Science
Foundation of China (31570797, 31270864, 30972675), andNatural Science Foundation of Liaoning Province (2015020253),
China.

SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be found online at
http://www.frontiersin.org/articles/10.3389/fimmu.2018.00078/
full#supplementary-material.
VIDEO S1 | Experimental autoimmune encephalomyelitis symptoms in Fut8+/+
mice.
VIDEO S2 | Experimental autoimmune encephalomyelitis symptoms in
Fut8−/− mice.

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Core Fucosylation in T-Cell Activation

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Copyright © 2018 Liang, Mao, Sun, Li, Li, Yu, Ma, Gu, Zhang, Taniguchi and Li.
This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does notcomply with these terms.

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.

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14

January 2018 | Volume 9 | Article 78

Published OnlineFirst March 16, 2018; DOI: 10.1158/1541-7786.MCR-17-0365

Molecular
CancerResearch

Oncogenes and Tumor Suppressors

b4-Integrin/PI3K Signaling Promotes Tumor
Progression through the Galectin-3–N-Glycan
Complex
Yukiko Kariya1, Midori Oyama1, Yasuhiro Hashimoto1, Jianguo Gu2, and Yoshinobu Kariya1

Abstract
Malignant transformation is associated with aberrant N-glycosylation, but the role of protein N-glycosylation in cancer progression remains poorly deﬁned. b4-integrin is a major carrierof N-glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N-glycosylation of b4-integrin contributes to the activation of signaling pathways that promote b4dependent tumor development and progression. Increased
expression of b1,6GlcNAc-branched N-glycans was found to becolocalized with b4-integrin in human cutaneous squamous cell
carcinoma tissues, and that the b1,6GlcNAc residue was abundanton b4-integrin in transformed keratinocytes. Interruption of
b1,6GlcNAc-branching formation on b4-integrin with the introduction of bisecting GlcNAc by N-acetylglucosaminyltransferase
III overexpression was correlated with suppression of cancer cell
migration and tumorigenesis. N-Glycan deletion on b4-integrin
impaired b4-dependent cancer cell migration, invasion, andgrowth in vitro and diminished tumorigenesis and proliferationin vivo. The reduced abilities of b4-integrin were accompanied

Introduction
Integrins are a large family of heterodimeric transmembrane
receptors comprising a and b subunits. The extracellular domains
of integrin subunits bind to extracellular matrix proteins, such as
collagen, ﬁbronectin, and laminin, and the cytoplasmic domains
interact with the actin cytoskeleton and signaling molecules. Theinteraction of integrin with its ligand can activate intracellularsignaling and reorganize the actin cytoskeleton (1). Such integrin
signaling regulates various cellular functions, such as cell adhesion, migration, and proliferation, not only in normal tissues but
also in tumor tissues (1, 2).
The a6b4-integrin (referred to herein as b4-integrin because theb4 subunit only pairs with the a6 subunit) is a receptor for

1
Department of Biochemistry, Fukushima Medical University School of Medicine,
Fukushima, Japan. 2Division of Regulatory Glycobiology, Institute of Molecular
Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical
University, Miyagi, Japan.

Note: Supplementary data for this article are available at Molecular Cancer
Research Online (http://mcr.aacrjournals.org/).
Corresponding Author: Yoshinobu Kariya, Fukushima Medical University Schoolof Medicine, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan. Phone:81-24-547-1144; Fax: 81-24-548-8641; E-mail: kariya@fmu.ac.jp
doi: 10.1158/1541-7786.MCR-17-0365
2018 American Association for Cancer Research.

with decreased phosphoinositol-3 kinase (PI3K)/Akt signals andwere restored by the overexpression of the constitutively active
p110 PI3K subunit. Binding of galectin-3 to b4-integrin via
b1,6GlcNAc-branched N-glycans promoted b4-integrin–mediated cancer cell adhesion and migration. In contrast, a neutralizing
antibody against galectin-3 attenuated b4-integrin N-glycan–
mediated PI3K activation and inhibited the ability of b4-integrin
to promote cell motility. Furthermore, galectin-3 knockdown by
shRNA suppressed b4-integrin N-glycan–mediated tumorigenesis. These ﬁndings provide a novel role for N-glycosylation of
b4-integrin in tumor development and progression, and theregulatory mechanism for b4-integrin/PI3K signaling via the
galectin-3–N-glycan complex.
Implications: N-Glycosylation of b4-integrin plays a functionalrole in promoting tumor development and progression throughPI3K activation via the galectin-3–N-glycan complex. Mol Cancer
Res; 16(6); 1024–34. 2018 AACR.

laminin-332 and is an essential component of the hemidesmosome, an anchoring structure in the basal membrane of stratiﬁedepithelial cells (2, 3). In contrast to such a static role in normal
epithelial cells, b4-integrin was originally identiﬁed as a tumorassociated antigen (4, 5). b4-integrin overexpression was found inseveral types of metastatic cancers, which correlates with poor
prognosis and reduced survival (6, 7). Recent studies have shownthat b4-integrin is required for tumorigenesis in several in vivo
mouse models (8–10). In addition, b4-integrin promotes cell
motility, invasion, and proliferation through activation of thephosphoinositol-3-kinase (PI3K) and ERK pathways (10–14).
Glycosylation is the most common posttranscriptional modiﬁcation of proteins, and it modulates the folding, stability,and function of glycoproteins (15). Malignant transformation
is associated with aberrant glycosylation of cell surface proteins, and the structural change of glycans is mainly due
to the altered activity and expression of multiple glycosyltransferases in cancer cells. In addition, some glycans are used astumor markers for cancer diagnosis (16). Overexpressionof b1,6GlcNAc-branched N-glycans, which is due to increased
activity of N-acetylglucosaminyltransferase V (GnT-V; Fig. 1A),
is often found in tumor tissues, and the increase inb1,6GlcNAc-branched N-glycans is directly associated withmalignancy and poor prognosis (17, 18). GnT-V knockout
mice exhibit reduced mammary tumor growth and metastasis
induced by the polyomavirus middle T oncogene (19). Furthermore, introduction of bisecting GlcNAc residues catalyzed

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N-Glycosylation of b4-Integrin Promotes Tumor Progression

Figure 1.
Increased expression of b1,6GlcNAc
residues on b4-integrin in humancarcinoma cells. A, Glycosylation
reactions catalyzed byglycosyltransferases, GnT-III and
GnT-V. b1,6GlcNAc-branchedN-glycans can be preferentially
modiﬁed by polylactosamine N-glycan
containing b-galactoside. Bisecting
GlcNAc N-glycans inhibit b1,6GlcNAcbranching formation catalyzed by GnTV. B, Immunohistochemical analysis of
bisecting GlcNAc and b1,6GlcNAc
expression in human cutaneous SCC
using E4-PHA and L4-PHA lectins.
C, Immunoﬂuorescence microscopic
analysis of b4-integrin (red) andb1,6GlcNAc (L4-PHA, green) in parafﬁnembedded sections of humancutaneous SCC. Merged images areshown with nuclear Hoechst staining(blue). Normal human skin was stained
as comparison. D, Western blot analysisof b4-integrin immunoprecipitatesfrom normal human keratinocytes(NHK) and Ras/IkB-transformed NHK(transformed NHK) with L4-PHA andE4-PHA lectins, and anti–b4-integrin
polyclonal antibody. Results of thedensitometric analysis are shown as theintegrated density of lectin-recognized
b4 to total b4 bands, which was
1.0 for NHK cells.

by N-acetylglucosaminyltransferase III (GnT-III) into N-glycans, which can disturb further processing and elongation of
N-glycans, such as the formation of b1,6GlcNAc-branchedstructures (Fig. 1A), suppresses cell migration and cancer
metastasis (20).
b1,6GlcNAc-branched N-glycans are often further modiﬁed byadditional sugars to form poly-N-acetyllactosamine (polylactosamine) for the elongation of N-glycans consisting of b-galactoside sugars (Fig. 1A). This polylactosamine structure is a preferred
ligand for one of the galectin isoforms, galectin-3 (21). Galectin-3
is widely expressed in epithelial and immune cells, and its
expression is associated with cancer aggressiveness and metastasis(22–24). Cross-linking between glycoproteins by the binding ofgalectin-3 to polylactosamine on proteins regulates diverse cellular functions in cancer cells (25–27). We previously reported
that N-glycosylation of b4-integrin plays a crucial role in celladhesion, migration, and galectin-3 binding in human keratinocyte cells (28). However, the contribution of N-glycosylation of
b4-integrin to tumor progression remains poorly deﬁned. In thepresent study, we investigated the contribution of N-glycosylation
to b4-integrin-dependent tumor progression.

Materials and Methods
Antibodies and reagentsThe following antibodies were used in this study: rat monoclonal antibodies speciﬁc for galectin-3 (M3/38; #sc-2393; Santa
Cruz Biotechnology), a6-integrin (GoH3; #sc-19622; Santa Cruz

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Biotechnology), and b4-integrin (439-9B; #555719; BD Transduction Laboratories); rabbit polyclonal antibodies against
human b4-integrin (H101; #sc-9090; Santa Cruz Biotechnology),
phospho-Akt (pSer473; #9271; Cell Signaling Technology), and
Akt (#9272; Cell Signaling Technology); and mouse monoclonal
antibodies against Ki-67 (#610968; BD Transduction Laboratories), and b4-integrin (3E1; #MAB1964; Merck Millipore). Alexa
Fluor 488-conjugated leukoagglutinating phytohemagglutinin(L4-PHA; #L-11270), Alexa Fluor 546-conjugated goat anti-rabbit
IgG secondary antibody (#A11035) and Alexa Fluor 546-conjugated goat anti-rat IgG secondary antibody (#A11081) werepurchased from Thermo Fisher Scientiﬁc. Biotinylated L4-PHA(#B-1115) was obtained from Vector Laboratories. Hoechst
33342 (#382065) was obtained from Merck Millipore. Biotinylated erythroagglutinating phytohemagglutinin (E4-PHA;#300425) and biotinylated Sambucus sieboldiana agglutinin (SSA;#300442) were purchased from J-OIL MILLS. Puriﬁed humanlaminin-332 and galectin-3 were prepared as described previously(27, 29).
Cell culture
The human cancer MDA-MB435S cell line, which lacks b4integrin, was purchased from the American Type Culture Collection. Modiﬁed human 293 phoenix cells were received as a
gift from Dr. M. Peter Marinkovich (Stanford University, Stanford, CA). The human epidermoid carcinoma cell line A431
(RCB0202) was provided by the RIKEN BRC through the
National Bio-Resource Project of the MEXT, Japan. These cells

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Kariya et al.

were cultured in DMEM supplemented with 10% FBS, penicillin and streptomycin sulfate at 37 C in a humidiﬁed 5% CO2
incubator. Primary human keratinocytes isolated fromnormal skin (NHK) and Ras/IkB-transformed NHK (a gift fromDr. M. Peter Marinkovich; ref. 8) were cultured in a 50:50
mixture of deﬁned keratinocyte serum-free medium (#10744019; Life Technologies) and medium 154 (#M-154-500; Life
Technologies) with human keratinocyte growth supplement(#S-001-5; Life Technologies) containing penicillin and streptomycin sulfate at 37 C in a humidiﬁed 5% CO2 incubator. All
cells were passaged fewer than 6 months after purchasing or
receiving them for all the experiments and tested for mycoplasma
contamination.
Immunohistochemistry/immunoﬂuorescence
Human formalin-ﬁxed parafﬁn-embedded sections were purchased from BioChain and US Biomax. Specimens were deparafﬁnized, rehydrated, and immersed in 0.3% hydrogen peroxidase-containing methanol for 20 minutes at room temperature
to inactivate the intrinsic peroxidase. After blocking with 5%
skim milk for 1 hour at room temperature, the sections wereincubated with each biotinylated lectin overnight at 4 C, followed by peroxidase-labeled streptavidin (#426061; Nichirei
Corp.) and 3,30 -diaminobenzidine detection using the Histoﬁne DAB substrate kit (#425011; Nichirei Corp.). The slides
were then counterstained with Mayer's hematoxylin solution(#131-09665; Wako). Images were obtained using a PROVIS
AX-80 microscope (Olympus). For immunoﬂuorescent staining, sections were deparafﬁnized, rehydrated, and treated with
0.05% protease XXIV in 50 mmol/L Tris-HCl (pH 7.5) for 20
minutes at room temperature. After blocking with 2% BSA in
PBS for 1 hour at room temperature, the sections were visualized using a b4-integrin antibody (H101) and Alexa Fluor 488conjugated L4-PHA. Fluorescence images were obtained using
an IX71 ﬂuorescent microscope (Olympus). Lectin reactivity
and b4-integrin expression were assessed as follows: positive,
10% positive tumor cells; negative, <10% positive tumor
cells. For cross-linking inhibitory assay, glass-bottom dishes(#3971-035; IWAKI) were coated with laminin-332 proteinsand blocked with 1% BSA in PBS. Cells (5  104 cells) wereincubated in serum-free medium in the presence of IgG or afunctional blocking antibody against galectin-3 for 20 minutes.
Then, the cells were plated to the glass-bottom dish andincubated for 1.5 hours at 37 C in a humidiﬁed 5% CO2
incubator. The cells were ﬁxed with 4% (w/v) paraformaldehyde in PBS for 10 minutes and blocked with 2% BSA in PBS for
1 hour at room temperature. The ﬁxed cells were stained with ab4-integrin antibody (H101) and Alexa Fluor 488-conjugatedanti-rabbit IgG antibody. Fluorescence images were obtainedusing an A1 confocal microscope (Nikon).
Preparation of cell lysates and immunoprecipitation
Cell lysates were prepared as follows. The cells were washedtwice with cold PBS and then lysed with a lysis buffer [1% Triton X100, 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L
EDTA] containing a protease inhibitor cocktail (#25955; Nacalaitesque) and a phosphatase inhibitor cocktail (#07575; Nacalaitesque). After incubation for 10 minutes on ice, the cell lysates
were clariﬁed by centrifugation at 15,000 rpm for 10 minutes at
4 C. The resulting supernatant was used in the following experi-

1026 Mol Cancer Res; 16(6) June 2018ments. The protein concentration was determined using a protein
assay kit (#29449-44; Nacalai tesque). For immunoprecipitation,
the primary antibody was added to the supernatant and rotatedfor 2 hours at 4 C. Then, protein G-Sepharose was added, followed by 3 hours of incubation at 4 C. Immunoprecipitates were
washed ﬁve times with a washing buffer [50 mmol/L Tris-HCl (pH7.5), 150 mmol/L NaCl, 2 mmol/L EDTA, 0.2% NP40 (v/v)],
suspended in a reducing sample buffer, and heated at 95 C for 5
minutes.
Western blot analyses
Protein samples were resolved by SDS-PAGE under reducing
conditions and then transferred to nitrocellulose membranes.
The blots were probed with each speciﬁc antibody or biotinylated lectin. Immunoreactive bands were detected using an
ImmunoStar Zeta (#297-72403; WAKO) or Trident femto-ECL(#GTX14698; GeneTex). Band intensity was calculated using
NIH ImageJ software.
Expression vectors
Retroviral expression vectors encoding b4-integrin were prepared as previously described (28). Retroviral expression vector
encoding active PI3K p110-CAAX was received as a gift from Dr.
M. Peter Marinkovich. The cDNA encoding human GnT-III was
ampliﬁed by PCR using a speciﬁc primer set and KOD Plus
polymerase (#KOD-201; TOYOBO) for cloning into pENTR-DTOPO (#K2400-20; Life Technologies) for the Gateway Conversion System according to the manufacturer's instructions. The
ﬁnal construct was recombined from pENTR-D-TOPO to the LZRS
blast retroviral vector, including a Gateway cassette, using the LR
clonase II Enzyme mix (#11791-020; Life Technologies) by arecombination reaction. The cDNA sequence was veriﬁed bysequencing.
Retrovirus infection
Retrovirus vectors were transfected into 293 phoenix cells
using FuGENE6 transfection reagent (#11814443001; Roche).
After transfection, cells were selected with 5 mg/mL puromycin(#P8833; Sigma-Aldrich). The retrovirus was then produced in
293 phoenix cells. One day before infection, 4  105 cells wereplated in 6-well plates. After incubation with 5 mg/mL polybrene (#10768-9; Sigma-Aldrich) for 15 minutes, media were
exchanged to 3 mL retroviral supernatant and another 5 mg/mL
polybrene was added. Plates were centrifuged at 1,200 rpm for
1 hour at 32 C using a Hitachi CR22N centrifuge machine.
After centrifuge, the retroviral supernatant was replaced withgrowth medium and the cells were maintained. To establish acell line, cells were selected with 5 mg/mL blasticidin S(#203350; Calbiochem).
Flow cytometry analysis
Cells were detached from a 10-cm dish using trypsin with 1
mmol/L EDTA. After quenching trypsinization with a mediumthat contained 10% FBS, the cells were washed twice with PBS that
contained 1 mmol/L EDTA and incubated with a primary antibody or control IgG on ice for 30 minutes. The cells were thenwashed thrice with PBS that contained 1 mmol/L EDTA, followed
by incubation for 15 minutes with the appropriate Alexa Fluorconjugated secondary antibodies. After washing thrice with PBS
containing 1 mmol/L EDTA, the cells were analyzed by ﬂow

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N-Glycosylation of b4-Integrin Promotes Tumor Progression

cytometry using a FACSCalibur and a CellQuest software program(BD Biosciences).
Cell migration assay
Cell migration was assayed using a 24-well chemotaxis chamber (BD Falcon cell culture companion plates; #353504 and 8.0mm insets; #353097; BD Biosciences). Two hundred microliters of
2.5  105 cells/mL (5  104 cells in serum-free DMEM) was
inoculated into each upper well of a 24-well chemotaxis chamber,and 750 mL of 10% FBS containing DMEM was placed in thebottom chamber to act as a chemoattractant. After incubating for
22 hours, the cells on the upper side of the membrane were
removed with a cotton swab, and the cells on the lower side of themembrane were ﬁxed with 4% paraformaldehyde and stainedwith 0.5% crystal violet in 20% methanol. Random ﬁelds were
photographed using a phase contrast microscope and the numberof migrated cells was counted.
In vivo tumorigenicity assay
All animal studies were performed in accordance with protocols approved by the Fukushima Medical University Animal Careand Use Committee. MDA-MB435S transfectants (1  106 cells/mouse) were injected subcutaneously along with Matrigel(#354234; BD Biosciences) into 6-week-old female nude mice.
Tumor volume was measured weekly for a total of 6 weeks.
Proliferating cells in 5-mm frozen sections were detected with
Ki-67 immunoﬂuorescent staining. Proliferation was quantiﬁed
as the ratio of Ki-67 staining to total nuclear staining.
Cell adhesion assay
Cell adhesion assays were performed as described previously(29). In brief, the wells of a 96-well ELISA plate (#3590; Corning)were coated with 50 mL of indicated concentration of laminin-332
proteins and blocked with 1% BSA in PBS. Cell suspensions inserum-free medium were plated to each well (5  104 cells/well)and incubated at 37 C for 20 minutes in a 5% CO2 incubator.
After removing the non-adherent cells, the adherent cells were
ﬁxed with 25% (w/v) glutaraldehyde for 10 minutes and stainedwith 0.5% crystal violet in 20% (v/v) methanol for 10 minutes.
Random ﬁelds were photographed using a phase contrast microscope, and the number of adherent cells was counted.
Cell proliferation assay
Cell suspensions in 1% serum-containing medium were plated
to each well of a 96-well ELISA plate (5  103 cells/well) andincubated at 37 C for 45 hours in a 5% CO2 incubator. Afterincubation, the number of growing cells was measured using a cellcounting kit-8 (#347-07621; DOJINDO) according to the manufacturer's instructions.
Cell invasion assay
One hundred microliters of Matrigel, diluted to a ﬁnal concentration of 1.6 mg/mL, was added to the upper chamber of 24well Transwell plates and dried for 24 hours in a hood. The wells
were reconstituted by incubation with 200 mL of serum-free
DMEM at 37 C for 1 hour. After removing the medium, 200 mLof 5  105 cells/mL (1  104 cells in 0.1% BSA containing serumfree DMEM) were inoculated into each upper well of a 24-wellchemotaxis chamber, and 750 mL of 10% FBS containing DMEM
was placed in the bottom chamber to act as a chemoattractant.
After 6 hours of incubation, the cells on the upper side of themembrane were removed with a cotton swab, and the cells on the

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lower side of the membrane were ﬁxed with 4% paraformaldehyde and stained with 0.5% crystal violet in 20% methanol.
Random ﬁelds were photographed using a phase contrast microscope and the number of invaded cells was counted.
Lentiviral short hairpin RNA (shRNA)
Lentiviral shRNA clones (Dharmacon RNAi consortium Lentiviral shRNA) targeting galectin-3 (#RHS3979-201759611, clone
ID: TRCN0000029304) 50 -ATTGTACTGCAACAAGTGAGC-30
and control shRNA (#RHS4459) 50 -TACAACAGCCACAACGTCTAT-30 were purchased from GE Healthcare. These vectors were
cotransfected with the packaging vectors into HEK293T cells using
the Trans-Lentiviral shRNA Packaging kit (#TLP5912; GE Healthcare) according to the manufacturer's instructions. After incubation for 15 hours, media were exchanged to 5% FBS containing
DMEM. After further incubation for 48 hours, viral supernatant
was harvested and then centrifuged at 1,600 g for 10 minutes at
4 C. The supernatant was ﬁltrated through a 0.45-mm ﬁlter.
Lentivirus was infected into MDA-MB435S cells using the same
method as retrovirus infection. To establish a cell line, cells wereselected with 5 mg/mL puromycin.
Statistical analysis
Results are given as mean  SEM and are representative of twoor three independent experiments. Statistical comparisons were
calculated between two groups using unpaired Student t test andamong the groups using one-way or two-way ANOVA followed bya Bonferroni posttest, with GraphPad Prism Version 5.0a software. A P value of <0.05 was considered statistically signiﬁcant.

Results
b1,6GlcNAc residue on b4-integrin was associated with cell
migration and tumorigenesis
Expression of b1,6GlcNAc is associated with metastasis andpoor prognosis, whereas bisecting GlcNAc suppresses the effect byinhibition of b1,6GlcNAc-branching formation (20). Human
cutaneous squamous cell carcinoma (SCC) showed positive
expression of b1,6GlcNAc (52 out of 75) and negative expressionof bisecting GlcNAc (72 out of 75) by immunohistochemistry
using L4-PHA and E4-PHA lectins (Fig. 1B, n ¼ 75), which isconsistent with previous observations that b1,6GlcNAc was more
likely to be associated with tumor malignancy (17). As overexpression of b4-integrin in SCC has been reported (6, 30), we
next examined the expression patterns of b4-integrin andb1,6GlcNAc in human cutaneous SCC. In skin cancer specimens(n ¼ 37), b4-integrin was abundantly present and colocalizedwith b1,6GlcNAc-branched N-glycans. Thirty-three b4-integrin
positive tumor cells, 33 (100%) colocalized b1,6GlcNAcbranched N-glycans (Fig. 1C). In contrast, b4-integrin was clearly
localized in the basement membrane, while the expression of
b1,6GlcNAc was almost undetectable in all tested normal humanskin samples (n ¼ 7). Taking these results into consideration, we
hypothesized that the amount of b1,6GlcNAc-branched N-glycans on b4-integrin increases in cancer cells. To test this hypothesis, we examined whether b1,6GlcNAc was attached to b4-integrin upon transformation of NHK cells. Ras/IkB-mediated transformation of NHK cells increased attachment of b1,6GlcNAc tob4-integrin while the extent of the bisecting GlcNAc modiﬁcationof b4-integrin was decreased after transformation (Fig. 1D). These

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results suggest that the b1,6GlcNAc modiﬁcation of b4-integrin is
increased in cancer cells.
GnT-III inhibits the formation of b1,6GlcNAc branching on Nglycans by the introduction of bisecting GlcNAc (Fig. 1A). To
address the role of b1,6GlcNAc modiﬁcation of b4-integrin intumor malignancy, we used MDA-MB435S cells, which do not
express b4-integrin, for preparing stable transfectants retrovirallytransduced with control lacZ, human b4-integrin, lacZ with GnTIII, or b4 with GnT-III. The FACS analysis exhibited that GnT-III
expression did not affect the cell surface expression of b4-integrin(Fig. 2A, lacZ versus lacZþGnT-III and b4 versus b4þGnT-III).
Overexpression of GnT-III in cells expressing b4-integrin significantly reduced b1,6GlcNAc-branched N-glycans on b4-integrin
accompanied with increased bisecting GlcNAc (Fig. 2B). Expression of b4-integrin exhibited enhanced cell migration (Fig. 2C)
and tumor formation (Fig. 2D) in the MDA-MB435S cells compared with the control cells, which were impaired by GnT-III
expression. These results suggest that the b1,6GlcNAc residue in
b4-integrin may be associated with primary tumor growth andmetastatic cancer cell behavior.
N-Glycosylation of b4-integrin drives proliferation, migration,and invasion through PI3K/Akt activation
GnT-III overexpression modiﬁes not only b4-integrin but
also other glycoproteins (Supplementary Fig. S1). b4-integrin
contains ﬁve potential N-glycosylation sites in its extracellulardomain (Fig. 3A). To directly address the biological role of Nglycosylation of b4-integrin in cancer cell behavior, we prepared two b4-integrin constructs; a full-length wild-type (WT)and a mutant lacking all ﬁve N-glycosylation sites (DN; ref. Fig.
3A). These constructs were retrovirally expressed in MDAMB435S cells that endogenously expressed no b4-integrin (Fig.
2A, lacZ). To conﬁrm the loss of N-glycans on DN, we performed lectin blot analysis using L4-PHA, E4-PHA, and SSA
lectins, which recognize b1,6GlcNAc, bisecting GlcNAc, anda2,6 sialic acid in N-glycans, respectively. The WT reacted withall tested lectins, but the DN did not react (Fig. 3B), suggesting
the loss of N-glycans in the DN. Similar to our previous reports
using keratinocytes (28), the deletion of N-glycans had noeffect on either cell surface expression (Supplementary Fig.
S2A) or heterodimer formation of b4-integrin with a6-integrin(Supplementary Fig. S2B) in the MDA-MB435S cells. These
results suggest that N-glycans on b4-integrin were not required
for either expression or heterodimer formation of a6b4-integrin. We next assessed whether N-glycosylation on b4-integrin
can affect cell adhesion and spreading, which are generally
associated with cancer progression, using the MDA-MB435S
transfectants. Typically, b4-integrin causes cellular signaling
through cell adhesion to the extracellular matrix protein laminins in cancer progression (2, 31). Compared with the WT cells,
the DN cells showed decreased cell adhesion and spreading on alaminin-332 substrate, which was comparable to those in thecontrol lacZ cells (Fig. 3C), suggesting that N-glycans on b4integrin are related to b4-integrin-dependent cell adhesion andspreading in MDA-MB435S cancer cells.
b4-integrin regulates cell proliferation, migration, and invasionin cancer cell lines (2, 6). Therefore, we examined the cellularfunctions of MDA-MB435S cancer cells expressing WT and DN.
The expression of b4-integrin markedly enhanced cell proliferation, migration, and invasion of the MDA-MB435S cells (Fig. 3D–
F, WT) compared with the control lacZ cells. The enhanced cellularfunctions were signiﬁcantly impaired by the loss of N-glycans on
b4-integrin (Fig. 3D–F, DN). Previous studies have shown that b4integrin–dependent cell proliferation, migration, and invasion
are closely associated with the PI3K pathway in several carcinoma
cell lines (9, 13). To address whether the decreased cellularfunctions due to the deletion of N-glycosylation were correlated
to the downregulation of PI3K signaling, we examined the activation of the PI3K signaling pathway by Western blot analysis
with an anti-phosphorylated Akt antibody. The DN cells, as wellFigure 2.
Decreased b1,6GlcNAc residues in b4integrin by GnT-III correlates with
suppression of cancer cell migrationand tumorigenesis. A, Cell surface
expression of b4-integrin in MDAMB435S cells expressing control lacZ,
lacZ with GnT-III (lacZþGnT-III), b4,
and b4 with GnT-III (b4þGnT-III).
B, Lectin blot analysis of b4-integrin
immunoprecipitates from MDAMB435S cells expressing b4 and b4
with GnT-III using L4-PHA(b1,6GlcNAc) and E4-PHA (bisecting
GlcNAc) lectins. Data arerepresentative of three independent
experiments. C, Effect of GnT-III
overexpression on b4-integrin–
mediated cell migration. Results are
the mean  SEM of three independent
experiments conducted in triplicate.
, P < 0.001. ns, not signiﬁcant (twoway ANOVA, Bonferroni posttest). D,
Tumor formation in nude mice injectedsubcutaneously with the indicatedcells. Results are the mean  SEM of
5 mice per group.  , P < 0.05 (two-way
ANOVA, Bonferroni posttest).

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N-Glycosylation of b4-Integrin Promotes Tumor Progression

Figure 3.
N-Glycosylation of b4-integrin drives tumor cell proliferation, migration, and invasion through PI3K/Akt activation. A, Schematic diagram of the human b4-integrin
cDNA constructs in this study. WT, wild-type b4-integrin; DN, N-glycosylation–defective b4-integrin mutant. The sites corresponding to the N-glycosylation sites on
b4-integrin subunit (Asn327, Asn491, Asn579, Asn617, and Asn695) are shown by ﬂags. Numbers indicate the number of amino acid residues. TM, transmembraneregion. B, Lectin blot analysis of b4-integrin immunoprecipitates from MDA-MB435S cells expressing lacZ, WT, and DN with L4-PHA, E4-PHA, and SSA lectins.
C, Cell adhesion and spreading of MDA-MB435S transfectants to laminin-332. Note that MDA-MB435S cells expressing no b4-integrin (lacZ) or DN showed decreased
cell adhesion and spreading compared with the cells expressing WT. Cell spreading morphology was examined under a phase-contrast microscope after
incubation for 20 minutes. D–F, Effect of the N-glycosylation defect in b4-integrin on cell proliferation (D), migration (E), and matrigel invasion (F). Note that cells
expressing DN showed decreased cell proliferation, migration, and matrigel invasion, which were rescued by overexpression of constitutively active PI3K p110a
subunit (DNþPI3K). In contrast, overexpression of the active PI3K subunit had no signiﬁcant effect on WT-b4–dependent cell proliferation, migration, or matrigel
invasion (WTþPI3K). Results are the mean  SEM for three independent experiments conducted in triplicate.   , P < 0.01;    , P < 0.001; ns, not signiﬁcant (one-way
ANOVA, Bonferroni posttest) versus WT. G, Western blot analysis of cell lysates from MDA-MB435S transfectants with phospho-Akt and total Akt antibody. Note thatcells expressing lacZ or DN showed decreased PI3K/Akt pathway activation, which was rescued by overexpression of constitutively active p110a PI3K
subunit (DNþPI3K). Results of the densitometric analysis are shown as the integrated density of the ratio of phosphorylated protein to total protein bands. All blots
are representative of at least three independent experiments.

as the lacZ cells, showed decreased phosphorylation of Akt compared with the WT cells (Fig. 3G). We also found that the
impairment of cellular functions in the DN cells was completely
restored through overexpression of the constitutively active PI3K
p110a subunit (Fig. 3D–F, DNþPI3K). In contrast, overexpression of the active PI3K subunit in WT cells did not signiﬁcantly
affect the WTb4-dependent cell proliferation, migration, andinvasion (Fig. 3D–F, WTþPI3K), suggesting that the WTb4-mediated PI3K/AKT activation is sufﬁcient for such functions in the

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cells and higher PI3K/AKT activation does not always promote
activities of cancer cells. Together, these ﬁndings demonstrate that
the N-glycosylation of b4-integrin plays a fundamental role inactivating the PI3K pathway, thereby promoting cell proliferation,
migration, and invasion.
N-Glycans of b4-integrin promote in vivo tumor growth.
To determine whether N-glycosylation of b4-integrin is related
to tumorigenesis in vivo, we subcutaneously injected MDA-

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Figure 4.
N-Glycosylation of b4-integrin
functionally contributes totumorigenesis in vivo. A, Tumor
growth after subcutaneous injectionof MDA-MB435S cells expressing theindicated proteins into mice (n ¼ 5
mice per group  SEM).   , P < 0.01;
, P < 0.001 (one-way ANOVA,
Bonferroni posttest). B, Hematoxylin/
eosin staining of the indicated tumors.
C, Immunoﬂuorescent analysis of
Ki-67 (green) and b4-integrin (red)expression in the indicated tumors.
Nuclei were stained with Hoechst
33342 (blue). D, Quantiﬁed
percentage of Ki-67-positive cells tototal cells in C. The bar graphrepresents the mean  SEM forthree independent experiments.
, P < 0.001; ns, not signiﬁcant(one-way ANOVA, Bonferroni
posttest) versus WT.

MB435S transfectants into nude mice and analyzed tumor growth.
Compared with the WT cells, the DN cells showed signiﬁcantly
impaired tumorigenesis, which was comparable with the b4
null (lacZ) cells (Fig. 4A and B). Impairment of the tumorigenesis
in the DN cells was fully restored through overexpression of theconstitutively active PI3K p110a subunit (Fig. 4A, DNþPI3K). In
comparison with the WT tumors, staining with anti–Ki-67 antibody demonstrated that the DN tumors included notably fewer
proliferating cells, which was fully increased to WT tumor levels byoverexpression of the constitutively active PI3K p110a subunit(Fig. 4C and D). These results demonstrate that the ability of b4integrin to drive tumorigenesis, and tumor proliferation is associated with activation of PI3K pathway via N-glycans.
b4-integrin–dependent tumor progression is associatedwith interaction between galectin-3 and b1,6GlcNAc
residue on b4-integrin
Galectin-3 is known to be associated with cancer aggressiveness and metastasis (22–24). To examine the role ofgalectin-3 in b4-integrin–mediated tumor progression, we
performed inhibition assays using a functional blocking antibody against galectin-3 (M3/38). The functional blocking
antibody against galectin-3 suppressed b4-integrin clustering
probably through inhibition of galectin-3 multimerization,suggesting that galectin-3 actually cross-links b4-integrin (Fig.
5A). The antibody suppressed WT cell motility, whereas noeffect was observed in the DN cells (Fig. 5B). Corresponding
with the decreased cell motility of the WT cells, the galectin-3
antibody reduced Akt phosphorylation compared with control
IgG in the WT cells (Fig. 5C). Furthermore, we tested thetumorigenicity of the WT cells expressing control shRNA or
galectin-3 shRNA to conﬁrm the importance of galectin-3

1030 Mol Cancer Res; 16(6) June 2018expression on tumor formation (Supplementary Fig. S3). The
result showed that reduced expression of galectin-3 in WT cells
suppressed tumor formation, suggesting that galectin-3 plays
an important role in b4-integrin–promoting tumor formation(Fig. 5D).
Galectin-3 has a high afﬁnity for b1,6GlcNAc-branched Nglycans modiﬁed by polylactosamine consisting of b-galactosidesugars. The oligomerization of integrins by galectin-3–mediatedcross-linking between the b1,6GlcNAc-branched N-glycans on
integrins promotes integrin function (25, 27, 32). To understandwhy deletion of N-glycosylation suppressed the ability of b4integrin to promote cancer cell progression, we examined therelationship between galectin-3 binding to b4-integrin and cellular functions of b4-integrin. b4-integrin immunoprecipitatesfrom the WT cells contained high amounts of galectin-3 comparedwith those from the lacZ and DN cells, indicating that N-glycans
on b4-integrin promote complex formation by the two molecules(Fig. 5E). The association of b4-integrin with galectin-3 wasinhibited by a competitive inhibitor of galectin binding, b-lactose but not by control sucrose, suggesting that galectin-3 isbound to b1,6GlcNAc-branched N-glycans modiﬁed by polylactosamine on b4-integrin (Fig. 5F). Similar results wereobtained from the squamous cell carcinoma cell line, A431
cells, which endogenously express b4-integrin. The addition of
galectin-3 signiﬁcantly enhanced cell adhesion of the WT cells
but not the lacZ or DN cells to a laminin-332 substrate (Fig.
5G). Furthermore, the addition of galectin-3 to the cell culture
signiﬁcantly promoted cell motility of the WT cells but not thatof either the WT with GnT-III cells or DN cells (Fig. 5H). Theseﬁndings suggest that galectin-3 binding to b4-integrin throughb1,6GlcNAc-branched N-glycans promotes the ability of b4integrin to drive cell adhesion and motility.

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N-Glycosylation of b4-Integrin Promotes Tumor Progression

Figure 5.
Galectin-3 is a key modulator of b4-integrin function through N-glycan. A, Inhibitory effect of a functional blocking antibody against galectin-3 (Ab) or control IgG on
b4-integrin clustering. Note that MDA-MB435S cells expressing WT showed decreased b4-integrin clustering by the treatment of a functional blocking antibody
against galectin-3. B, Inhibitory effect of a functional blocking antibody against galectin-3 on N-glycosylation of b4-integrin–mediated cell migration. Note that
MDA-MB435S cells expressing WT but not DN showed decreased cell migration by the treatment of a functional blocking antibody against galectin-3.
C, MDA-MB435S cells expressing WT and A431 cells were incubated in serum-free medium in the presence of IgG or a functional blocking antibody against galectin-3(Ab) for 20 minutes. Cell lysates were analyzed by Western blot with phospho-Akt and total Akt antibody. D, Effect of galectin-3 (gal3) knockdown on tumor growth
in MDA-MB435S cells expressing WT. Results are the mean  SEM of six mice per group.   , P < 0.001 (unpaired Student t test). E, Western blot analysis of b4-integrin
immunoprecipitates from MDA-MB435S transfectants and A431 cells. F, MDA-MB435S cells expressing WT and A431 cells were serum-starved for 2 hours
and were then incubated in serum-free medium in the presence of conditioned medium of MDA-MB435S cells and saccharides (0.2 mol/L) for 30 minutes.
The b4-integrin immunoprecipitates from the cells were analyzed by Western blot. Note that the binding of galectin-3 to b4-integrin was inhibited by a competitive
disaccharide, b-lactose, but not a noncompetitive disaccharide, sucrose. Results of the densitometric analysis are shown as the integrated density of the ratio of
galectin-3 protein to b4-integrin protein bands. G, Effect of galectin-3 on N-glycans of b4-integrin–mediated cell adhesion. Note that galectin-3 enhanced
cell adhesion of MDA-MB435S cells expressing WT but not either lacZ or DN to laminin-332. H, Effect of galectin-3 on N-glycosylation in b4-integrin–mediated cell
migration. Note that MDA-MB435S cells expressing WT, but not either WT with GnT-III or DN, showed increased cell migration in the presence of galectin-3.
Results are the mean  SEM for two or three independent experiments conducted in triplicate.  , P < 0.05;    , P < 0.001; ns, not signiﬁcant (one-way ANOVA,
Bonferroni posttest).

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Figure 6.
Hypothetical model of Nglycosylation–mediated b4integrin–dependent tumor
progression. Galectin-3 cross-links
between b1,6GlcNAc-branchedN-glycans, which in turn promotes
b4-integrin clustering. The complex
formation activates PI3K/Akt
signaling, thereby promoting tumor
progression by stimulating cell
migration, invasion, proliferation, andtumorigenesis.

Discussion
In the current study, b4-integrin was colocalized with
b1,6GlcNAc-branched N-glycans in tumor tissues. The suppression of the modiﬁcation of b4-integrin by GnT-III was associatedwith reduced cancer cell migration and tumorigenesis in MDAMB435S cells expressing b4-integrin. Deletion of N-glycosylationsites in b4-integrin, which was accompanied with downregulationof the PI3K signaling pathway, inhibited b4-integrin–dependent
cancer cell migration, invasion, proliferation, and tumor formation. Furthermore, loss of association between galectin-3 and b4integrin via b1,6GlcNAc-branched N-glycans abolished galectin3–promoting cancer cell adhesion and migration. These results
provide evidence that N-glycosylation of b4-integrin plays afunctional role in promoting tumor development and progression through the PI3K activation.
b4-integrin promotes cell proliferation, migration, and invasion (31, 33), and plays pivotal roles in tumorigenesis (8). In thepresent study, deletion of N-glycosylation sites in b4-integrin
suppressed those cellular functions in vitro as well as cell proliferation and tumorigenesis in vivo. GnT-V knockout mice havebeen reported to suppress polyomavirus middle T oncogeneinduced mammary tumor growth and metastasis (19). Previous
studies have shown that an increase of b1,6GlcNAc in the a3-, a5-,
or b1-integrin subunit resulted in increased cancer cell migration(34, 35). Our ﬁndings indicate that modiﬁcation of b4-integrin

1032 Mol Cancer Res; 16(6) June 2018with b1,6GlcNAc may be upregulated in tumor tissue. In addition,
suppression of the b1,6GlcNAc modiﬁcation in b4-integrin byoverexpression of GnT-III reduced b4-integrin–dependent cancer
cell migration and tumor formation. Therefore, this loss of b4integrin function by the N-glycosylation defect may be mainly due
to a lack of b1,6GlcNAc modiﬁcation in b4-integrin.
Galectin-3 has a high afﬁnity for b1,6GlcNAc-branched Nglycans through multivalent binding, and thereby cross-links
glycoproteins on the cell surface and in the extracellular matrix
to form molecular complexes (24). The formation of the macromolecular complexes on the cell surface by galectin-3 affects the
distribution of glycoproteins and cellular signaling. Furthermore,
integrin clustering mediated by galectin-3 promotes integrin
activation (25, 27). In our study, we found that the deletion of
N-glycans of b4-integrin, resulting in impaired galectin-3 binding
to b4, suppressed b4-integrin functions mainly through downregulation of the PI3K pathway. These results support the hypothesis that the cross-linking between b1,6GlcNAc-branched N-glycans of b4-integrins by galectin-3 directly or indirectly affects PI3K
activation and cellular functions such as cell motility and tumorigenesis (Fig. 6); however, further investigation is required.
The association of b4-integrin with laminin-332 is known to
induce PI3K activation, thereby promoting cell adhesion andmigration (2). Our previous study showed that a decreased levelof b1,6GlcNAc-branched N-glycans on laminin-332 by

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N-Glycosylation of b4-Integrin Promotes Tumor Progression

introduction of bisecting GlcNAc suppressed its cell adhesion andmigration activity as well as galectin-3–mediated b4-integrin
clustering (27). The present study showed that a defect of Nglycosylation in b4-integrin suppressed the association with laminin-332 and PI3K activation, suggesting that N-glycosylation of
b4-integrin played a critical role in PI3K activation through theinteraction with laminin-332. These results suggest that the association between laminin-332 and b4-integrin throughb1,6GlcNAc-branched N-glycans may also be important for b4integrin–dependent tumor progression.
In the present study, we cannot exclude the potential effect of
the lack of N-glycosylation on b4-integrin protein folding. NGlycosylation on integrins is important for such protein folding,which is required for heterodimer formation. In fact, the lack of Nglycans on a5-integrin causes its misfolding and loss of heterodimer formation with b1-integrin (36). In contrast, DNb4-integrin
could form a heterodimer with a6-integrin (Supplementary Fig.
S2B). Furthermore, FACS analysis showed that DNb4 expressedon the cell surface, which is comparable with WTb4 (Supplementary Fig. S2A). In essence, noncomplexed integrin is degraded
immediately or remains in the endoplasmic reticulum (37).
Therefore, the lack of N-glycans on b4-integrin seems to affectits function and the association with other molecules, rather thanb4-integrin folding.
In conclusion, our study suggests that N-glycosylation of b4integrin is associated with tumor development and progression
through b4-integrin/PI3K signaling via the galectin-3–N-glycan
complex. N-Glycosylation of b4-integrin may therefore represent
a potential therapeutic target for cancer.

Disclosure of Potential Conﬂicts of Interest
No potential conﬂicts of interest were disclosed.

Authors' Contributions
Conception and design: J. Gu, Y. Kariya
Development of methodology: Y. Kariya, Y. Kariya
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): Y. Hashimoto, Y. Kariya
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): Y. Kariya, J. Gu, Y. Kariya
Writing, review, and/or revision of the manuscript: Y. Kariya, J. Gu, Y. Kariya
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): Y. Kariya, M. Oyama, Y. Kariya
Study supervision: J. Gu, Y. Kariya

Acknowledgments
This work was supported by the Japan Society for Promotion of Science
KAKENHI grant number 25860243, 17K08665 (Y. Kariya), 15H04354(J. Gu), and the Fukushima Medical University Research Project from
Fukushima Medical University No. KKI28001 (Y. Kariya).
The authors are grateful to Dr. M. Peter Marinkovich (Stanford University,Standford, CA) for providing the cells. We thank the Fukushima Medical
University English Editing Service for their English language review.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received July 11, 2017; revised February 7, 2018; accepted March 12, 2018;
published ﬁrst March 16, 2018.

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Received: 24 July 2018

| Revised: 13 December 2018 | Accepted: 27 December 2018

DOI: 10.1002/mc.22971

RESEARCH ARTICLE

Core fucosylation of copper transporter 1 plays a crucial rolein cisplatin-resistance of epithelial ovarian cancer byregulating drug uptake
Xiaoxue Lv1 | Jiazhe Song1 | Kai Xue1 | Zhi Li2 | Ming Li1 | Danishi Zahid1 |Hongyu Cao3 | Lu Wang1 | Wanli Song1 | Tonghui Ma1 | Jianguo Gu4 |Wenzhe Li1
1 College of Basic Medical Sciences, Dalian

Medical University, Liaoning, China

Core fucosylation catalyzed by core fucosyltransferase (Fut8) contributes to the2 Clinical Laboratory, Dalian Municipal Central

progressions of epithelial ovarian cancer (EOC). Copper transporter 1 (CTR1), which

Hospital, Dalian, Liaoning, China
3 College of Life Science and Technology,

contains one N-glycan on Asn15, mediates cellular transport of cisplatin (cDDP), andDalian University, Liaoning, China

plays an important role in the process of cDDP-resistance in EOC. In the present study,

4 Institute of Molecular Biomembrane andwe found that the core fucosylation level elevated significantly in the sera of cDDP-

Glycobiology, Tohoku Medical andPharmaceutical University, Miyagi, Japan

treated EOC patients. The in vitro assays also indicate that core fucosylation of CTR1
was significantly upregulated in cDDP-resistant A2780CP cells compared to theCorrespondence
Wenzhe Li and Kai Xue, College of Basic
Medical Sciences, Dalian Medical University,
9-Western Section, Lvshun South Road,
Liaoning 116044, China.
Email: liwenzhe@dlmedu.edu.cn (W.L.);
nkx123xuekai@dmu.edu.cn (K.X.)
Funding information
National Nature Science Foundation of China,
Grant numbers: 31570797, 31870797;
Nutrition and Care of Maternal & Child
Research Fund Project of Guangzhou
Biostime Institute of Nutrition & Care,
Grant number: 2018BINCMCF39; Liaoning
Provincial Program for Top Discipline of Basic
Medical Sciences, China

cDDP-sensitive A2780S cells. Intriguingly, the hyper core fucosylation suppressed the
CTR1-cDDP interactions and cDDP-uptake into A2780CP cells. Conversely, contrast
to the Fut8+/+ mouse ovarian epithelial cells, the Fut8-deleted (Fut8−/−) cells obviously
showed higher cDDP-uptake. Furthermore, the recovered core fucosylation induced
the suppression of cDDP-uptake in Fut8-restored ovarian epithelial cells. In addition,
the core fucosylation could regulate the phosphorylation of cDDP-resistanceassociated molecules, such as AKT, ERK, JNK, and mTOR. Our findings suggest thatthe core fucosylation of CTR1 plays an important role in the cellular cDDP-uptake andthus provide new strategies for improving the outcome of cDDP based chemotherapyof EOC.
KEYWORDS

cDDP-resistance, core fucosylation, CTR1, EOC, Fut8

1 | INTRODUCTION

paclitaxel yields a response rate of more than 80%. Despite an
initial high response rate, EOC has a poor prognosis due to theEpithelial ovarian cancer (EOC) is the most lethal female genital
1high proportion of chemo resistance (approximately 90%),2 andtract cancer in the world. The standard therapy scheme for thethe 5-year survival rate for advanced ovarian cancer is less thaninitial management of ovary cancer is based on cytoreductive

30%. Reduced cellular accumulation of platinum-based drugs,

surgery, followed by primary chemotherapy with a platinum-

enhanced detoxification capability, aberrant apoptosis pathways,

2

based regimen. The first-line chemotherapy with platinum andand increased DNA repair ability from DNA damage areassociated with the chemo resistance,3,4 however, the precise
mechanisms of how EOC cells achieve resistance to platinum

Xiaoxue Lv and Jiazhe Song contributed equally to this work.

Molecular Carcinogenesis. 2019;1–14.

drugs have not been fully identified.
wileyonlinelibrary.com/journal/mc© 2019 Wiley Periodicals, Inc.

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The resistance to platinum drugs usually occurs during the ovarian

2 | MATERIALS AND METHO DS

tumor progressions, and the treatment for recurrent ovarian cancer isoften difficult due to resistance to chemotherapeutic agents.4,5
Aberrant glycosylation is a universal feature of cancer malignancy
and metastatic progress including EOC.1,5–12. Recent studies havereported that core fucosylation could participate in the EOC
progression.1,5,12 Core fucosylation is catalyzed by core fucosyltransferase (Fut8) through transferring a fucose residue from GDP-fucose
to the innermost N-acetylglucosamine (GlcNAc) residue of N-glycans
via α1,6-linkage (Figure 1A).13 It plays important roles in various
cellular biological events.14 In our previous studies, we found that lackof core fucosylation in transforming growth factor β1 (TGFβ1) receptor
and epidermal growth factor receptor results in a marked dysregulation of their activation that is due to a decreased ligand affinity for the
receptor.15 Moreover, core fucosylation of B cell receptor is required
for antigen recognition.16 These findings indicate that core fucosyla-

2.1 | Clinical samples
The ovarian cancer patients newly diagnosed and treated in Dalian
Municipal Central Hospital from September 2016 to January 2017
were analyzed retrospectively. Pathology records were reviewed
including morphology, immunophenotype, and cytogenetic andmolecular findings. All cases met the criteria published by the World
health Organization (WHO). Serum samples were obtained fromhealthy people (n = 20) and ovarian cancer patients (n = 21). All ourinvestigations were undertaken either for diagnostic purposes or withresidual material obtained through diagnostic procedures. All procedures had been approved by the ethical committee of Dalian Municipal
Central Hospital. Patient clinical characteristics were listed in
Supplemental Table S1.

tion is necessary for many molecular interactions on cell membrane.
Since the drug uptake of cancer cell is based on the molecularinteraction between cell surface proteins and drugs,17–20 the
relationship between core fucosylation and the membrane protein-

2.2 | Mice
Fut8−/− mice were backcrossed eight times to the BALB/cA

dependent drug uptake should be a novel point for the studies on the

background. Homozygous wild (Fut8+/+) and knockout (Fut8−/−) micedrug-resistance of EOC.

were obtained by crossing heterozygous Fut8+/− mice. Mice were

The inhibition of cisplatin (cDDP) uptake is a main reason for the

maintained in a room illuminated for 12 h (08:00-20:00) and kept at

cDDP-resistance of EOC, which usually occurs during the malignant

24 ± 1°C with free access to food and water in the specific pathogen-

The transporters on the cell

free laboratory animal facility of Dalian Medical University. All animal

membrane play a key role in the cellular accumulation of platinum-

procedures complied with the institutional animal protocol guidelines.

transformation in ovarian tumors.

3,4,21,22

based drugs, and several transporters of platinum compounds areglycoproteins, including the efflux ATPases (MRPs, ATP7A/B), and the

2.3 | Reagents

solute carrier importers (SLCs, AQP2, and AQP9).3 Copper transporter
1 (CTR1) on the cell membrane contains three membrane-spanning

Antibodies for AKT, phospho-AKT (Ser473), mTOR, phospho-mTOR

segments. CTR1 is a transporter that play a key role in regulating(Ser2448), CTR1, phospho-ERK (Thr202/Tyr204), JNK, phospho-JNK

copper homeostasis, and is also responsible for the uptake of cDDP in(Thr183/Tyr185) were purchased from Cell Signaling Technology

yeast and mammals.

3,4,23–26

Over-expression of CTR1 in humanovarian tumor cells increases their sensitivity to platinum drugs.

3,4,23–(Beverly, MA), and rabbit polyclonal anti-GAPDH antibodies werepurchased from Abcam (Cambridge, UK). Anti-cytokeratin 14 (K14)

CTR1-mediated cDDP-uptake was dependant on its metal-binding

antibody and cDDP were purchased from Sigma (St. Louis, MO). Lens

ecto-domain, which is linked with the N-glycan (Asn15) and the O-

Culinaris Agglutinin (LCA) and Aspergillus oryzae lectin (AOL) were

25

27 19

glycan (Thr ).

Blocking CTR1 ecto-domain O-linked glycosylation by

27

27

mutating Thr , increased the abundance of truncated CTR1.

purchased from Vector Laboratories, Inc. (Burlingame, CA).

In

mammalian cells the N15Q mutant protein trafficked to the plasma
membrane and copper transport was modestly reduced by the loss of

2.4 | Western blot and lectin blot analysis

glycosylation.27 However, the role of core fucosylation of CTR1 in

Cells were washed with phosphate buffer solution (PBS) and then lysedcDDP-uptake and in platinum-resistance in ovarian cancer has notwith ice-cold buffer (50 mM Tris-HCl, 150 mM of NaCl, 1% Triton-X
100, 5 mM EDTA, 10 mM NaF, 0.1 mM Na3VO4, supplemented with

been elucidated.
In order to investigate the role of core fucosylation in CTR1-

0.1 mM phenylmethylsulfonylfluoride, 1 mM DTT). Total protein

mediated cDDP-uptake and cDDP-resistance of EOC, the core

concentrations were determined by bicinchoninic acid protein assay

fucosylation level in the serum of cDDP-treated EOC patients was(BCA assay kit, Pierce, Rockford, IL). Ten microgram proteins from eachanalyzed. Furthermore, we compared the ability of cDDP-uptake via

sample were separated by 10% sodium dodecyl sulfate polyacrylamide

CTR1 between different human EOC cell lines. By applying the Fut8-

gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting for−/−) mouse ovary epithelial cell model, we provide

first antibody or the biotin-labeled LCA, which preferentially recognizes

evidence that the core fucosylation of CTR1 is involved in the cDDP-

the core fucose,28 followed by incubation with a HRP-conjugateduptake, followed by cell apoptosis or survival. Our data suggest that

secondary antibody or HRP-conjugated streptavidin for 1 h at room

down-regulation of core fucosylation could improve the cDDP efficacy

temperature. After washing, the membranes were visualized by

in EOC chemotherapy.

chemiluminescence using an ECL kit (Pierce, Rockford, IL).

deleted (Fut8LV ET AL.

| 3

FIGURE 1 High level of core fucosylation frequently occurs in cDDP-treated EOC patients. A, The biological reaction of Fut8. Fut8transfers fucose to the innermost GlcNAc residue of complex N-glycans via α 1, 6-linkage (core fucosylation) in the Golgi apparatus of
mammal cells. Asn, asparagine; Fuc, fucose; GDP-Fuc, GDP-fucopyranoside; Man, mannose. B, Comparative analysis of the core fucosylationlevels in sera of EOC patients and healthy controls. The core fucosylation levels in serum samples (5 μg) were detected by LCA lectin blot.
The represent image was shown. C, The statistical analysis of core fucosylation levels in the serum samples. Serum samples are from healthy
control (n = 20), cDDP-treated (n = 15), and untreated (n = 3) EOC patients. The significantly difference was shown as *P < 0.05 or **P < 0.01.
D, Analysis of core fucosylated molecules and CTR1 expressions in EOC patient specimens using H&E (left), AOL (middle), and anti-CTR1(right) staining. The paraffin sections were deparaffinized and hydrated through a graded series of ethanol to PBS and then incubated withbiotin-conjugated AOL antibody or anti-CTR1 antibody. Finally, the slides were visualized with 3, 3′-diaminobenzidine. The original
magnification of lower panel is x 400 (n = 3). E, The comparative analysis by Cancer Genome Atlas (TCGA) demonstrated that the expressionof Fut8 was significantly increased in EOC patients (n = 185). The significantly difference was shown as ***P < 0.001. F, The overall survival
between high Fut8 expressed and low Fut8 expressed patients were analyzed by TCGA dataset. [Color figure can be viewed atwileyonlinelibrary.com]

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LV ET AL.

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2.5 | Immunohistochemistry

2.9 | Immunoprecipitation

Formalin-fixed ovarian tissue specimens were paraffin-embedded. For

For immunoprecipitation (IP), 500 μg of cell extracts were mixed withimmunohistochemical analysis, the specimens were deparaffinized

10 μL anti-CTR1 antibody and incubated at 4°C for 1 h withtwice in xylene and hydrated through a graded series of ethanol to PBS.

continuous rotation. Then 10 μL of a 50% suspension of Protein G-

The endogenous peroxidase activity was blocked with 3% H2O2 for

Sepharose (Amersham Pharmacia Biotech AB) was added and5 min. Specimens were pretreated with avidin-biotin blocking andincubated at 4°C overnight with gentle agitation. The beads werehydroxygen blocking for 10 min at 37°C, and then incubated with

washed four times with lysis buffer. The immunoprecipitated samples

primary antibody (1:50 dilution) for 16 h at 4°C. Then the specimens

were eluted from the protein G-Sepharose by heating at 100°C for

were incubated with appropriate peroxidase-conjugated secondary

5 min in 2 x loading buffer without 2-mercaptoethanol. After

antibody for 1 h at 37°C. The slides were visualized with 3, 3′-centrifugation, the supernatant were resolved by SDS-PAGE anddiaminobenzidine. Stained slides were mounted in anti-fade medium

Western blotted.

(PermaFluor Mamtant Medium; Immunon,Pittsburgh, PA). Finally, theslides were examined under a fluorescence microscope (Leica, UK) at
an x200 magnification using the appreciate filter. The specimens wereanalyzed by hematoxylin-eosin (H&E) staining.

2.10 | Remove of fucosylation on the CTR1
The CTR1 from A2780CP and A2780S cells was purified by IP assay,
and then purified CTR1 were treated with 100 mU Glyko® α (1-2, 3, 4,
6) Fucosidase (GKX-5006, Prozyme) for 16 h at 37°C to remove the

2.6 | Cell culture

core fucosylation.

Two EOC cell lines, cisplatin-sensitive parental cell line (A2780S) andits cisplatin-resistant variant (A2780CP) were kindly provided in 2015

2.11 | Molecular interactions

by Chun Peng (York University, Toronto, Canada) and Dr Shiying Cui(Dalian Medical University), and were cultured in Dulbecco's modified

For molecular interaction analysis, the purified CTR1 proteins were

Eagle's medium (DMEM) containing 10% fetal bovine serum andmixed with different concentrations of cDDP, and detected bymaintained at 37°C with 5% CO2 as previously described.2,29 Cell line

fluorescence measurement. The fluorescence measurements wereauthentication and pathogen testing was performed upon receipt of

performed on an FP-6500 fluorescence spectrophotometer (Jasco,

cell lines. All experiments were performed on cells cultured for lessJapan) equipped with a 150 W xenon lamp source. The fluorescencethan 2 months. The morphology of A2780S and A2780CP cells wasemission spectra were recorded with 1.0 cm quartz cell in theshown in Supplemental Figure S1.

wavelength of 290-400 nm upon excitation at 280 nm at a scanningspeed of 500 nm per min. The bandwidths for both excitation andemission were 3 and 5 nm, respectively. All synchronous fluorescence

2.7 | Quantitative gene expression analysis by realtime PCR and RT-PCR
Total RNAs were extracted from ovarian cancer cell lines with the
TRIzol™ reagent (Invitrogen, Carlsbad, CA). First-strand cDNA wassynthesized using SuperScript II reverse transcriptase (Invitrogen)spectra were measured according to Δλ = 20 nm and Δλ = 60 nm, while
the width of the excitation and emission slits were adjusted at 3 and
5 nm, respectively. The scanning speed of the synchronous fluorescence was 500 nm/min and the response time was 0.5 s with a medium
sensitivity.

and the oligo (dT) 18 primer. Samples were analyzed in a triplicate
20 μL reaction (20 μmol/L of primers, 10 μL of Master Mix), which

2.12 | Measurement of intracellular platinum content

was adapted from the standard protocol provided by SYBR GreenPCR Master Mix. The default PCR procedure was used on an

Platinum contents were then measured by inductively coupled plasma-

Applied Biosystems Prism 7000 Sequence Detection System

mass spectrometry (ICP-MS, Optima 2100dv) in Dalian Research(Applied Biosystems, Tokyo, Japan). The primers used for PCR

Institute of Product Quality Testing as previously described.17,24 Cells

were listed in Supplemental Table S2. Using the 2-ddCt method,were plated in dish at a density of 5 × 106 cells/dish followed byrelative internal mRNA expression of target genes was normalized

incubation with different concentration of cDDP for 4 h. Then cells

to GAPDH.

were collected and digested in 1 mL of concentrated nitric acid andconcentrated hydrochloric acid (1:3).

2.8 | Flow cytometry analysis
Cells were collected and blocked in 3% BSA for 10 min. Primary

2.13 | Cell proliferation assay

antibodies were incubated on ice for 30 min, and then stained withFor MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro-

FITC-labeled secondary antibodies. Flow cytometry was performed onmide] assay, cells were cultured in 96-well plates, and added cDDP at

a FACS-Calibur (Becton Dickinson, Mountain View, CA), and the data

the indicated concentrations for 0, 24, 48, 72, 96 h. Ten microliter of

was analyzed with Flowjo software (Treestar, San Carlos, CA).

5 mg/mL of MTT was added in the each well. After incubation for 4 h,

| 5

LV ET AL.

DMSO (Sigma) was added to each well to dissolve the precipitate. The

primary cells. Transfectants were screened in the presence of 400 μg/

plate was read at 490 nm on a Benchmark microplate reader (BioRad,mL zeocine, and Fut8+/+, Fut8+/− and Fut8−/− mouse ovarian epithelial

Hercules, CA). The percentage growth inhibition was calculated using

cells were established. The primers for Fut8+/+, 5′-TTC CAG AGA GAT

following(%) = (Ac-At)/(Ac-TAG CCT GTC T-3′ and 5′-TCT CGA ACC AAA TGA CCA CCT A-3′,

Ab) × 100%. At = Absorbance value of cDDP-treated; Ab = Absor-

and for FUT8−/−, 5′-TTG CCG TCT TTT GGC AAT GTG A-3′ and 5′-

bance value of blank; Ac = Absorbance value of control.

CCA GAT CAG ATC CCA TAC AAT G −3′, were used for PCR to detect

formula,cellsurvival

inhibition

mouse genotyping.

2.14 | CCK8 cell proliferation assay

To prepare Fut8-reintroduced cells, pLHCXsi-U6-Fut8 mutant
expression vectors were prepared. The pLHCXsi-U6-Fut8 mutant

Cell IC50 were measured by using CCK8 assay (APExBIO). Briefly, thevector was transduced into Fut8−/− mouse ovary epithelial cells by

A2780 and A2780-cp cells were plated in 96-well plates (5000 cells/

Lipofectamine, and Fut8-restored (Re) mouse ovary epithelial cells

well). After pre-culture for 12 h, the cells were treated with various

were generated after selection with 200 μg/mL hygromycin.

concentrations of cisplatin (0, 10, 20, 30, 40, and 50 μg/mL) for 24 h,and 10 μL of CCK-8 solution (APExBIO) was subsequently added toeach well of the plate. The bubbles were prohibited into the cells. Then

2.18 | Immunofluorescence assay

the plate was incubated at 37°C for 3 h. The optical density (OD) value

Cells were seeded onto 6-well chamber microscope slides and allowed

was measured at 450 nm using a microplate reader (BioTek Instru-

to adhere overnight. Then cells were washed and fixed in methanol:

ments, VT).

acetone (1:1) for 30 min followed by block out in rabbit serum for
30 min. Primary antibody was incubated for 1 h at room temperature,

2.15 | Cell cycle and apoptosis analysis

then the secondary antibody was covered for 1 h and followed by
DAPI staining for 5 min. Cell immunostaining was imaged on a

For cell cycle analysis, cells were incubated with and without cDDP for

fluorescence microscopy (Leica, Cambridge). The images were

48 h. After extensive washing, the cells were fixed with 70% ethanol

projected by using the Nikon Ez-C1 and Adobe Photoshop 8.0

overnight at 4°C and incubated with propidium iodide (PI) (Sigma-

software.

Aldrich) and RNaseA (Sigma-Aldrich) for 1 h at room temperature, andanalyzed using a FACS calibur instrument. The cell-cycle phase
distribution was analyzed with the use of the CellQuest software.

2.19 | Statistical analysis

For cDDP-induced cell apoptosis analysis, cells were harvested

Each experiment was performed at least three times and the mean wasand fixed. Then, annexin V-APC/PI-FITC double staining wereused to calculate significance. Data was expressed as mean

performed according to the manufacturer's instructions.

values ± standard error mean (SEM). Data analysis was performed
with the statistical software package Graph Pad Prism 5. The statistical

2.16 | Phosphorylation assay

significance was assessed by one-way analysis of variance (ANOVA).
The level of differences among groups was analyzed by Student's test.

Serum-starved cells were treated with indicated concentration ofDifferences were considered to be statistically significant at *P < 0.05,

cDDP for 0, 5, 15, and 30 min. Stimulations were terminated by**P < 0.01, and ***P < 0.001.

washing the cells once with ice-cold PBS supplemented with 0.4 mM
sodium orthovanadate and then harvested by scraping. Cells werecollected and solubilized for 30 min at 4°C in lysis buffer. After lysis,
the lysate was centrifuged at 10 000g for 15 min at 4°C to precipitate
insoluble materials, and the supernatant were subjected toimmunoblotting.

3 | RESULTS
3.1 | Core fucosylation of CTR1 is associated withthe cDDP-resistance of EOC
During the therapy process of EOC, about 90% of patients exhibited

2.17 | Establishment of FUT8+/+, FUT8+/−, FUT8−/−,and Fut8-restored (Re) ovary epithelial cells

chemo resistance.2,31 To investigate the role of core fucosylation in the

Ovary epithelial cells were obtained from homozygous wild typecDDP-based primary therapy, and the other three were not received(FUT8+/+), heterozygous (FUT8+/−) and Fut-8 gene-knockout (FUT8−/

any chemotherapy (Supplementary Table S1). LCA staining demon-−cDDP-resistance of EOC, we evaluated the sera of 21 patients, who
had received primary surgery. Among those, 18 EOC patients received) mice.16,30 In detail, ovarian tissues were removed from the micestrated that the core fucosylation levels were dramatically up-

following by incubation in hyaluronidase (100 U) and collagenases

regulated in cDDP-treated EOC patients, compared to the healthy(300 U) for 30 min. After centrifugation at 1000g, the cells wascontrols and none cDDP-treated EOC patients (Figures 1B and 1C),

collected and cultured in DMEM supplemented with 10% FCS andindicating that high level of core fucosylation frequently occurs inincubated at 37°C. To obtain immortal cells, zeocine-resistant vector

cDDP-treated EOC patients. Moreover, the ovarian specimens of(pcDNA3.1) containing the SV40 gene was introduced to thesecDDP-treated EOC patients were analyzed by H&E staining and6

LV ET AL.

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immunohistochemical assay using AOL, which is another lectin specific(28.3 ± 0.06 μg/mL) was significantly higher than that of the A2780S

for recognizing core fucosylation.32 As shown in Figure 1D, corecells (20.5 ± 0.08 μg/mL). These results indicated that the hyper corefucosylation was markedly increased in ovarian carcinoma tissues

fucosylation of CTR1 in A2780CP cells is important for their survival

contrast to the adjacent normal tissues. Here we also investigate CTR1

against cDDP-treatment. Considering the colocalization of CTR1

expressions of the specimens of ovary tissue from cDDP-treated EOCwith hyper core fucosylation in the ovarian carcinoma tissues ofpatients and found that CTR1 was mainly concentrated on the cell

cDDP-treated EOC patients (Figure 1D) and the high proportion of

membrane of ovarian carcinoma (Figure 1D). Moreover, the Cancer

cDDP-resistance in EOC patients,2,31 core fucosylation of CTR1 is

Genome Atlas (TCGA) Bonome Ovarian Statistics demonstrates thatassociated with the cDDP-resistance of EOC.

the expression of Fut8 was significantly increased in EOC patients(n = 185, ***P < 0.001) (Figure 1E), although there are no significant
difference of the overall survival between high Fut8 expressed and low
Fut8 expressed patients (Figure 1F, data from Oncomine). The

3.2 | Core fucosylation of CTR1 plays a crucial role inthe cDDP-uptake of EOC cells

colocalization of CTR1 with the core fucosylation in the cDDP-treated

CTR1 on cell membrane is the most important cDDP-transporting

ovarian carcinoma tissues suggests that core fucosylation of CTR1 mayprotein with the N-linked glycosylation on its metal-binding ecto-

be associated with the cDDP-resistance of EOC.

domain.3,4,19,23–25,33 The colocalization of CTR1 with core fucosyla-

Next, we compared the core fucosylation levels of human EOC cell
2,29tion of N-glycans on the cell membrane of ovarian carcinoma implied

The LCAthat the function of CTR1 may be correlated with core fucosylation inblot indicated that core fucosylation levels of A2780CP cells are

cDDP-treated EOC (Figure 1D). Given that the CTR1 plays a crucial

significantly higher than those of A2780S cells (Figure 2A). The Fut8role in regulating the influx of platinum drugs into the cells,3,4,23–26 wemRNA and protein expressions are dramatically upregulated insuspected that the core fucosylation is involved in the CTR1-mediatedA2780CP cells compared with A2780S cells (Figure 2B). No apparent

cDDP-uptake of cells. To determine the influence of core fucosylation

changes were found in the expressions of other glycosyltransferaseon the interaction between CTR1 and cDDP, the CTR1 from A2780CP

genes, such as N-acetylglucosaminyltransferase III (GnT III), GnT IV,

and A2780S cells was purified (Figure 3A) and treated with 100 mU

GnT V, and α2,6-sialyltransferase I (ST6Gal1). These findings were

Fucosidase, which can cleave α1,6 fucose linkages more efficiently

line A2780S and its cDDP-resistance variants A2780CP.

suggesting that the up-

than other α-fucose linkages.32 As shown in Figure 3B, the core fucose

regulation of core fucosylation is positively correlated to cDDP-

was completely removed on the CTR1 by fucosidase treatment fromresistant in EOC cells. Then we detected CTR1 expressions in theA2780CP (A2780CP + E) and A2780S (A2780S + E). Surprisingly, loss

A2780S and A2780CP cells (Figures 2C and 2D). No significant

of core fucosylation on the CTR1 exhibited no binding activity to cDDP

difference of the CTR1 expression on the cell membrane was found(Figure 3C), suggesting that the fucosylation of CTR1 is essential to thebetween A2780S and A2780CP cells (Figure 2C). However, the coreinteraction between CTR1 and cDDP. Indeed, the CTR1 of A2780S

fucosylation level of CTR1 in A2780CP cells is dramatically higher thancells exhibited stronger binding activity to cDDP than those ofthat in A2780S cells (Figure 2D). These findings also indicated that core

A2780CP cells with a dose-dependent manner (Figure 3C). The signals

fucosylation of CTR1 were associated with the cDDP-resistance ofof CTR1-cDDP complex reached a threshold at 100 nM of cDDP,

EOC cells.

evidenced by no change of the signals at 140 nM (Figure 3C). Since theconsistent with other previous report,

15

To further explore the relationship between core fucosylation ofcore fucosylation of A2780CP cells are significantly higher than those

CTR1 and the cDDP-resistance of EOC cells, the cDDP-treatment

of A2780S cells, it is reasonable to consider that the hyper core

assays were performed. The cell survival inhibition assay showedfucosylation of CTR1 suppressed the CTR1-cDDP interaction inthat the A2780CP cells were more cDDP-resistant than that of

A2780CP cells.

A2780S cells during treatment with 10, 20, and 35 μM cDDP

ICP-MS analysis is usually applied for detecting the cellular(Figure 2E). To further determine whether cDDP influences cell

accumulation of platinum.23,33 To further clarify the association of coresurvival via cell-cycle arrest, the cell-cycle was analyzed by flow

fucosylation with the CTR1-mediated cDDP-uptake, we measured thecytometry. In the cell-cycle profiles the cDDP-treatment in A2780S

cellular accumulation of platinum in the cells by ICP-MS analysis. The

cells induced higher proportion of G2/M arrest than those incellular accumulation of platinum in A2780S cells was higher thanA2780CP cells, albeit the cDDP-treatment increased the G2/M

those in A2780CP cells (Figure 3D). These results suggested that thearrest in both cell lines (Figures 2F and S2). We next analyzed thehyper core fucosylation of CTR1 could suppress the CTR1-cDDP

effect of cDDP on cell apoptosis using annexin V and PI staining. Asinteractions and the following cDDP-uptake of EOC cells.

shown in Figure 2G, after cDDP-treatment, the percentages of early
apoptotic cells (PI− annexin V+ cells) in A2780S and A2780CP cells
were 2.30% and 1.10%, respectively; the percentages of late
apoptotic cells (PI+ annexin V+ cells) were 7.53% and 2.14%,
respectively. Obviously, the rates of cell apoptosis in the A2780CP

3.3 | Lack of core fucosylation weakens thecDDP-resistance of mouse ovarian epithelial
cells by promoting cellular cDDP-uptake

cells were significantly lower than those in A2780S cells.

Since Fut8 is the sole glycosyltransferase for core fucosylation, theMoreover, in the CCK8 assay the IC50 of the A2780CP cells

Fut8−/− mice, which were generated in our laboratory, were applied

LV ET AL.

| 7

FIGURE 2 Core fucosylation are involved in the cDDP-resistance and survival of EOC cells. A, LCA staining of A2780S and A2780CP cells.
The core fucosylation levels of A2780S and A2780CP cell lysates were detected using LCA staining. Coomassie brilliant blue (CBB) staining ofgels showed the comparable amounts of protein. B, Fut8 expressions of A2780S and A2780CP cell lines were analyzed by real time PCR andWestern blot. The mRNA and protein levels of different genes were normalized with GAPDH expressions. The significantly difference wasshown as *P < 0.05. C, Expressions of CTR1 on the surface of A2780S and A2780CP cells. Cells were stained with anti-CTR1 Ab for 30 min
on ice, and the expression were analyzed by flow cytometry assay. D, Comparison of core fucosylation levels of CTR1 in A2780S and
A2780CP cells. Whole lysates were immunoprecipitated by anti-CTR1 Ab, and followed by subjected to CTR1 Western blot and LCA blot
analysis. E, The survival inhibition assay. A2780S and A2780CP cells were treated with 10, 20, and 35 μM of cDDP for 24, 48, 72, and 96 h,
and cell proliferation was analyzed by MTT assay. OD values were measured at 490 nm using microplate reader. Cell survival inhibition(%) = (Ac-At)/(Ac-Ab) x 100%. Ab, absorbance value of blank; Ac, absorbance value of control; At, absorbance value of cDDP-treated. The
significantly difference was shown as *P < 0.05 or **P < 0.01 or ***P < 0.001. F, Cell cycle analysis of A2780S and A2780CP cells. Cells weretreated with 20 μM cDDP for 48 h, the untreated cells were served as control. The PI staining was performed to measure the DNA content ofcells; then the cell cycles were measured on a FACS Calibur cytometer. The percentages of cells were shown in different cell cycle phases(G1, S, or G2/M). G, The flow cytometry analysis was performed to measure the apoptosis of A2780S and A2780CP cells after PI andannexin V staining. Cells were exposed to 20 μM cDDP for 48 h. [Color figure can be viewed at wileyonlinelibrary.com]

8

LV ET AL.

|

FIGURE 3 Core fucosylation regulates the CTR1-mediated cDDP uptake. A, Purification of CTR1. The proteins from A2780S and
A2780CP cells were isolated, and CTR1 was further purified by the immunoprecipitation. B, Remove of fucose on the CTR1. The purified
CTR1 was treated with 100mU Fucosidase. C, Molecular interactions between CTR1 proteins and cDDP. The cDDP solution (0, 50, 100, and
140 nM, was titrated into a fixed concentration of CTR1. Significant increase was observed in the fluorescence intensity, when the cDDP wasinteracted with CTR1. D, Cellular Pt accumulation after exposure to cDDP. A2780S and A2780CP were incubated with 50 μM cDDP for 4 h.
Cellular Pt accumulation was then measured by ICP-MS. The significantly difference was shown as *P < 0.05. [Color figure can be viewed atwileyonlinelibrary.com]

here to establish a Fut8-deleted mouse ovarian epithelial cellrole of core fucosylation in the cDDP-uptake of cells, we

model.16,30 Immunohistochemistry assays with LCA show that coretransduced Fut8 cDNA to Fut8−/− mouse ovarian epithelial cells

fucosylation was abolished in Fut8−/− mouse ovary (Figure 4A). The

and established Fut8 restored cells (Re cells) (Figure 5D). As shown

purified ovarian epithelial cells were transfected with SV40 cDNA

in Figure 5E, compared to Fut8+/+ cells, cDDP-uptake wasfollowed by screening with G418. Then the genotypes of immortalized

significantly increased in Fut8−/− cells, and that was restored in

−/−

+/+

cells were proved by PCR assay

the Re cells. These results suggested that the lack of core

(Figure 4B) and the epithelial type was identified by immunostaining

fucosylation increased the CTR1-mediated cDDP-uptake ofof cytokeratin 14 (K14) in these three cell lines (Figure 4C).

ovarian epithelial cells.

Fut8, Fut8+/−, and Fut8

Furthermore, the CTR1 expressions in these three cell lines were

To further confirm the role of core fucosylation in cell survival

examined by flow cytometry and western blot analysis. No significant

after cDDP-treatment, we examined the proliferation of Fut8−/−,difference of CTR1 expressions was found in Fut8−/−, Fut8+/−, and

Fut8+/−, and Fut8+/+ mouse ovarian epithelial cells by MTT assay.

+/+

Fut8

mouse ovarian epithelial cells (Figures 5A and 5B), however,−/−Compared to Fut8+/+ cells, the survival rate of Fut8−/− cells wasmouse ovarian epithelial cells was

dramatically reduced upon 10 and 20 μM cDDP-treatment (Figure 5F).

abolished, and the core fucosylation level of Fut8+/− cells was about

Moreover, the flow cytometry analysis shows that Fut8−/− cells

the core fucosylation of Fut8

half of Fut8+/+ cells (Figure 5B).

exhibited obviously increased apoptosis rate under 20 μM cDDP-

The cellular accumulation of platinum in Fut8−/− cells weretreatments (Figure 5G). Taken together, lack of core fucosylation

notably higher than that in Fut8+/− and Fut8+/+ cells after

weakens the cDDP-resistance of ovarian epithelial cells by promoting

incubation with 30 μM of cDDP (Figure 5C). To determine thethe cellular cDDP-uptake.

LV ET AL.

| 9

FIGURE 4 Establishment of the Fut8+/+, Fut8+/−, and Fut8−/− ovary epithelial cells. A, Core fucosylation of Fut8+/+ and Fut8−/− ovarianspecimens by immunohistochemical analysis with LCA blot. Red arrows indicate antral follicles. Black arrows indicate primary follicles. B, PCR
analysis of Fut8 gene in the Fut8+/+, Fut8+/−, and Fut8−/− cells. DNA were extracted from the Fut8+/+, Fut8+/−, and Fut8−/− cells and were
subjected to PCR assay. C, Identification of Fut8+/+, Fut8+/−, and Fut8−/− ovarian epithelial cells. Cells were immunostained by antibody specificfor epithelial marker (K14, red color, and DAPI, blue color). The magnification is 400-fold. [Color figure can be viewed at wileyonlinelibrary.com]

3.4 | Core fucosylation is involved in thecDDP-induced signal transduction in EOC cells

apoptosis, metabolism, and autophagy.3,34–37 To verify the impacts
of core fucosylation on cell signaling, the A2780S and A2780CP cells
were treated with 20 μM of cDDP for 0, 5, 10, 15 min. Compared to

Several previous studies have proved that the uptake of cDDP initiates

low-core fucosylated A2780S cells, the phosphorylation levels of Akt,different signaling events, which modulating cell proliferation,

mTOR, ERK, and JNK was significantly enhanced in high-core10|

LV ET AL.

FIGURE 5 Loss of core fucosylation promotes CTR1-mediated cDDP uptake. A, Core fucosylation did not influence the CTR1 expressions
on the surface of the Fut8+/+, Fut8+/−, and Fut8−/− mouse ovarian epithelial cells. The CTR1 expressions on the cellular surface were analyzed
by flow cytometry analysis. B, Immunoblot analysis. The CTR1 expressions were measured by Western blot. The core fucosylation of Fut8+/+,Fut8+/−, and Fut8−/− cells were detected by LCA blot. GAPDH expressions were served as a control. C, Cellular Pt accumulation after
exposure to cDDP. Fut8+/+, Fut8+/−, and Fut8−/− mouse ovary epithelial cells were incubated with 30 μM cisplatin for 4 h. Platinum contents
were then measured by ICP-MS. Data was expressed as mean ± SEM (n = 4). The significantly difference was shown as *P < 0.05. D,
Establishment of Re cells. The Re cells were generated by introduction of Fut8 cDNA to Fut8−/− cells. The core fucosylation of Fut8+/+, Fut8−/−, and Re cells were detected by LCA blot. E, Cellular Pt accumulation of Fut8+/+, Fut8−/−, and Re cells by ICP-MS. Data was expressed asmean ± SEM (n = 4). The significantly difference was shown as *P < 0.05. F, MTT assay for cells viability. Fut8+/+ and Fut8−/− cells wereincubated with 10 and 20 μM of cDDP for 24 and 48 h. The significantly difference was shown as *P < 0.05 or **P < 0.01. G Flow cytometry
analysis for cell apoptosis. After exposure to 20 μM cDDP for 48 h, Fut8+/+ and Fut8−/− cell apoptosis was detected by double staining with PI
and annexin V. [Color figure can be viewed at wileyonlinelibrary.com]

| 11

LV ET AL.

fucosylated A2780CP cells at 5, 15, and 30 min (Figures 6A and 6B). In

necessary for the molecular interactions such as receptor-ligand

addition, cDDP-induced cell signaling was also detected in Fut8−/−,interaction on cell membrane,15,16 it is conceivable that core

+/−

Fut8

+/+

, and Fut8−/−mouse ovary epithelial cells. Compared withfucosylation is likely to be important in all three proposed stages

ovary epithelial cells the enhanced activation of JNK and ERK

involved in the cDDP-uptakes via CTR1. First, core fucosylation issignaling was found in Fut8+/+ cells under cDDP-treatment

essential for the conformation of metal-binding ecto-domain of CTR1.

(Figures 6C and 6D). These results suggested that the core fucosylation

Second, core fucosylation of CTR1 could regulate the recognition ofis associated with the cDDP-induced signaling of EOC cells.

cDDP and affect the interaction between cDDP and CTR1. Third, core

Fut8

fucosylation is involved in the cDDP-uptake via CTR1 and the followed
cell signaling.

4 | DISCUSSION

CTR1 undergoes constitutive endocytosis and recycling to theplasma membrane. The removal of Asn15-linked glycan by mutation

Altered glycosylations contribute to the pathophysiological events(N15Q) does not affect trafficking of the transporter to the cell

during EOC progressions.1,5–12 In ovarian cancer, the mRNA expres-surface.27 Several studies on EOC cells reported that the expression

sion and activity of the glycosyltransferase GnT-III, the enzyme

levels of CTR1 are not correlated to the amount of cDDP

responsible for the addition of bisecting N-linked glycans, wereaccumulation.17,3939 Conversely, it has been reported that theincreased. The forced expression of ST6Gal-I, which catalyzes theelevation of CTR1 expression in the tumor cells increased theiraddition of sialic acid to the termini of N-linked glycans, confers

sensitivity to platinum drugs and the survival of cancer patients.4,19,40

resistance to cisplatin, whereas ST6Gal-I knockdown conversely

We found that there is no significant difference in CTR1 expressions ofsensitizes cells to cisplatin.11 The elevated fucosylation was also

A2780S and A2780CP cells (Figure 2C), the CTR1 expression is notdetected by AAL staining in ovarian cancer.6 Taniguchi and co-workers

changed in A2780S and A2780CP cells after cDDP-treatment. It also

discovered that the mRNA levels and activity of FUT8 were increased

reported that cDDP-treatment did not even change membrane CTR1

in serous adenocarcinoma ovarian tissues compared with normal

levels.17,39 However, cDDP induced down-regulation of human CTR1

38

These studies suggested the involvement of glycans

expression in the CTR1−/−/WT, CTR1−/−/M2, and CTR1−/−/Truncatedin the ovarian cancer processes, but there are only a few reports

cells.19,41 The discrepancy from our results may be caused byavailable regarding the involvement of core fucosylation in resistancedifferences in the experimental procedure and the cell models.

to platinum drugs in ovarian cancer. In the present study, we show that

Although the changes of core fucosylation have no influence on thethe level of core fucosylation was significantly increased in sera andexpression of CTR1 in Fut8+/+, Fut8+/−, and Fut8−/− ovarian epithelial

cDDP-treated ovarian carcinoma tissues of cDDP-treated EOC

cells, loss of core fucosylation would give rise to the increased CTR1-

patients. The elevated core fucosylation attributed to the cDDP-

cDDP interaction, thereby promotes cDDP-uptake, indicated that coreresistance of EOC by suppressing the binding of CTR1 with cDDP and

fucosylation could sterically hinder cDDP-uptake within the ecto-

the following cDDP-uptake, of which also affects cell signal transduc-domain. Therefore, it is reasonable to conceive that cDDP-uptake istion, survivals, and apoptosis (Figure 6E).

mainly regulated by the core fucosylation of CTR1 rather than theovarian tissue.

CTR1 transports cDDP via direct binding it by the Met-rich motifs

cDDP expression.

within the ecto-domain.18 Thus the Met-rich motifs within the CTR1

The cDDP could initiate activation of different signaling

ecto-domain are especially critical for cDDP acquisition of cells.18,19

pathways, such as ERK, MAPK, JNK, and AKT.3,34–37 which affectContrast to the CTR1−/−/WT cells, the Pt content of CTR1−/−/M2cell survival and apoptosis. Here we found that the different core

(40MXXXM45 was deleted) and CTR1−/−/Truncated (the first N-terminal

fucosylations of CTR1 and distinct cDDP-uptakes existed between

45 amino acids were deleted) variants were increased by 2.5- and 2.3-

A2780S and A2780CP cells, implying that core fucosylation may befold, respectively.18 CTR1 contains one N-glycosylation site Asn15 on

associated with cDDP-induced cell signal transductions by regulating

its metal-binding ecto-domain in mammalian cells. Fut8-catalyzed core

CTR1-mediated cDDP-uptake. On the cell survival, cDDP caused thefucosylation is one of the most prevalent glycosylations in mammalian

activation of ERK and JNK pathways that contribute to the increased

N-glycans. Core fucosylation contributes to the folding, stability, andproliferative rate of tumor cells.3,35,42 In our present study, thebiological function of the molecules.

15,30

Recently, Zhao et al5signaling in cDDP-resistant A2780CP cells was notably stronger thandetected a high level of core fucosylation in A2780CP cells by lectin

in cDDP-sensitive A2780S cells. In addition, compared with Fut8−/−array analysis. In agreement with this observation, we found that thecells, the activation of JNK and ERK signaling were increased incore fucosylation levels in A2780CP cells are significantly higher than

Fut8+/+ cells under cDDP-treatment. On the apoptosis, AKT and its

those of A2780S cells (Figure 2A) and the hyper core fucosylation

downstream molecular, mTOR, are correlated to the process of

suppressed the CTR1-cDDP interactions and cDDP-uptake in

cDDP-resistance of ovarian cancer cells.3,34,36,37 In agreement with

A2780CP cells (Figures 3C and 3D). Moreover, the capability ofthe previous study, the anti-apoptosis AKT-mTOR signaling was

cDDP-uptake was obviously increased in the Fut8−/− cells contrast to

stronger in A2780CP cells than in A2780S cells, and the cell

the Fut8+/+ cells, and suppressed in Re cells by re-introduction of Fut8

apoptosis in the A2780CP cells were significantly lower than thosegene. Since the cDDP-uptake of cancer cells is based on the molecularin A2780S cells, suggesting that the low apoptosis rate is correlated

interaction between CTR1 and cDDP,4 and core fucosylation isto the signal transductions via AKT-mTOR signaling. Beside the12|

LV ET AL.

FIGURE 6 Core fucosylations regulate the cDDP-induced cell signal transduction. A, Phosphorylation assays. Phosphorylation assays for
AKT, mTOR, JNK, and ERK. A2780S and A2780CP cells were serum starved for 24 h. The cells were harvested and treated with cDDP
(20 μM, for 0, 5, 15, and 30 min. Cell lysates were subjected to Western blot using anti-AKT, anti-pAKT, anti-mTOR, anti-pmTOR, anti-JNK,
anti-pJNK, anti-ERK, and anti-pERK Abs. Data are representative of three independent experiment. B, The density of phosphorylated protein
bands of A2780S and A2780CP cells was normalized to the corresponding density of total protein bands and the signal-activation are shown
as Relative density. C, Phosphorylation assays. Fut8+/+ and Fut8−/− mouse ovary epithelial cells were serum starved overnight. Then theharvested cells were incubated with cDDP (20 μM, for 0, 5, and 15 min, and subjected to Western blot using anti-JNK, anti-pJNK, anti-ERK,
and anti-pERK Abs. Data are representative of three independent experiment. D, The density of phosphor-protein bands of Fut8+/+ andFut8−/− mouse ovary epithelial cells was normalized to the corresponding density of total protein bands and the signal-activation are shown as
relative density. E, Core fucosylation of CTR1 regulates the cDDP uptake and followed cell signaling and survival. [Color figure can be viewed
at wileyonlinelibrary.com]

| 13

LV ET AL.

cDDP uptake of EOC cells, CTR1 also regulate the cellular
pharmacology and sensitivity to platinum drugs.3,4,19,23–25,33 Similar
to Cu+, platinum binds to Met-rich motifs within the CTR1 ectodomain,19,43 and CTR1 delivers platinum into cells via receptor
mediated endocytosis rather than acting as a membrane channel as it
does for Cu+.19,20 In this study, the cDDP-induced cellular signaling
occurs by a short time (5 min), suggesting that the activation of signal
pathway may depend on the receptor-mediated approaches. The
precise biochemical mechanisms of how core fucosylation affects
cDDP-induced cell signaling with or without CTR1 pathway remains
to be elucidated in future study.
To our knowledge, this is the first attempt to show the alteration ofcore fucosylation in cDDP-resistance of EOC. The most important
finding of the present study was the identification of the regulationrole of core fucosylation in cDDP-uptake, which in turn, regulates
several parameters of cDDP related events, such as cell survival, signaltransduction, and cell apoptosis. Because the cDDP-uptake is a criticalprocess during cDDP-resistance and the chemotherapy for the
EOC,3,4,17,18 our results may have far-reaching implications that canenhance cDDP efficacy in resistant tumors or decrease the therapeutic
dose needed for clinical efficacy by de-core fucosylation. An advanced
understanding of the underlying mechanism of core fucosylation
would provide new strategies for the treatment and diagnosis formalignant ovarian tumors from aspects of glycobiology.

ACKNOWLEDGMENTS
We gratefully acknowledge Chun Peng (York University, Toronto,
Canada) and Dr. Shiying Cui (Dalian Medical University) for theA2780S and A2780CP cell lines and Silethukuthula Natalie Ncube(Dalian Medical University) for critical reading of the manuscript. This
work was supported by the National Nature Science Foundation ofChina (31570797, 31101717 and 31870797), and the Nutrition andCare of Maternal & Child Research Fund Project of Guangzhou
Biostime Institute of Nutrition & Care (2018BINCMCF39), China. This
work was also supported by Liaoning Provincial Program for Top
Discipline of Basic Medical Sciences, China.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest with thecontents of this article.

ORCID
Wenzhe Li

http://orcid.org/0000-0001-7725-5325

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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.

How to cite this article: Lv X, Song J, Xue K, et al. Core
fucosylation of copper transporter 1 plays a crucial role incisplatin-resistance of epithelial ovarian cancer by regulating
drug uptake. Molecular Carcinogenesis. 2019;1–14.
https://doi.org/10.1002/mc.22971

molecules
ArticleSialic Acid-Binding Lectin from Bullfrog Eggs
Exhibits an Anti-Tumor Effect Against Breast Cancer
Cells Including Triple-Negative Phenotype Cells
Takeo Tatsuta 1 , Shoko Sato 2 , Toshiyuki Sato 3 , Shigeki Sugawara 1 , Tsuneyoshi Suzuki 2 ,Akiyoshi Hara 3 and Masahiro Hosono 1, *
1

2
3

*

Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical
and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan; t-takeo@tohoku-mpu.ac.jp (T.T.);
ssuga@tohoku-mpu.ac.jp (S.S.)
Department of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Sendai,
Miyagi 981-8558, Japan; shoko-s@tohoku-mpu.ac.jp (S.S.); tusuzuki@tohoku-mpu.ac.jp (T.S.)
Department of Clinical Pharmacotherapeutics, Tohoku Medical and Pharmaceutical University, Sendai,
Miyagi 981-8558, Japan; hachikuma81@gmail.com (T.S.); hara-a@tohoku-mpu.ac.jp (A.H.)
Correspondence: mhosono@tohoku-mpu.ac.jp; Tel.: +81-022-727-0114

Academic Editors: Tomohisa Ogawa and Hiroaki Tateno
Received: 18 September 2018; Accepted: 19 October 2018; Published: 21 October 2018


Abstract: Sialic acid-binding lectin from Rana catesbeiana eggs (cSBL) is a multifunctional protein thathas lectin and ribonuclease activity. In this study, the anti-tumor activities of cSBL were assessedusing a panel of breast cancer cell lines. cSBL suppressed the cell growth of all cancer cell lines tested
here at a concentration that is less toxic, or not toxic at all, to normal cells. The growth suppressive
effect was attributed to the cancer-selective induction of apoptosis. We assessed the expressions ofseveral key molecules associated with the breast cancer phenotype after cSBL treatment by western
blotting. cSBL decreased the expression level of estrogen receptor (ER) α, while it increased thephosphorylation level of p38 mitogen-activated protein kinase (MAPK). cSBL also suppressed theexpression of the progesterone receptor (PgR) and human epidermal growth factor receptor type 2(HER2). Furthermore, it was revealed that cSBL decreases the expression of the epidermal growth
factor receptor (EGFR/HER1) in triple-negative breast cancer cells. These results indicate that cSBL
induces apoptosis with decreasing ErbB family proteins and may have great potential for breast
cancer chemotherapy, particularly in triple-negative phenotype cells.
Keywords: lectin; sialic acid-binding lectin; ribonuclease; breast cancer; ErbB family

1. Introduction
Lectins are proteins that bind to speciﬁc carbohydrate structures. They exist universally in plants,
microorganisms, and animals, and have great potential for cancer therapy [1]. Even though only
lectins play a role in recognizing sialic acids, i.e., sialic acid-binding lectins (SBLs), there are several
lectins that reportedly have anti-tumor effects, such as Maackia amurensis seed lectin (MASL) [2],Polygonatum odoratum lectin (POL) [3], and Haliotis discus discus lectin (HddSBL) [4]. Sialic acids on theplasma membrane are generally observed to be linked to the terminal position of the carbohydrategroups of glycoproteins and glycolipids and have roles in the conformation, recognition, or binding ofglycomolecules [5]. Given that altered sialylation is closely associated with malignant phenotypes,
including metastasis and invasiveness [6,7], exploration of the effects of SBLs in cancer therapy is aﬁeld of great interest for basic studies, and also for clinical researchers.
The 12 kDa protein isolated from Rana catesbeiana oocytes was found to be a cell agglutinin [8] ofmany kinds of cancer cells, but not normal cells. These agglutinations were shown to be inhibited by
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the sialic acid-containing complex, but not by their asialo-derivatives and, thus, the protein was named
R. catesbeiana sialic acid-binding lectin (cSBL) [9]. Subsequent analyses revealed that it is homologous
to the ribonuclease (RNase) A superfamily and it has substantial RNase activity [8,10,11]. An RNase
puriﬁed from R. catesbeiana oocytes collected in Taiwan by Liao et al., and named RC-RNase, was foundto be identical to cSBL [12,13]. Therefore, this interesting SBL is now consequently also called a leczyme(lectin + enzyme) [14,15].
Breast cancer is a molecularly heterogeneous disease [16]. Currently, the classiﬁcation of breast
cancer is based mainly on the expression of the estrogen receptor (ER), progesterone receptor (PgR),
and the overexpression or ampliﬁcation of human epidermal growth factor receptor 2 (HER2/c-ErbB2).
In addition, tumors are characterized by grade and proliferative fraction (most commonly assessed by
Ki-67). The intrinsic molecular subtypes of breast cancer are known as luminal A-like (strongly ER
and PgR positive, HER2 negative, with lower proliferation markers), luminal B-like (variable degrees
of ER/PgR expression, with higher proliferative fraction), HER2-enriched (ER and PgR negative,and HER2 positive) and basal-like (ER, PgR, and HER2 negative), and these are routinely usedclinically to classify patients for prognostic predictions and to select treatments [17]. The basal-like
subtype includes triple-negative breast cancer [18]. Patients diagnosed with triple-negative breast
cancer have a poorer prognosis than HER2 and/or hormone receptor positive groups [19]. Recently,
the three additional members of the HER/ErbB family of receptor tyrosine kinases (epidermal growth
factor receptor (EGFR)/HER1/c-ErbB1, HER3/c-ErbB3 and HER4/c-ErbB4) have been of particularinterest because of their ability to interact with HER2 [20]. Members of ErbB family are criticallyinvolved in the development and progression of breast cancer. The overexpression of HER1/EGFR
is signiﬁcantly associated with poor prognosis [21,22]. EGFR is well known as a treatment target forcolorectal, head and neck, and non-small cell lung cancers, and is also a therapeutic target for breast
cancer [23].
Since 2011, the efﬁcacy of cSBL on breast cancer cells has been reported; however, the selectivityof cSBL to some cell lines is controversial. Tseng et al. showed that cSBL induces cell death selectively
on ER-positive breast cancer cell lines (MCF7 and ZR-75-1), but not on ER-negative breast cancer celllines (MDA-MB-231 and ZR-75-30) [24]. Their report indicates that ER is an important target of the
RNase activity of cSBL. In contrast, our group has demonstrated that cSBL induces cell death in all celllines tested in the report including MCF7 (ER-, PgR- and HER2-positive), SK-BR-3 (HER2-positive)
and MDA-MB-231 (triple-negative) [25]. Here, we tested the effects of cSBL on a larger number ofcell lines that represent distinct phenotypes, and also on a normal breast-derived cell line. It was
revealed that cSBL exerts its pro-apoptotic effects on all cancer cells, but not on normal breast cells.
Furthermore, we found that treatment with cSBL leads to the decrement of HER2 expression, and thisreduced expression was also observed with regard to other ErbB family proteins expressed in each cellline. Our results suggest a potential application of cSBL in the treatment of breast cancers, including
triple-negative breast cancer.
2. Results
2.1. Effects of cSBL on Breast Cancer Cell Growth
To evaluate the impact of cSBL on breast cancer cell growth, we ﬁrst examined the effects ofcSBL on cell proliferation in several breast cancer cell lines and a normal breast cell line by WST
assay. The immortalized human mammary epithelial cell line, MCF 10A, was used as the normal
breast cell line due to its non-tumorigenic origin [26]. The characteristic features of each cell lines
used in this study are summarized in Table 1. As shown in Figure 1A, treatment with cSBL resulted
in a signiﬁcant reduction in the proliferation of breast cancer cells. In contrast, cSBL hardly showed
any inhibitory effect on the proliferation of normal breast cells. Although statistical differences weredetected at 10 and 20 μM treatments with cSBL in MCF 10A cells, the viabilities were kept as 91%
and 85%, respectively. The viabilities of each cell line with treatment of 20 μM cSBL were as follows:Molecules 2018, 23, 2714

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ZR-75-1, 69%; BT-474, 51%; MCF7, 45%; SK-BR-3, 46%; MDA-MB-231, 52%; MDA-MB468, 40%; andMCF 10A, 85%. MCF7 cells were shown to be the most sensitive to cSBL and the inhibitory effect wasobserved from relatively low concentrations, such as 1 μM (viability: 50%).
Table 1. Classiﬁcation of breast cancer cell lines [16,27].
Cell Line

ER 1

PgR 1

HER2 2

Subtype

ZR-75-1
BT-474
MCF7
SK-BR-3
MDA-MB-231
MDA-MB-468
MCF 10A

+
+
+
-

+
+
+
-

2+
3+
0-1+
3+
0-1+
0-1+

Luminal A
Luminal B
Luminal A
HER2
Basal
Basal
Basal

1 Data from mRNA, protein levels [16], and/or immunohistochemical (IHC) analysis [26]; 2 Data from IHC analysis[26]. The Allred score was used for HER2.

Figure 1. Cont.

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Figure 1. Anti-proliferative effects of R. catesbeiana sialic acid-binding lectin (cSBL) against breast cancer
cell lines (ZR-75-1, BT-474, MCF7, SK-BR-3, MDA-MB-231, and MDA-MB-468) and a non-tumorigenic
epithelial cell line (MCF 10A). (A) Viability of cells treated with cSBL. Cells were treated with cSBL
(0.2–20 μM) for 72 h. Each data point represents the mean ± SD of three independent WST-8 assays
conducted in triplicate. (B) The effects of cSBL on the clonogenic potential of the cells. Colony assays
were performed on the cells in the absence or presence of cSBL (1, 5, and 10 μM) for 72 h followed byincubation in cSBL-free medium for 7–28 days. Then, the cells were ﬁxed with 2% paraformaldehyde
and stained with crystal violet. Representative images from three independent experiments are shown.
Cell growth (C) and viability were determined by trypan blue assay (D) of the cells treated with orwithout cSBL. Cells were treated with cSBL (10 μM) for 48–96 h. Both live and dead cell numbers werecounted by TC10. Each data point represents the mean ± SD of three independent assays. For bothgraphs, * p < 0.05. ** p < 0.01. *** p < 0.001.

Next, we assessed the effects of cSBL on a clonogenic assay. At ﬁrst, 5 × 103 cells were seeded,
and then, the cells were treated with cSBL. cSBL markedly impaired the colony formation of breast
cancer cells, whereas it had no profound effect on the growth of normal breast cells (Figure 1B).
Untreated control cells of MCF7, MDA-MB-231, and MCF 10A exhibited higher colony forming rates,
and ZR-75-1 and BT-474 exhibited low colony forming rates. Since the initial seeding concentration

Molecules 2018, 23, 2714

5 of 17of the cells affected the colony formation efﬁciency, we also performed the clonogenic assay withdifferent cell seeding concentrations (Supplementary Figure S1). MCF7, SK-BR-3, MDA-MB-231,
MDA-MB468, and MCF 10A cells were seeded with 2.5 × 103 cells per well (Figure S1A), ZR-75-1
and BT-474 cells were seeded with 1 × 104 cells per well (Figure S1B). As shown in Figure 1B and
Figure S1A and B, similar tendencies were observed in all conditions. Namely, MCF7 showed thehighest sensitivity; moderate effects were found in MDA-MB-231 and MDA-MB-468; SK-BR-3, ZR-75-1
and BT-474 exhibited relatively lower levels of sensitivity, but their effects on MCF 10A were limited.
Furthermore, lower concentrations of cSBL were assessed with MCF7 and the signiﬁcant inhibitory
effects were conﬁrmed for all cell seeding concentrations tested here, even with treatment of 0.1 μM(Figure S1C,D).
To obtain further evidence that cSBL only reduced the proliferation of breast cancer cells, we alsomeasured the cell viability with the trypan blue dye exclusion assay (Figure 1C,D), since the assay
counts the live and dead cells directly. All untreated control cells continued to proliferate overthe experimental period; however, the proliferations of the cancer cells were impaired with cSBL
treatment (Figure 1C). cSBL decreased the viability of all cancer cells signiﬁcantly in a time- dependent
manner. In MCF 10A, the effects of cSBL on cell growth and viability were not signiﬁcant. In summary,cSBL showed concentration- and time-dependent cancer cell growth-suppressing effects. All assays
showed a similar tendency, whereby MCF7 exhibited the highest, and MCF 10A exhibited the lowest
sensitivity to cSBL. There were no correlations between cSBL effectiveness and the expression statusesof ER, PgR, or HER2. These results suggest that treatment with cSBL selectively leads to a cell growth
inhibitory effect on breast cancer cells, regardless of the cancer cell phenotype.
2.2. cSBL-Induced Apoptosis
To investigate further insight into the growth inhibitory effects of cSBL, we assayed apoptotic
cell death by detecting morphological and biochemical apoptotic changes. We analyzed nuclear
morphological changes by Hoechst staining. As shown in Figure 2A, similar to the effects on cellproliferation, treatment with cSBL led to the induction of chromatin condensation and nuclear collapse
in breast cancer cells, whereas it had almost no pro-apoptotic effects on normal cells. Since MCF7 is a
type of caspase-3 deﬁcient cell [28], and since the pro-apoptotic effects of cSBL are mainly initiatedby caspase-9 activation [29], we analyzed the cleavage of procaspase-9 as an initiative event and thatof poly-(ADP-ribose) polymerase (PARP) as an executive event. Treatment with cSBL cleaved the
pro-caspase-9 and PARP in breast cancer cells, although the effects were relatively low in ZR-75-1(Figure 2B). The cleaved form of PARP was detected slightly in MCF 10A treated with cSBL, but thelevel was comparable with the control. Taken together, these results suggest that cSBL induces
apoptotic cell death via caspase cascade activation, particularly in cancer cells. Considering that cSBL
decreased viabilities as well as cell numbers of breast cancer cells (Figure 1C,D), it is suggested thatcSBL causes the death of cancer cells rather than the arrest of the cell cycle, and the induction of
apoptosis is responsible for the cell death.

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Figure 2. Apoptosis induction in breast cancer cell lines (ZR-75-1, BT-474, MCF7, SK-BR-3,
MDA-MB-231, and MDA-MB-468) treated with cSBL. (A) Morphological changes of nuclei in
cSBL-treated cells. Cells were treated with cSBL (10 μM) for 72 h and stained with Hoechst 33342.
Nuclei were observed using a ﬂuorescent microscope. Arrows indicate apoptotic nuclei. Magniﬁcation,
20×. (B) Cells were treated with cSBL (10 μM) for 72 h, and caspase-9 and PARP cleavage were detectedby western blotting.

2.3. Effects of cSBL on Cell Survival-Related Molecules
We previously reported that cSBL induces p38 mitogen-activated protein kinase (MAPK) andconsequently, activates caspase-3/7 in SBL-induced cell death [25]. Furthermore, Tseng et al., reported
that cSBL induces cell death in ER-positive breast tumors through down-regulation of Bcl-2 andER [24]. To conﬁrm these phenomena and to further investigate cSBL-induced alterations on cell
survival-related molecular expressions, we analyzed the phosphorylation of p38, JNK, and ERK
MAPKs and protein expressions of the anti-apoptotic Bcl-2 family, as well as ERα, PgR, and HER2
in ER-positive ZR-75-1 and MCF7 cells. As shown in Figure 3A, consistent with previous reports,
cSBL induced p38 phosphorylation. A slight phosphorylation of JNK was also observed in ZR-75-1
cells—whereas total JNK expression decreased in MCF7 cells, the phosphorylated JNK level was kept
the same as control cells, and relative phosphorylation tended to increase. The phosphorylation status
of ERK was not affected by cSBL treatment in both cell lines. Regarding the Bcl-2 family, althoughstatistical signiﬁcance was not observed, consistent with previous reports, cSBL tended to decrease theexpression of Bcl-2 in MCF7, and decrements of Bcl-xL and Mcl-1 were detected in the cells (Figure 3B).
In ZR-75-1 cells, Bcl-2 expression was very faint, and BCL-xL and Mcl-1 expression levels were slightly

Molecules 2018, 23, 2714

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increased. Thus, the effects of cSBL on the anti-apoptotic Bcl-2 family seem to vary depending on
the cell line. ERα expression drastically decreased after exposure to cSBL in both cells, as expected(Figure 3C). Interestingly, cSBL also suppressed PgR in MCF7 and HER2 protein expression in bothcell lines. These results indicate that cSBL works in an inhibitory manner concerning the protein
expressions of key molecules that are implicated in breast cancer phenotypes.

Figure 3. Effects of cSBL on the activations of mitogen-activated protein kinases (MAPKs) (A),and expressions of the anti-apoptotic Bcl-2 family (B), and breast cancer-related proteins (C) in ZR-75-1
and MCF7 cells. Cells were treated with cSBL (10 μM) for 72 h, and the phosphorylation and expression
levels of each molecule were detected by western blotting. The values relative to the controls arepresented as the mean ± SD of three independent experiments (right graphs). * p < 0.05, ** p < 0.01,*** p < 0.001.

Molecules 2018, 23, 2714

8 of 172.4. Expressions of ErbB Family Protein in Breast Cancer Cells Treated with cSBL
Since the ErbB family proteins and their signals were generally attractive therapeutic targets,
we evaluated the effects of cSBL on ErbB family expression in all breast cancer cell lines tested here
(Figure 4). EGFR/HER1 was predominantly expressed in the triple-negative breast cancer cell lines,
MDA-MB-231 and MDA-MB-468, and a faint band was also detected in SK-BR-3. HER2 expression was
clearly observed in ZR-75-1, BT-474, SK-BR-3, and slightly in MCF7. HER3 expression was detected
in MDA-MB-468 in addition to the four cell lines mentioned above. Slight expression of HER4 wasfound only in ZR-75-1 and MCF7. Then, the effects of cSBL on the levels of protein expression ofthese proteins were assessed in each cell line. Surprisingly, treatment with cSBL resulted in decreased
protein levels of all ErbB proteins in each breast cancer cell line.

Figure 4. Effects of cSBL on the expression levels of ErbB family proteins in breast cancer celllines (ZR-75-1, BT-474, MCF7, SK-BR-3, MDA-MB-231, and MDA-MB-468). Cells were treated withcSBL (10 μM) for 72 h and the expression levels of ErbB family in each cell line were detected bywestern blotting.

2.5. Expressions of EGFR and Its Signaling in Triple-Negative Breast Cancer Cells Treated with cSBL
Even though some drugs tailored to the breast cancer cell types, such as hormonal agents for
ER-positive cells and HER2 targeted agents for HER2 positive cells, have achieved clinical effects tosome extent, chemotherapeutic options for triple-negative phenotype breast cancers are very limited.
Targeting EGFR/HER1, which is expressed in most patients of triple-negative breast cancer [23], or its
signaling is a novel and attractive strategy. Since cSBL was shown to decrease EGFR/HER1 protein
expression in triple-negative cell lines, we next sought to explore the downregulation mechanism of
EGFR/HER1. Statistically signiﬁcant reductions in EGFR/HER1 were observed after 24-h and 72-h oftreatment with cSBL in MDA-MB-231 and MDA-MB-468, respectively (Figure 5A).

Molecules 2018, 23, 2714

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Figure 5. Effects of cSBL on EGFR/HER1 expression and downstream signaling in MDA-MB-231 and
MDA-MB-468 cells. (A) Cells were treated with cSBL (10 μM) for 24 to 72 h, and the expression levelsof EGFR/HER1 in each cell line were detected by western blotting. Densitometric quantiﬁcation of
three independent experiments (mean ± SD) was performed using Image J. (B) Cells were treatedwith cSBL (10 μM) for 24 to 72 h, and the phosphorylation levels of AKT, ERK, and p38 were detectedby western blotting. (C) Cells were treated with cSBL (10 μM) for 3–24 h and the expression levelsof EGFR/HER1 and the phosphorylation levels of AKT and ERK were detected by western blotting.
The values relative to the controls are presented as the mean ± SD from three independent experiments(right graphs). For both graphs, * p < 0.05; ** p < 0.01; *** p < 0.001.

Molecules 2018, 23, 2714

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In an effort to determine the contribution of EGFR/HER1 reduction to the cSBL-induced
pro-apoptotic effect, we investigated the activation status of the EGFR signaling pathway. No change
was observed in ERK activation in either MDA-MB-231 or MDA-MB-468 cells (Figure 5B).
In MDA-MB-231, AKT phosphorylation seemed to slightly decrease after 48-h of treatment with
cSBL, but increased after 72-h of treatment compared to the control. The phosphorylation level of AKT
was unchanged in MDA-MB-468 cells. In contrast, phosphorylation of p38 increased in both cell lines.
Since the AKT activation status in MDA-MB-231 cells treated with cSBL was uncertain, we thenfurther observed these events at an earlier time period (from 3 to 24 h of treatment). Even though low
EGFR/HER1 levels were observed compared to 24-h untreated control throughout the time points,
the level of phosphorylation of AKT gradually increased, and the level at 24-h treatment with cSBL was
the same level as that of the 24-h untreated control in MDA-MB-231 (Figure 5C). Thus, it is suggested
that AKT phosphorylation increased during the early period in our experimental culture period on
cSBL-treated MDA-MB-231, although EGFR/HER1 expression was affected by the treatment. On theother hand, EGFR/HER1 expression was slightly decreased by 24-h of cSBL treatment. The AKT
phosphorylation status tended to increase from 3 to 12-h of treatment even though statistical differenceswere not detected at all time points.
Taken together, the effects of cSBL treatment on ERK and AKT activation in MDA-MB-231 and
MDA-MB-468 cells were not considered to be signiﬁcant. Similarly, ERK activation was unchanged in
ZR-75-1 and MCF7 cells (Figure 3A). Therefore, the EGFR signaling pathway might not be implicated incSBL-induced apoptosis. P38 phosphorylation, which has been proven to, at least partially, contributeto caspase activation in cSBL-induced apoptosis [25], was observed, and this also supports thisprediction. These results indicate that cSBL induces apoptosis to breast cancer cells, including
triple-negative cells, accompanied by a decrease in the levels of ErbB family proteins, whereas EGFR
signaling is not affected.
3. Discussion
In this study, the effects of cSBL on the breast cancer cell growth and on the expression of moleculesthat play key roles in breast cancer prognosis were evaluated. cSBL signiﬁcantly suppressed the cell
growth of six cancer cell lines representing a variety of phenotypes in ERα, PgR, and HER2 (Table 1 andFigure 1) through the induction of apoptosis (Figure 2). However, the effects of cSBL on normal breast
cells were very limited; the viability of MCF 10A stayed over 85% in all concentration ranges tested,and pro-apoptotic changes were not detected under conditions that cause rigid apoptotic changes tocancer cells (Figures 1 and 2). We measured not only the activation of p38 and downregulation ofERα, consistent with previous reports, but also found varied effects on the expression of anti-apoptotic
Bcl-2 family proteins depending on the cell types as well as the downregulation of PgR and HER2
in multiple cancer cell lines (Figure 3). Further investigation revealed that cSBL causes a reduction
in all ErbB family proteins that are expressed in the breast cancer cell lines (Figure 4). The analysis
of EGFR downstream signaling showed that the inhibition of EGFR signaling did not occur in thecSBL treatments (Figure 5). Accordingly, we propose that cSBL triggers apoptosis induction without
affecting EGFR signaling, but the downregulation of ERα, PgR, and ErbB receptors are accompaniedwith cSBL-induced cell death under the experimental conditions used in the current study.
ER and PgR are nuclear hormone receptors whose activation is controlled by ligand binding,kinase activators, or phosphatase inhibitors [30–35]. The degradation of ER and PgR has been shown
to be under the control of the ubiquitin proteasome system [35–39], and recent research revealed
that the phosphorylation of ERα via p38 activation promotes ERα turnover in breast cancer cells [40].
The activation of p38 by cSBL treatment has been observed in leukemia cells [41], mesothelioma
cells [42], and some breast cancer cells [25], as well as the cells tested here. Thus, the reduced expression
levels of ERα and PgR in cSBL-treated cells may be associated with p38 activation. In the meantime,interaction and stabilization of these hormonal receptors with/by heat shock proteins (HSPs) has
also been reported [36,43,44]. We previously found that reducing HSP70 expression attenuates theMolecules 2018, 23, 2714

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apoptosis-inducing effects of cSBL [45]. Moreover, treatment with cSBL causes remarkable changes
in the localization of HSP70 and HSC70; cSBL treatment has been shown to evoke increases ofthese proteins in the cytosol prior to the execution of apoptosis in mouse leukemia P388 cells [45].
This implies that HSP70 and HSC70 have important roles in the effects of cSBL, and, also, there isa possibility that cSBL decreases the expression of ERα and PgR through the regulation of HSPs.
The implications of proteasome degradation and stabilization by HSPs are common in ErbB receptor
turnover. EGFR/HER1 is known to be internalized after ligand binding and part of it is degradedby lysosomes or by the proteasome [46,47]. HSP70 and HSC70 act as key co-chaperones for HSP90
machinery, at least in part, by aiding in client protein recruitment [48]. The HSP70 and HSP90
chaperones work together to target certain client proteins, including EGFR/HER1 for degradationby the ubiquitin-proteasome system, and inhibitory agents targeting HSPs are one of the potential
approaches for cancer therapeutics today [49,50]. Further works should be performed to determine
the mechanisms by which p38 activation and/or HSPs are involved in the reduction of ERα, PgR, and
ErbB receptors in cSBL-treated cells.
It has been reported that cSBL acts as an anti-cancer agent against various human cancer cells
including carcinoma (cervical, oral, hepatocellular, and breast), leukemia, lymphoma, mesothelioma,
and glioblastoma [24,41,42,51–57]. Interestingly, although cSBL induces apoptosis in those cancer
cells, normal tissue-derived cells, such as ﬁbroblasts, melanocytes, keratinocytes, and mesothelial
cells, are relatively insensitive to this agent [53,56,58–60]. Moreover, our recent study using humanmalignant mesothelioma xenograft model mice showed that cSBL exerts signiﬁcant anti-tumor effects
without adverse effects in vivo [61]. It is also noteworthy that the members of the pancreatic-type
RNase super-family, including cSBL, show high thermal stability and strong resistance to protein
denaturants and to proteases [62,63]. Moreover, they are expected to have lower immunogenicity
due to their compact structures and homology to human pancreatic RNases [64]. However, only
limited information has been obtained regarding its cancer-selectivity so far. The known factors that
affect cell susceptibility are summarized in our previous review [65]. To date, differences in the cSBL
binding to the cell surface and its internalization into cytosol were considered to be the main reasonsfor the selectivity. In the present study, pro-apoptotic effects were observed irrespective of breast
cancer cell type, and this is not consistent with previous reports; cSBL is effective towards breast
cancer cells, including triple-negative breast cancers. It can be speculated that one of the reasons forthis contradiction may be the variance of cell culture conditions. The elements that inﬂuence the celldependency on particular hormones or growth factors, such as FBS, composed of complete medium
may be an explanation. Our results also revealed obvious differences in sensitivity between MCF7
and MCF 10A to cSBL. Comparative studies on cSBL binding and/or internalization to these cells
would give us evidence for the cancer selectivity of cSBL that could promote the development ofnovel anti-cancer agents. Furthermore, growth signals, such as the AKT and ERK activation of the celllines investigated here, seem to be independent of EGFR/HER1 expression. Although involvement ofalternative pathways such as PLCγ or STATs activated by EGFR/HER1 in the effects of cSBL needs tobe elucidated, our results suggest that applications of cSBL against the cancer cells that depend on
the ErbB receptors for growth may provide great beneﬁt as a type of chemotherapy. Moreover, recent
studies on triple-negative breast cancer therapeutics have been shown to affect the crucial parts of
ErbB receptors [48,66,67]. Tao et al. reported that single treatment with an AKT inhibitor (GDC-0068)
or a PI3K inhibitor (GDC-0941), inhibitors for EGFR downstream signaling, activates EGFR/HER1
and HER3 [68]. The blockade of EGFR and HER3 combined with drugs results in superior anti-tumor
activity in vitro and in vivo, suggesting the importance of inhibition on both the upstream activationof key molecules and the downstream signaling pathway, due to the cellular response of activating
an alternative pathway for cancer cell survival. Thus, the combination with cSBL that reduces theexpressions of all ErbB family proteins with drugs such as AKT inhibitors could have a great potential
for therapy in breast cancers, including triple-negative phenotype cells.

Molecules 2018, 23, 2714

12 of 174. Materials and Methods
4.1. ReagentscSBL was isolated using sequential chromatography with Sephadex G75, DEAE-cellulose,hydroxyapatite, and SP-Sepharose, as previously described [9]. The anti-caspase-9 antibody waspurchased from Medical and Biological Laboratories Co., Ltd. (MBL; Nagoya, Japan). The anti-PARP(46D11), phospho-p38 (Thr180/182) (D3F9), p38 (D13E1), Phospho-ERK (Thr202/Tyr204) (F13.14.4E),
ERK (137F5), Bcl-2 (D55G8), Bcl-xL (54H6), Mcl-1 (D35A5), EGFR/HER1 (D38B1), HER2 (29D8),
HER3 (D22C5), HER4/ErbB4 (111B2), phospho-AKT (Ser473) (D9E), AKT (11E7), and phospho-AKT(Ser473), horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG antibodies
were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-β-actin antibody
was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The anti-phospho-JNK(pT183/pY185), and JNK antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA).
The anti-ERα (E115) and PgR (ERP5489) antibodies were purchased from Abcam Biotechnology(Cambridge, UK).
4.2. Cell Culture
The breast cancer cell lines (ZR-75-1, BT-474, MCF7, SK-BR-3, MDA-MB-231, and MDA-MB-468),
and a non-tumorigenic epithelial cell line, MCF 10A, were purchased from American Type Culture
Collection (ATCC; Manassas, VA, USA). The cancer cells were cultured in Dulbecco’s modiﬁed Eagle’s
medium (WAKO Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine
serum (Biosera, Nuaille, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies,
Carlsbad, CA, USA). MCF 10A cells were cultured in mammary epithelial cell growth medium (Lonza,
Basel, Switzerland) supplemented with bovine pituitary extract, hydrocortisone, hEGF, insulin (Bullet
Kit, Lonza) and 100 ng/mL cholera toxin (WAKO Pure Chemical Industries, Ltd.). All cell lines were
cultured in a humidiﬁed atmosphere in 5% CO2 at 37 ◦ C.
4.3. WST Assays
WST-8 assay was performed to determine the cell viability. Cells (5 × 104 cells/mL) cultured in
96-well plates (100 μL/well) were treated with cSBL (1, 5, 10, and 20 μM) for 72 h. Then, the cells wereincubated with Cell Count Reagent SF (Nacalai Tesque Inc., Kyoto, Japan) for 1–4 h. The absorbanceof the resulting product at 450 nm was measured, and the background absorbance at 650 nm was
subtracted. Experiments were conducted in triplicate.
4.4. Clonogenic Assay
The cells were seeded in six-well plates (5 × 103 cells/well) and allowed to adhere overnight.
The cells were treated with 1–10 μM cSBL for 72 h. The culture medium was subsequently replacedwith cSBL-free culture medium and the cells were incubated for 7–28 more days (7 days: MCF7,
MDA-MB-231, MCF 10A; 14 days: SK-BR-3, MDA-MB-468; 28 days: ZR-74-1, BT-474). The colonies
were ﬁxed with 2% paraformaldehyde and stained with crystal violet.
4.5. Trypan Blue Dye Exclusion Assay
A trypan blue dye exclusion assay was also performed to assess the effects of cSBL on cell
viability. Cells (5 × 104 cells/mL) cultured in a 24-well plate (500 μL/well) were treated with cSBL
(10 μM) for 48, 72, and 96 h. Then, the cells were harvested, and the numbers of live or dead cells
were counted by TC10 (Bio-Rad Laboratories, Inc. Hercules, CA, USA) in accordance with to the
manufacturer’s instructions.

Molecules 2018, 23, 2714

13 of 174.6. Observation of Nuclear Morphology
Cells (5 × 104 cells/mL) cultured in a Cell Carrier-96 Ultra Microplate (100 μL/well) weretreated with cSBL (10 μM) for 72 h. Then, cells were stained with 2 μg/mL Hoechst 33342 (Dojindo
Laboratories, Kumamoto, Japan) for 1 h. The resulting images were acquired with the high-content
analysis system Operetta CLS™ with NA 20× objectives.
4.7. Western Blotting
The cells (5 × 104 cells/mL) were cultured in six-well plates (4 mL/well) and were treated withcSBL (10 μM) for the indicated time. Whole cell lysates were prepared using extraction buffer (150 mM
NaCl, 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, and 0.1%
sodium dodecyl sulfate) supplemented with cOmplete™ Mini EDTA-free protease inhibitor cocktail
tablets and PhosSTOP phosphatase inhibitor tablets (each 1 tablet/10 mL; Roche Applied Science,
Indianapolis, IN, USA). Soluble proteins were collected, and the protein concentration was measured
using a BCA protein assay kit (Thermo Fisher Scientiﬁc, Inc., Waltham, MA, USA) according to the
manufacturer’s instructions. The proteins were separated using 10% or 14% SDS-PAGE and transferred
onto Immobilon-P transfer membranes (EMD Millipore, Billerica, MA, USA). The membranes weresequentially incubated with primary and secondary antibodies diluted in Can Get Signal solution(Toyobo Co., Ltd., Osaka, Japan). The protein bands were detected using ECL Prime Western Blotting
Detection Reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The relative density of theprotein bands was measured by ImageJ 1.51s software (National Institutes of Health, Bethesda, MD,
USA). The experiments were repeated three times.
4.8. Statistical Analysis
Results are expressed as means ±SE or SD and are the representative of at least three independent
experiments. Statistical comparisons of two groups were made using Student’s t-tests and amonggroups using one-way ANOVA, followed by Bonferroni’s post hoc tests. All calculations were
performed using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA, USA).
The signiﬁcance threshold was p < 0.05.
5. Conclusions
In summary, our founding suggestion is that cSBL exhibits an apoptosis-inducing effect regardlessof the breast cancer cell type and has high cancer selectivity. These effects are accompanied by adecrease in the ErbB family receptors. To explore the effects of cSBL on cancer cells showing ErbB
family-dependent growths, a combinatorial study with EGFR signal-targeted drugs, such as AKT
inhibitors, should be performed.
Supplementary Materials: The following are available online, Figure S1. Effects of cSBL on clonogenic potentialof the cells seeded in distinct cell densities. Colony assays on the cells were performed in the absence or presenceof cSBL for 72 h followed by incubation in cSBL-free medium for 7–28 days. The cells were ﬁxed with 2%paraformaldehyde and stained with crystal violet. (A) 2.5 × 103 cells were seeded and allowed to adhere
overnight, and then the cells were treated with 1–10 μM cSBL. (B) 1 × 104 cells were seeded and allowed to adhere
overnight, and then the cells were treated with 1–10 μM cSBL. 5 × 103 (C) or 2.5 × 103 (D) cells were seeded andallowed to adhere overnight, and then the cells were treated with 0.1 and 0.5 μM cSBL. Representative images
are shown.
Author Contributions: T.T. conceived, designed, and performed the experiments, analyzed the data,
and wrote the paper; S.S. (Shoko Sato) designed the experiments and wrote the paper; T.S. (Toshiyuki Sato)
designed and performed the experiments; S.S. (Shigeki Sugawara) contributed reagents and analysis tools.
T.S. (Tsuneyoshi Suzuki) and A.H. provided the laboratory support. M.H. provided the laboratory support,
wrote the paper, and improved the manuscript.
Funding: This study was supported by the ‘Strategic Research’ Project (2012–2017) for Private Universities from
the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and funded by a Grant-in-Aid forYoung Scientists (B), grant number 17K15029 to Takeo Tatsuta.

Molecules 2018, 23, 2714

14 of 17Conﬂicts of Interest: The authors declare that there is no conﬂict of interest.

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Serra, V.; Scaltriti, M.; Prudkin, L.; Eichhorn, P.J.A.; Ibrahim, Y.H.; Chandarlapaty, S.; Markman, B.;
Rodriguez, O.; Guzman, M.; Rodriguez, S.; et al. PI3K inhibition results in enhanced HER signaling andacquired ERK dependency in HER2-overexpressing breast cancer. Oncogene 2011, 30, 2547–2557. [CrossRef][PubMed]Rodrik-Outmezguine, V.S.; Chandarlapaty, S.; Pagano, N.C.; Poulikakos, P.I.; Scaltriti, M.; Moskatel, E.;
Baselga, J.; Guichard, S.; Rosen, N. mTOR kinase inhibition causes feedback-dependent biphasic regulationof AKT signaling. Cancer Discov. 2011, 1, 248–259. [CrossRef] [PubMed]
Tao, J.J.; Castel, P.; Radosevic-robin, N.; Elkabets, M.; Aceto, N.; Weitsman, G.; Barber, P.; Vojnovic, B.;
Morse, N.; Viola-villegas, N.T.; et al. Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of
the PI3K-Akt Pathway in Triple-Negative Breast Cancer. Sci. Signal. 2015, 7, ra29. [CrossRef] [PubMed]

Sample Availability: Samples of cSBL are not available from the authors.
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

RESEARCH ARTICLE

Sialic acid-binding lectin from bullfrog eggs
inhibits human malignant mesothelioma cell
growth in vitro and in vivo
Takeo Tatsuta1ᄊ, Toshiyuki Satoh1,2ᄊ, Shigeki Sugawara1, Akiyoshi Hara2,
Masahiro Hosono1
1 Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical
and Pharmaceutical University, Aobaku, Sendai, Japan, 2 Department of Clinical Pharmacotherapeutics,
Tohoku Medical and Pharmaceutical University, Aobaku, Sendai, Japana1111111111
a1111111111
a1111111111
a1111111111
a1111111111

23(1 $&&(66Citation: Tatsuta T, Satoh T, Sugawara S, Hara A,
Hosono M (2018) Sialic acid-binding lectin frombullfrog eggs inhibits human malignant
mesothelioma cell growth in vitro and in vivo. PLoS
ONE 13(1): e0190653. https://doi.org/10.1371/
journal.pone.0190653
Editor: Irina V. Lebedeva, Columbia University,
UNITED STATES
Received: September 19, 2017
Accepted: December 18, 2017
Published: January 3, 2018
Copyright:  2018 Tatsuta et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper.
Funding: This study was supported by the‘Strategic Research’ Project (2012-2017) for
Private Universities from the Ministry of Education,
Culture, Sports, Science and Technology of Japan,
and a Grant-in-Aid for Young Scientists (B) [grantno. 17K15029 (to Takeo Tatsuta)]. The funders had
no role in study design, data collection andanalysis, decision to publish, or preparation of theᄊ These authors contributed equally to this work.
* mhosono@tohoku-mpu.ac.jp

Abstract
Malignant mesothelioma is an aggressive cancer that results from exposure to asbestos. The
therapeutic options for this type of cancer are limited; therefore, the development of novel therapeutic agents is urgently required. Sialic acid-binding lectin isolated from Rana catesbeiana
oocytes (cSBL) is a novel therapeutic candidate for cancer, which exhibits antitumor activity
mediated through RNA degradation. In the present study, we evaluated the effect of cSBL in
vitro and in vivo. Xenograft-competent H2452 and MSTO human mesothelioma cell lines weretreated with cSBL, and the pathway by which cSBL induces apoptosis was analyzed. In vivo
studies were performed using nude mice inoculated with one of the two cell lines, and theeffects of cSBL and pemetrexed were monitored simultaneously. Furthermore, the pharmacological interactions between the three agents (pemetrexed, cisplatin and cSBL) were statistically assessed. It was demonstrated that cSBL treatments caused morphological andbiochemical apoptotic changes in both cell lines. Caspase cascade analysis revealed that an
intrinsic pathway mediated cSBL-induced apoptosis. The administration of cSBL significantly
inhibited tumor growth in two xenograft models, without any adverse effects. Furthermore, thecombination index and dose reduction index values indicated that the cSBL + pemetrexed
combination showed the highest synergism, and thus potential for reducing dosage of eachdrug, compared with the other combinations, including the existing pemetrexed + cisplatin regimen. cSBL exerted prominent antitumor effects on malignant mesothelioma cells in vitro and invivo, and showed favorable effects when combined with pemetrexed. These results suggestthat cSBL has potential as a novel drug for the treatment of malignant mesothelioma.

Introduction
Malignant mesothelioma is an aggressive cancer of the mesothelial cells of serous membranes,
involving the pleural and peritoneal spaces, which results from exposure to asbestos [1–3]. The
mechanisms underlying the induction of DNA damage by asbestos fibers in mesothelial cells
remain unclear. The production and use of asbestos is now forbidden in the majority of

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In vitro/vivo antitumor effect of cSBL

manuscript. There was no additional externalfunding received for this study.
Competing interests: The authors have declaredthat no competing interests exist.

industrialized countries; however, it is still actively used in many developing countries [4]. As
the time between asbestos exposure and disease diagnosis is >40 years on average, the incidence of mesothelioma is increasing and is projected to peak in the late 2020s, even in developed countries [5].
Malignant mesothelioma may have an epithelioid, sarcomatoid or biphasic morphology[6]. The epithelioid type is associated with a longer survival time compared with the biphasic
and sarcomatoid types [7,8]. Although chemotherapeutic approaches are limited for malignant
mesothelioma, it is generally accepted that patients with the epithelioid subtype respond betterto treatment [9,10]. Additionally, other factors, including sex, performance status, diseasestage, serum lactate dehydrogenase level, anemia and leukocytosis, reportedly influence survival in malignant mesothelioma [11]. However, it is difficult to identify the biological factorsthat clearly differentiate between patients with a poor prognosis and those with a more favorable prognosis, as long-term survival is rare in malignant mesothelioma [6].
There are few therapeutic options (surgery, radiation therapy, and chemotherapy) for
mesothelioma [1]. The folate antimetabolite pemetrexed is a chemotherapeutic agent, which istypically used in combination with platinum-containing drugs, such as cisplatin [12,13]. This
combination therapy improves the response rate, progression-free survival, overall survival,and quality of life of patients with mesothelioma [12]; however, any treatment-induced regression observed is typically transient, and local tumors rapidly relapse due to the high chemoresistance of this cancer type [4]. So far, some progresses were obtained by multimodality
therapy [14]. A median survival time of up to 29 months has been reported for those who complete a trimodal therapy including chemotherapy, surgery, and hemithoracic radiation therapy[15–18]. Moreover, pleurectomy/decortication with intraoperative photodynamic therapy andadjuvant pemetrexed-based chemotherapy demonstrated 36 months median survival [19].
However, even with these aggressive approaches, the prognosis of malignant mesothelioma
remains poor. Considering the predicted incidence peak and poor prognosis, as well as the factthat intrinsic and acquired resistance to existing drugs is common, further research into developing therapeutic agents for mesothelioma is essential.
There is growing interest in the use of naturally derived molecules as potential cancer therapeutics. Lectins, carbohydrate-binding proteins that occur in all organisms, are representativeof such natural compounds that have great potential for cancer therapy. Among them, several
sialic acid-binding lectins (SBLs), including mistletoe lectin (ML1) [20], Maackia amurensis
seed lectin (MASL) [21], Polygonatum cyrtonema lectin (POL) [22] and Haliotis discus discus
lectin (HddSBL) [23], have been reported to have antitumor effects. SBL isolated from Ranacatesbeiana oocytes (cSBL) is a unique compound that has multifunctional activity with lectin[24,25] and ribonuclease (RNase) [26], as well as antitumor activity [25]. cSBL exerts potent
cytotoxicity in various cancer cell types, but low cytotoxicity in normal cells [27]. Rana catesbeiana RNase (RC-RNase), an RNase purified from R. catesbeiana oocytes collected in Taiwan
by Liao et al. is identical to cSBL [28,29]. cSBL consists of 111 amino acid residues with four
disulfide bonds [29], and belongs to the vertebrate-secreted RNase family (RNase A superfamily) [30]. It has high thermal stability and strong resistance to protein denaturants [31]. Thesefeatures are considered one reason for the potent antitumor activity, and provide benefits forcommercialization. Furthermore, that cSBL does not associate with endogenous mammalian
RNase inhibitors, and that it exerts cytotoxicity in human cancer cells via its RNase activity[25], has facilitated further research into its antitumor effects. The mechanism of cSBLinduced cytotoxicity is proposed to be as follows: cSBL binds to the cancer cell surface and isinternalized. It subsequently degrades RNA in the cytosol, leading to the induction of apoptotic signaling [27]. In human leukemia Jurkat cells, cSBL was found to activate p38 and JNK
mitogen-activated protein kinase (MAPK) signals and induce apoptosis via the intrinsic

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In vitro/vivo antitumor effect of cSBL

(mitochondrial) pathway [32]. The RNase activity was also determined to be critical for apoptosis induction in MDA-MB231 human breast cancer cells, as an amino acid-replaced mutant
of cSBL that lacked RNase activity did not exhibit the apoptosis-inducing effect, even wheninternalized into the cells like native cSBL [33]. The efficacy of cSBL on malignant mesothelioma cells has previously been reported [34,35]; Even though cSBL hardly show cytotoxicity tonormal mesothelial cell Met5A, it efficiently reduced the viability of H28 malignant mesothelioma cells, and exhibited synergistic effects with TRAIL and pemetrexed on these cells.
In our previous study, in vivo experiments with cSBL were performed using mice transplanted with related ascites carcinoma, Ehrlich, Mep II and Sarcoma 180 cells. cSBL prolonged
their survival at non-toxic dose levels [25]. However, to date, the effect of cSBL on humanmalignant mesothelioma cells in vivo has not been elucidated. In the present study, to assess
the therapeutic potential of cSBL on malignant mesothelioma, we conducted an in vivo studyof cSBL using human malignant mesothelioma cell xenografts, and analyzed its antitumor
effects on these xenograft-competent cells.

Materials and methods
Cell culture
The human malignant mesothelioma cell lines NCI-H2452 (H2452, #CRL-5946) and MSTO211H (MSTO, #CRL-2081) were purchased from the American Type Cell Culture Collection(ATCC; Manassas, VA, USA). The cells were cultured in RPMI-1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS, Biosera, Nuaille,
France), 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA,
USA) at 37˚C in an atmosphere of 95% air and 5% CO2.

Animals
Eggs-bearing bullfrogs (domestically caught) and 5-week-old male nude mice (BALB/c nu/nuSlc) were purchased from Japan SLC, Inc (Shizuoka, Japan). All animal experiments were carried out in accordance with the Guidelines for Animal Experiments of the Tohoku Medical
and Pharmaceutical University (permission number: A16012-cn). Housing condition of themice was kept under standard conditions approved by the institutional guidelines with freefood- and water-consumptions.

Reagents
cSBL was isolated using sequential chromatography with Sephadex G75, DEAE-cellulose,hydroxyapatite and SP-Sepharose, as previously described [24]. Pemetrexed disodium heptahydrate was purchased from LC Laboratories (Woburn, MA, USA). The caspase-3 and caspase-8 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
The caspase-9 antibody was purchased from Medical & Biological Laboratories Co., Ltd.
(MBL; Nagoya, Japan). The Č-actin antibody was obtained from Sigma-Aldrich (Merck KGaA,
Darmstadt, Germany) and a horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody was purchased from Zymed Laboratories (Thermo Fisher Scientific, Inc., Waltham, MA,
USA). An HRP-conjugated anti-rabbit IgG antibody was purchased from Cedarlane Laboratories (Burlington, Ontario, Canada).

Annexin V staining assay
To investigate the induction of apoptosis, we evaluated Annexin V binding using an MEBCYTO apoptosis kit (MBL, Nagoya, Japan) according to the manufacturer’s instructions. Cells

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In vitro/vivo antitumor effect of cSBL

(5×104 cells/mL) were cultured in 6-well plates (2 mL/well) and treated with cSBL (H2452:
1 μM; MSTO: 0.4 μM) for 24–72 h at 37˚C in an atmosphere of 95% air and 5% CO2. Fluorescence intensity was subsequently detected using a FACSCalibur™ flow cytometer, and the data
was analyzed using CELLQuest™ software version 6.0 (BD Biosciences, Franklin Lakes, NJ,
USA).

Detection of nuclear fragmentation
Cells (5×104 cells/mL) cultured in a Cell Carrier-96 Ultra Microplate (100 μL/well) weretreated with cSBL (H2452: 5 μM; MSTO: 2 μM) for 6, 24, 48 and 72 h, in triplicate. Then, cells
were stained with 2 μg/mL Hoechst 33342 (Dojindo Laboratories, Kumamoto, Japan) for 1 h.
The resulting images were acquired with the High-Content Analysis System Operetta CLS™
with NA 20X or 40X objectives, and the fragmentation index was calculated using Harmony™
Imaging and Analysis Software 4.6 (PerkinElmer Japan Co., Ltd., Kanagawa, Japan).

Detection of caspase activity
The protein expression levels of activated caspase-3, -8, and -9 were analyzed using western
blot assays. Cells (1×105 cells/mL) cultured in 6-well plates (2 mL/well) were treated with cSBL
(H2452: 5 μM; MSTO: 2 μM) for 1, 3, 6, 24, 48, and 72 h. Whole cell lysates were preparedusing extraction buffer [150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1% Nonidet
P-40, 0.1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate] supplemented with cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail tablets and PhosSTOP phosphatase inhibitor tablets (each 1 tablet/10 mL; Roche Applied Science, Indianapolis, IN, USA). Soluble
proteins were collected, and the protein concentration was measured using a BCA protein
assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. The
proteins were separated using 10 or 14% SDS-PAGE and transferred onto Immobilon-P transfer membranes (EMD Millipore, Billerica, MA, USA). The membranes were sequentially incubated with primary and secondary antibodies diluted in Can Get Signal1 solution (Toyobo
Co, Ltd., Osaka, Japan). The protein bands were detected using ECL Prime Western Blotting
Detection Reagent (GE Healthcare, Little Chalfont, USA) or Chemi-Lumi One Super (Nacalai
Tesque Inc., Kyoto, Japan).
Caspase enzymatic activity was measured using a Cell Meter™ Multiplexing Caspase-3/7, -8
and -9 Activity Assay Kit (AAT Bioquest, Inc., Sunnyvale, CA). Cells (5×104 cells/mL) cultured
in black 96-well plates (100 μL/well) were treated with cSBL (H2452: 5 μM; MSTO: 2 μM) for
1, 3, 6, 24, 48, and 72 h in triplicate. Substrate solution (100 μL) was added to each well, andthe contents of the wells were mixed using a plate shaker for 30 sec. The cells were incubated at
37˚C in a 5% CO2 atmosphere for 1 h. The luminescence in each well was measured using Infinite™ 200 PRO and i-control™ software (Tecan Japan Co., Ltd., Kanagawa, Japan).

In vivo experimentH2452 (5×106 cells) and MSTO (2×106 cells) cells were mixed with an equal volume of icecooled Corning™ Matrigel™ Basement Membrane Matrix (Corning, NY, USA), and an aliquot(100 μL) of suspended cells was subcutaneously injected into the lower backs of the mice. 2–4
weeks after inoculation (day 1), mice bearing tumors of 100–150 mm3 in volume were randomly divided into three groups, with 10 mice in each group. Group 1 was injected with 1 mL/kg vehicle (PBS) as the control. Group 2 was daily injected intraperitoneally with 100 mg/kg
pemetrexed dissolved in sterile PBS on days 1–5 and 15–19. The method of administration the
dosage of pemetrexed selected were based on the previously reported maximum tolerated dosage [36,37]. Group 3 was injected intratumorally with 2.5 mg/kg cSBL, twice weekly for four

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In vitro/vivo antitumor effect of cSBL

Fig 1. A schematic overview of the cancer cell injection and drug administration schedule. The cell suspension (100 ȝL/
mouse, H2452; 5î106 cells, MSTO; 2î106 cells) was injected subcutaneously into the lower backs of mice. After 2–4 weeks,
mice bearing tumors of 100–150 mm3 were randomly divided into 3 groups with 10 mice per group. Group 1 was administered
PBS as control. Group 2 was injected intraperitoneally with pemetrexed (100 mg/kg) dissolved in sterile PBS on days 1–5 and
15–19. Group 3 was injected intratumorally with cSBL (2.5 mg/kg) twice per week for 4 weeks. Body weights and tumor sizes
were measured twice per week. The endpoint of experiment was when the tumor diameter exceeded 200 mm3.
https://doi.org/10.1371/journal.pone.0190653.g001

weeks. Body weights and tumor sizes were measured twice weekly. Solid tumor volumes were
calculated as follows: 0.4×A×B2, with A and B representing the long and short tumor diameters(measured in mm), respectively. Tumor growth and body weight changes were evaluated as
the ratio of each value against the baseline (day 1). Fig 1 shows the administration schedule
used in the experiments. Mice were sacrificed by neck dislocation under ether anesthesia whentumor volumes reached 200 mm3.

Drug combination studies
The effect of combination treatment on cell viability was determined using a WST-8 assay.
Cells (5×104 cells/mL) were cultured in 96-well plates (100 μL/well). The concentration ofpemetrexed, cisplatin, or cSBL was based on the IC50 values obtained in the single-treatment
experiments conducted in our prior study [35]. After 72 h, the cells were incubated with Cell
Count Reagent SF (Nacalai Tesque Inc., Kyoto, Japan) at 37˚C in a 5% CO2 atmosphere for
1–4 h. The absorbance of the resulting product at 450 nm was measured, and the backgroundabsorbance at 650 nm was subtracted. Combination Index (CI) and Dose Reduction Index(DRI) values were calculated using CompuSyn software (ComboSyn, Inc., Paramus, NJ), as
described by Chou et al [38]. The experiments were conducted in triplicate. CI = 1 indicatedan additive effect; CI<1 indicated a synergistic effect; CI>1 indicated an antagonistic effect.
DRI = 1 indicated no dose reduction, whereas DRI>1 and <1 indicated favorable and unfavorable dose reductions, respectively.

Statistical analysis
The results from 3 independent experiments, each performed in triplicate, are expressedas the mean ± standard deviation. Statistical analyses were conducted using GraphPad Prism
5.0, and comparisons were made using one-way analysis of variance (ANOVA) or two-way
ANOVA followed by Bonferroni’s post hoc test. A P-value of <0.05 was considered statisticallysignificant.

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In vitro/vivo antitumor effect of cSBL

Results
cSBL induces apoptosis to H2452 and MSTO cells
In order to investigate the antitumor activity of cSBL on xenograft-competent malignant
mesothelioma cells, H2452 and MSTO cells were treated with cSBL and the antitumor mechanisms were analyzed. The percentage of Annexin V-positive cells was significantly increased inboth H2452 (16.13%, 72 h) and MSTO (40.05%, 72 h) cells (Fig 2A and 2B). In addition, chromatin condensation and nuclear collapse were observed in the two cell types treated with cSBL

Fig 2. cSBL induced apoptotic changes in H2452 and MSTO cells. Cells were treated with cSBL for theindicated times. (A, B) Rate of apoptosis as indicated by the percentage of annexin V-positive cells. (C) Nuclear
fragmentation images were captured using 40X objective; a false-colored image of the nuclei (blue) is shown.
White arrowheads indicate the cells with condensed or fragmented nuclei. (D, E) Fragmentation index indicating
the degree of nuclear fragmentation; a higher index indicates greater occurrence of fragmentation, calculatedusing the High-Content Analysis System. All data are expressed as the mean  SD of three independent
experiments. The statistical significance of these experiments compared with the control is shown as follows:*P0.05, **P0.01, ***P0.001.
https://doi.org/10.1371/journal.pone.0190653.g002

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In vitro/vivo antitumor effect of cSBL

(Fig 2C). Alterations to nuclear morphology were detected by High-Content Analysis Systems
and numerically output as fragmentation indexes. As shown in Fig 2D and 2E, cSBL provokedsignificant nuclear morphology changes in time-dependent manner.

cSBL-induced apoptosis is mediated by the intrinsic pathway
To obtain further insight into the mechanisms of cSBL-induced apoptosis in H2452 andMSTO cells, the activation of three key caspases was analyzed chronologically. The expression
levels of activated caspase-9, -8 and -3 were detected by western blotting, and the substantial
enzymatic activities of these caspases were evaluated by fluorometric analysis. As shown in Fig
3A and 3B, increased levels of activated caspase-9 were observed from 6 h and 1 h in H2452
and MSTO cells, respectively. After that, activated caspase-8 began to be observed from 24 h in
H2452 and 6 h in MSTO cells. The appearance of activated caspase-3 was recorded from 48 hin H2452 and 24 h in MSTO cells. Consistently, the enzymatic activity of caspase-9 was significantly enhanced from 1 h in H2452 and MSTO cells, and the levels of caspase-8 and -3/7
increased almost simultaneously (H2452: 48 h; MSTO: 24 h; Fig 3C and 3D). Thus, caspase-9
was activated prior to caspase-8 and -3, indicating that the intrinsic apoptotic pathway was
involved.

cSBL inhibits cancer cell proliferation without inducing weight loss inxenograft models
To examine the effects of cSBL on tumor growth in vivo, nude mice were inoculated with
H2452 and MSTO cells. cSBL was administered intratumorally and the effects of pemetrexed
were also assessed according to previously reported experimental conditions [36,37] (Fig 1).

Fig 3. cSBL induced apoptosis in H2452 and MSTO cells via activation of the caspase pathway. Caspase-3, -8,
and -9 activation was detected by western blotting (A, B) or fluorometry (C, D). Fluorometry was performed
independently three times and data are expressed as the mean  SD. The statistical significance of these experiments
compared with the control is shown in as follows: *P0.05, **P0.01, ***P0.001.
https://doi.org/10.1371/journal.pone.0190653.g003

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In vitro/vivo antitumor effect of cSBL

Fig 4. cSBL showed in vivo cytotoxicity without inducing loss of body weight. Mice were randomly
divided into 3 groups with 10 mice in each group. Groups 1, 2, and 3 were injected PBS, pemetrexed (100 mg/
kg, intraperitoneally), and cSBL (2.5 mg/kg, intratumorally), respectively. Body weights and tumor sizes were
measured twice per week. Tumor volumes were calculated as follows: 0.4îAîB2, where A and B represent
the long and short diameters (in mm) of the tumor, respectively. Relative body weight (A, B) and relative tumor
volume (C, D) are plotted as the mean of each group  SD at each timepoint. The statistical significance of
these experiments compared with PBS (*) or pemetrexed (†) is shown as follows: *P0.05, **P0.01,***P0.001, †P0.01, †††P0.001.
https://doi.org/10.1371/journal.pone.0190653.g004

The rate of changes to animal weights and tumor volumes were monitored following theadministration of each agent. As shown in Fig 4A and 4B, body weight changes were notobserved in any of the groups. In the H2452 xenograft model, cSBL and pemetrexed each significantly inhibited tumor growth compared with the PBS group (P<0.05), and the cSBL
group showed a growth inhibition effect earlier (after 36 days of treatment) than that of thepemetrexed group (after 47 days of treatment) (Fig 4C). Conversely, for the MSTO xenograftgroup, significant inhibition of cancer growth was observed only in the cSBL-treated group
after 29 days of treatment (P<0.05; Fig 4D).

cSBL and pemetrexed exhibit a strong synergistic effect in H2452 andMSTO cells
Finally, we performed the in vitro combination study of cSBL with other reagents. In addition
to pemetrexed, cisplatin, an existing drug for malignant mesothelioma usually used in combination with pemetrexed, was chosen for the test reagent. Pharmacological interactions
between these three agents were investigated by evaluating the viability of H2452 and MSTO
cells treated with pemetrexed + cisplatin, cSBL + pemetrexed, and cSBL + cisplatin. The drugconcentration in each combination regimen was based on the IC50 value for each agent previously determined via single treatments [35]. The viability curves for each drug in single or

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In vitro/vivo antitumor effect of cSBL

Fig 5. Viability curves of H2452 and MSTO cells treated with pemetrexed, cisplatin, and cSBL, either
alone or in combination. Each group of cells was treated with fixed concentration ratios of pemetrexed:
cisplatin:cSBL as follows: 800:100:1 (for H2452 cells) or 3:20:2 (for MSTO cells). Each data point represents
the mean  SD of at least three independent WST-8 assays. Each sample was plated in triplicate. The y-axis
indicates the viability of cells. The x-axis indicates the concentration of pemetrexed (upper), cisplatin (middle),
or cSBL (lower).
https://doi.org/10.1371/journal.pone.0190653.g005

combination treatments are presented in Fig 5. In H2452 cells, all combinations decreased cell
viability to a greater extent than each single treatment over the whole concentration range.
Similar tendencies were observed in MSTO cells over a wide concentration range, although
the combination effects appeared to saturate at a higher concentration. To evaluate the synergistic effect of each drug combination, CI values were calculated. At each experimental concentration (Fig 6A) in H2452 cells, the CI values for all combinations were <1, indicating that
all combinations were synergistic. cSBL + pemetrexed showed the highest synergistic effect at
all concentration points. In MSTO cells, CI values at the highest two concentration points of
all combinations exhibited antagonism rather than synergism; however, cSBL-containing
combinations (cSBL + pemetrexed; cSBL + cisplatin) exhibited high synergism in the mid-low
concentration range. The pemetrexed + cisplatin combination in MSTO cells showed dispersion and high CI values at various concentrations. Furthermore, we calculated DRI values, representing the index of the fold-number that each drug combination dose could be reduced bycompared with that of each drug alone (Fig 6B). In H2452 cells, all combinations had high
DRI values. In MSTO cells, high DRI values were observed in the cSBL-containing combinations, particularly in the low concentration range. However, the pemetrexed + cisplatin combination had low DRI values at all concentrations.

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In vitro/vivo antitumor effect of cSBL

Fig 6. Pharmacological interactions between pemetrexed, cisplatin and cSBL in H2452 and MSTO
cells. (A) CI values of each combination (CI = 1 indicates an additive effect; CI1 indicates a synergistic
effect; and CI!1 indicates an antagonistic effect). (B) DRI values of each reagent (DRI = 1 indicates no dose
reduction; whereas DRI !1 and 1 indicate favorable and unfavorable dose-reductions, respectively). Pem orP, pemetrexed; Cis or C, cisplatin; S, cSBL.
https://doi.org/10.1371/journal.pone.0190653.g006

Discussion
We previously demonstrated that cSBL induces apoptosis in H28 (sarcomatoid histologicaltype), MESO-1 and MESO-4 (epithelioid type) cells, but not in normal Met5A mesothelial
cells, by detecting elevated proportions of Annexin V positive cells following cSBL treatment[34]. Furthermore, from the investigations in which H2452 (epithelioid type) and MSTO
(biphasic type) were utilized, in addition to aforementioned cell lines, higher cancer-selectivityof cSBL was observed compared with either pemetrexed or cisplatin in their antiproliferative
effects [35]. The antitumor mechanism of cSBL in malignant mesothelioma has been well-documented in H28 cells; it was revealed that cSBL treatment activates the caspase cascade, theproapoptotic Bcl-2 family proteins Bik and Bim, as well as JNK and p38 MAPKs, consequently
inducing apoptosis in these cells. However, although the effectiveness of cSBL against mesothelioma in vitro has been reported, the in vivo efficacy of cSBL has not been investigated to date.
Although H28, MESO-1, and MESO-4 cells did not show tumorigenicity in the nude mice
used, we succeeded in establishing malignant mesothelioma xenografts with H2452 andMSTO cells. First, the antitumor effects of cSBL on these two cell lines were investigated invitro. cSBL induced typical apoptotic changes, such as phosphatidylserine externalization,

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In vitro/vivo antitumor effect of cSBL

nuclear condensation and fragmentation, in both cells in a time-dependent manner (Fig 2).
Moreover, caspase-9 was activated by cSBL treatment earlier and more strongly than caspase8, indicating that apoptosis was induced through the intrinsic pathway (Fig 3). In the in vivo
studies, no obvious toxicities or body weight changes were observed during the experimental
period in any group (Fig 4A and 4B). In both types of xenograft, significant tumor growth suppression was observed in cSBL-treated groups compared with control groups. In H2452 xenografted groups, cSBL showed a tumor-suppressive effect earlier than that of the pemetrexedtreated group, and the antitumor effect of pemetrexed was not observed in the MSTO xenografts (Fig 4C and 4D). The reason for the lack of effect by pemetrexed is uncertain; we speculate that the high growth rate of MSTO cells in the xenograft model may contribute to thisphenomenon. We were unable to compare the effects of cSBL and pemetrexed directly due tothe differences in the dosing conditions; however, our observations indicate that cSBL could
potentially inhibit the tumor growth of mesothelioma without any toxicity, even if previously
established pemetrexed administration had little or no effect. From these results, it was suggested that cSBL had the capability to inhibit tumor growth in xenografted mice. Thus, cSBL
may be safely used, and further studies are required to determine the maximal tolerated doseof cSBL in order to optimize its efficacy.
Combination therapy, a treatment modality that combines two or more therapeutic agentsto reduce the risk of drug resistance or adverse effects while simultaneously providing therapeutic anti-cancer benefits, is a mainstay of current cancer therapy [39]. In fact, combination
treatments comprising pemetrexed and cisplatin are used for the treatment of mesothelioma
as a standard regimen. We previously demonstrated that the cSBL + pemetrexed combination
exerted stronger cytotoxicity and synergism compared with the pemetrexed + cisplatin combination in H28 cell lines. The cytostatic effect of pemetrexed and the cytotoxic effect of cSBL
cooperated without any repulsion, although the effects of pemetrexed and cisplatin on cyclin A
expression were counteractive when used in combination [35]. In the present study, we evaluated the generality of the prominent synergistic effect of the cSBL + pemetrexed combination,utilizing H2452 and MSTO cells, by calculating CI and DRI values. The cSBL + pemetrexed
combination exhibited the highest synergism of the three combinations tested in both celllines (Fig 5B). Surprisingly, in MSTO cells, the pemetrexed + cisplatin combination appeared
to be antagonistic rather than synergistic or additive at the most of concentration points tested.
High DRI values (Fig 5C) were calculated for all combinations, except for pemetrexed + cisplatin in MSTO cells. These results suggest that cSBL + pemetrexed may be a rational treatment combination for several types of malignant mesothelioma. On the other hand, thecurrent gold-standard regimen for malignant mesothelioma, pemetrexed + cisplatin, may beineffective, depending on the cell type, with respect to synergism (i.e., undesired adverse effects
may easily occur in some circumstances, depending on the patient).
Although the combination of pemetrexed and cisplatin has been demonstrated to prolong
the survival of patients with malignant mesothelioma, the median survival is only 12 months,
and the response rate is ~40% [12]. Thus, almost half of all mesothelioma patients are initiallyresistant, and all eventually develop resistance [40]. Therefore, researches to improve themalignant mesothelioma therapy have been actively attempt. The combinations of carboplatin
and pemetrexed, or gemcitabine and cisplatin showed comparable outcomes with pemetrexed
and cisplatin combination in phase- II trials [41–43]. The French Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS) demonstrated a statistically significant improvement in the
median overall survival time using a combination of cisplatin, pemetrexed and bevacizumab, amonoclonal antibody that binds VEGF and blocks its interaction with the VEGF receptor [44].
In addition, several other molecular targeting and immunotherapeutic agents, such as antiEGFR signaling agent and anti-programmed cell death 1 (PD-1) antibodies, are currently

PLOS ONE | https://doi.org/10.1371/journal.pone.0190653 January 3, 201811 / 15

In vitro/vivo antitumor effect of cSBL

being investigated in clinical trials [43]. In this study, cSBL was demonstrated to induce apoptosis and inhibit tumor cell growth in xenografted mice. CI analysis also evidenced a prominent combinatory effect of cSBL with pemetrexed. As cSBL is a novel candidate anti-cancer
agent that exerts antitumor activity through targeting RNA (which represents a novel class ofpotential therapeutic targets), it may provide a new option for the chemotherapeutic treatmentof malignant mesothelioma. This is particularly true among patients with pemetrexed resistance, as cSBL was effective in pemetrexed-resistant cells, as shown in the current study and inprevious report [35].

Conclusions
cSBL induced apoptosis in H2452 and MSTO cells via the intrinsic apoptotic pathway. In vivo,cSBL treatment inhibited tumor growth in multiple xenograft models without any undesirable
adverse effects. A higher efficacy was achieved by the use of cSBL + pemetrexed in mesothelioma cells compared with pemetrexed + cisplatin. To the best of our knowledge, this is the firstreport to demonstrate the antitumor efficacy of cSBL in human malignant mesothelioma
xenograft models. cSBL has potential as a novel treatment for malignant mesothelioma.

Author Contributions
Conceptualization: Takeo Tatsuta, Masahiro Hosono.
Data curation: Takeo Tatsuta, Toshiyuki Satoh, Masahiro Hosono.
Formal analysis: Takeo Tatsuta, Toshiyuki Satoh.
Funding acquisition: Takeo Tatsuta, Akiyoshi Hara, Masahiro Hosono.
Investigation: Takeo Tatsuta, Toshiyuki Satoh, Shigeki Sugawara.
Methodology: Takeo Tatsuta, Toshiyuki Satoh, Shigeki Sugawara.
Project administration: Akiyoshi Hara, Masahiro Hosono.
Resources: Takeo Tatsuta, Toshiyuki Satoh, Shigeki Sugawara.
Supervision: Akiyoshi Hara, Masahiro Hosono.
Validation: Takeo Tatsuta, Masahiro Hosono.
Visualization: Takeo Tatsuta, Toshiyuki Satoh, Shigeki Sugawara.
Writing – original draft: Takeo Tatsuta, Toshiyuki Satoh.
Writing – review & editing: Takeo Tatsuta, Toshiyuki Satoh, Masahiro Hosono.

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