ࡈᣵᣜ ᖹᡂ㸱㸮ᖺ㸦2018 ᖺ㸧ࡣࠊ࠾࠿ࡆࡉࡲ࡛➨㸱㸵ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍ࢆ௝ྎ࡛
↓஦㛤ദ࡛ࡁࡲࡋࡓࡇ࡜ࠊࡇࢀࡶࡦ࡜࠼࡟ⓙᵝ࠿ࡽࡢࡈຓຊ࣭ࡈᨭ᥼ࢆ࠸ࡓࡔ࠸
ࡓ㈷≀࡜ࠊᨵࡵ࡚ឤㅰ⏦ࡋୖࡆࡲࡍࠋ⩣ᖺࡢᖹᡂ㸱㸯ᖺࡣࠊ㸳᭶㸯᪥࡟ඖྕࡀ௧
࿴࡜࡞ࡾࠊᕼᮃࢆᣢࡗ࡚᪂ࡓ࡞᫬௦ࡢᖥ㛤ࡅࢆ㏄࠼ࡲࡋࡓࠋࡑࡢ⣙༙ᖺᚋࡢ 11
᭶࡟Ⓨ⏕ࡋࡓ᪂ᆺࢥࣟࢼ࢘࢖ࣝࢫ(COVID-19)ࡣࠊ࠶ࡗ࡜࠸࠺㛫࡟ୡ⏺୰ࢆそ࠸ᑾࡃࡋࠊ་⒪ᔂቯࡸ⤒῭άື࡬ࡢ⏒኱࡞⿕ᐖࡢࡳ࡞ࡽࡎࠊ኱Ꮫࡢᩍ⫱࣭◊✲άືࡶ኱ࡁ࡞ᙳ㡪ࢆཷࡅ࡚ࠊ⌧ᅾ࡟⮳ࡗ࡚࠸ࡲࡍࠋCOVID-19 ࡣࠊᑡ࡞ࡃ࡜ࡶ௒ᚋ
ᩘᖺ࡟ࢃࡓࡗ࡚ே㛫♫఍࠿ࡽᾘ࠼ࡿࡇ࡜ࡣ࡞࠸࡛ࡋࡻ࠺ࠋே㛫࡜㔝⏕ື≀ࡀ᥋ゐࡍࡿᶵ఍ࡀቑ࠼ࠊࡇࡢࡼ࠺࡞඲ࡃணᮇ࡛ࡁ࡞࠸ࣃࣥࢹ࣑ࢵࢡࡀ㉳ࡇࡾࡸࡍࡃ࡞ࡗ
࡚࠸ࡿࡑ࠺࡛ࡍࠋ⮫ᗋ⑕≧ࡢண

ࡸ἞⒪ἲࡀ↓࠸ឤᰁ⑕ࡢ๓࡟ࠊᆅ⌫࡟࠾ࡅࡿᡃࠎ⮬㌟ࡢ⏕άࡢ࠶ࡾ᪉ࡢኚ㠉ࡀ㏕ࡽࢀ࡚࠸ࡲࡍࠋୡ⏺୰ࡢேࠎࡀࠊࡇࡢ᫬ࡔ࠿
ࡽࡇࡑᆅ⌫ୖࡢ࠶ࡾ࡜࠶ࡽࡺࡿ⏕≀ࡢࡓࡵ࡟ே㢮ࡀ࡞ࡍ࡭ࡁࡇ࡜ࡣఱ࠿ࢆ⮬ศ⮬㌟࡟ၥ࠸࠿ࡅࠊồࡵ࡚⾜ືࡍࡿࡇ࡜࡛ࠊࡼࡾⰋ࠸ᮍ᮶ࡢᆅ⌫ࡀ๰㐀ࡉࢀ࡚࠸ࡃ࡜
ಙࡌ࡚࠸ࡲࡍࠋ
௧࿴ඖᖺࡢ 2019 ᖺᗘ࡟ࡣࠊ⌮໬Ꮫ◊✲ᡤ࠿ࡽ᪂ࡓ࡟ᒣཱྀⰾᶞ༤ኈࡀศᏊ⏕య⭷◊✲ᡤࡢᩍᤵ(⸆ရ≀⌮໬Ꮫᩍᐊ)࡜ࡋ࡚╔௵ࡉࢀࠊ႐ࡤࡋ࠸ࡇ࡜࡟⌧ᅾࡢ㸲㒊㛛࠿ࡽ㸳㒊㛛యไ࡜࡞ࡾࡲࡋࡓࠋᮾ໭་⛉⸆⛉኱ᏛࡢศᏊ⏕య⭷◊✲ᡤࡀࠊ᪥ᮏ
ࡢ⢾㙐⏕≀Ꮫ◊✲ࡢⓎᒎ࡟ࠊࡇࢀࡲ࡛௨ୖ࡟㈉⊩࡛ࡁࡿࡇ࡜ࢆࠊ௝ྎࡢᆅ࠿ࡽᡤ
ဨ୍ྠ㢪ࡗ࡚ࠊ◊✲࡟໅ࡋࢇ࡛࠾ࡾࡲࡍࠋ
௒ᅇࡢᖺሗ࡛ࡣࠊྛ◊✲㒊㛛ࡢ◊✲άືሗ࿌࠾ࡼࡧ➨ 3 ᅇ⟽Ᏺ௝୍㑻㈹ࡢዡບ
㈹࠾ࡼࡧඃ⚽ㄽᩥ㈹ࡢࡈ⤂௓ࢆ࠸ࡓࡋࡲࡍࠋⓙᵝ࠿ࡽࡢ࡞࠾୍ᒙࡢࡈᣦᑟࠊࡈ㠴᧡ࡢ࡯࡝⾺ᚰࡼࡾ࠾㢪࠸⏦ࡋ࠶ࡆࡲࡍࠋ
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ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤ࣭ᡤ㛗ᮾ໭⢾㙐◊✲఍఍㛗஭ࣀཱྀ ோ୍

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ᡤ㛗ᣵᣜ࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸯⟽Ᏺ௝୍㑻㈹࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸲ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤࠕ⟽Ᏺ௝୍㑻㈹ࠖつ⣙௧࿴ඖᖺᗘ ⟽Ᏺ௝୍㑻㈹
ዡບ㈹    ఀᕥ἞ ▱ᘺ㸦ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤ⣽⬊ไᚚᏛᩍᐊ㸧ඃ⚽ㄽᩥ㈹  బ㔝 ຍⱑ㸦⩌㤿኱Ꮫ⌮ᕤᏛᗓ≀㉁࣭⏕࿨⌮ᕤᏛ㡿ᇦ༤ኈᚋᮇㄢ⛬஧ᖺ㸧ඃ⚽ㄽᩥ㈹  ஧⎼ ΅       㸦኱㜰኱Ꮫ኱Ꮫ㝔⌮Ꮫ◊✲⛉໬Ꮫᑓᨷ≉௵◊✲ဨ㸧◊✲ሗ࿌࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭࣭㸯㸱ᶵ⬟⑓ែศᏊ㒊㛛ࠕ⢾ᒀ⑓ᛶ⭈⑕࡟࠾ࡅࡿࢢࣟ࣎⣔⢾⬡㉁ࡢ⑓ែ⏕⌮Ꮫⓗᙺ๭ࠖ஭ࣀཱྀ ோ୍ࠊ✄᳃ ၨ୍㑻ࠊỌ⚟ ṇ࿴ࠊ⊁㔝 ⿱⪃ࠊ᪂⏣ ᪸኱⏕య⭷᝟ሗ㒊㛛
ࠕGࢱࣥࣃࢡ㉁ඹᙺཷᐜయ㸦GPCR㸧࡟ࡼࡿ⢾㙐ྜᡂไᚚ࡜⢾㙐࡟ࡼࡿ
GPCRࡢάᛶไᚚࠖᮾ ⚽ዲࠊ୰ᕝ ဴேࠊ㯮⏣ ႐ᖾ⣽⬊ไᚚ㒊㛛
ࠕ⢾㙐ಟ㣭ࡢ≉␗ᛶ࡜ཷᐜయࡢᶵ⬟ไᚚࠖ㢳 ᘓᅜࠊ⚟⏣ ཭ᙪࠊఀᕥ἞ ▱ᘺศᏊㄆ㆑㒊㛛
ࠕࣞࢡࢳࣥࡢᢠ⭘⒆࣓࢝ࢽࢬ࣒ࡢゎ᫂࡜ࡀࢇ⸆≀἞⒪࡬ࡢᛂ⏝ࠖ⣽㔝 㞞♸ࠊⳢཎ ᰤ⣖ࠊ❧⏣ ᓅ⏕⸆ရ≀⌮໬Ꮫ㒊㛛ࠕ⢾㙐ᵓ㐀ゎᯒࡢࡓࡵࡢᢏ⾡ᇶ┙ࡢᵓ⠏ࠖᒣཱྀ ⰾᶞࠊ┿㘠 ἲ⩏ࠊ኱㔝 リṌ3ᮾ໭་⛉⸆⛉኱ᏛศᏊ⏕య⭷◊✲ᡤࠕ⟽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௚
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஦ົᒁࡣศᏊ⏕య⭷◊✲ᡤෆ࡟⨨ࡃࠋ
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㸦㸯㸧ᛂເ㈨᱁࠾ࡼࡧᛂເ᪉ἲ  ᮾ໭ 6 ┴ࠊ᪂₲┴࠾ࡼࡧ⩌㤿┴㸦ᮾ໭⢾㙐◊✲఍࢚ࣜ࢔㸧࡛◊✲ࢆ⾜ࡗ
࡚࠸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∧ࠊࡲࡓࡣᥖ㍕ࡀỴᐃࡉࢀ࡚࠸ࡿཎⴭㄽᩥ㸦accepted ࡛ࡶྍ㸧࡜ࡋࠊᛂເ⪅ࡀ➹㢌ⴭ⪅࡜࡞ࡗ࡚࠸ࡿࡶࡢ࡜ࡍࡿࠋせ᪨࿴ヂ࡜ㄽᩥ PDF ࢆῧ࠼࡚᪤ᐃࡢ࢚
ࣥ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࡟ཧຍ
ࡋ࡞࠸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
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ホ౯᭱㧗Ⅼ
⊂๰ᛶ   
10 Ⅼ࢖ࣥࣃࢡࢺ㸦㔜せᛶ㸧 10 Ⅼㄽᩥᵓᡂ  
10 Ⅼᑗ᮶ᛶ   
10 Ⅼ

 Impact factor 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5௧࿴ඖᖺᗘ ⟽Ᏺ௝୍㑻㈹㸦Sen-itiroh Hakomori Glycoscience Award㸧⡿ᅜ࣡ࢩࣥࢺࣥ኱Ꮫྡ㄃ᩍᤵࡢ⟽Ᏺ௝୍㑻༤ኈࡣ㸪ᮏᏛศᏊ⏕య⭷◊✲ᡤࡢྡ㄃ᡤ㛗࣭㢳ၥࢆ㛗ᖺົࡵࡽࢀ࡚࠾ࡾࡲࡍࠋ2017 ᖺ࡟⟽Ᏺඛ⏕ࡀ㸶㸶ṓࢆ㏄࠼ࡽࢀࡓࡇ࡜ࢆグᛕࡋ࡚㸪ࡲࡓඛ⏕ࡢ⢾㙐⏕࿨⛉Ꮫ◊✲ศ㔝࡟࠾ࡅࡿୡ⏺ⓗ࡞ࡈຌ⦼ࢆ㛗ࡃᚋୡ࡟ఏᢎࡍ࡭ࡃ㸪ศᏊ⏕య⭷◊✲ᡤ࡛ࡣᚋ㐍◊✲⪅ࡢ⫱ᡂࢆ┠ⓗ࡜ࡋࡓࠕ⟽Ᏺ௝୍㑻㈹ࠖ㸦Sen-itiroh Hakomori Glycoscience Award㸧ࢆ๰タ⮴ࡋࡲࡋࡓࠋᑐ㇟ࢆᮏᏛෆ࡟␃ࡵࡎᗈࡃᮾ໭࢚ࣜ࢔ࡢ⢾㙐⛉Ꮫࡢ᣺⯆ࢆ┠ⓗ࡜ࡋ㸪ᮾ໭⢾㙐◊✲఍㸦http://tohokut-tousa.strikingly.com㸧ࡢୡヰே࡟ࡼࡿཝṇ࡞ᑂᰝࡢ⤖ᯝ㸪➨㸱ᅇ⟽Ᏺ௝୍㑻㈹ࡢ◊✲ዡບ㈹ཬࡧඃ⚽ㄽᩥ㈹ࡢཷ㈹⪅ࡀỴᐃࡋ㸪᪂₲࡛⾜ࢃࢀࡓ➨㸯㸱ᅇᮾ໭⢾㙐◊✲఍࡛ᤵ㈹ᘧཬࡧዡບ㈹ཷ㈹ㅮ₇ࡀ⾜ࢃࢀࡲࡋࡓࠋ
➨ 3 ᅇ⟽Ᏺ௝୍㑻㈹ዡບ㈹ఀᕥ἞ ▱ᘺ
ᮾ໭་⛉⸆⛉኱Ꮫ ศᏊ⏕య⭷◊✲ᡤ ⣽⬊ไᚚᏛᩍᐊ
A complex between phosphatidylinositol 4 kinase IIĮ and integrin Į3ȕ1 is required for
N-glycan sialylation in cancer cells
ඃ⚽ㄽᩥ㈹
బ㔝 ຍⱑ
⩌㤿኱Ꮫ⌮ᕤᏛᗓ ≀㉁࣭⏕࿨⌮ᕤᏛ㡿ᇦ ༤ኈᚋᮇㄢ⛬஧ᖺ
Fluorescence quenching-based assay for measuring Golgi endo-Į-mannosidase஧⎼ ΅኱㜰኱Ꮫ኱Ꮫ㝔 ⌮Ꮫ◊✲⛉ ໬Ꮫᑓᨷ ≉௵◊✲ဨ
NPC1L1-dependent intestinal cholesterol absorption requires ganglioside GM3 inmembrane microdomains

6

ዡບ㈹A complex between phosphatidylinositol 4 kinase IIǂ and integrin ǂ3ǃ1
is required for N-glycan sialylation in cancer cells.
ఀᕥ἞ ▱ᘺ㸦ᮾ໭་⛉⸆⛉኱Ꮫ ศᏊ⏕య⭷◊✲ᡤ ⣽⬊ไᚚᏛ࣭ㅮᖌ㸧ᡃࠎࡢࢢ࣮ࣝࣉࡣࡀࢇ⣽⬊࡛⏕ࡌࡿ␗ᖖ࡞⢾㙐ኚ໬ࡢ࣓࢝ࢽࢬ࣒࡟ὀ┠ࡋ࡚࠸ࡿࠋࡇࢀࡲ࡛࡟ࠊࡀࢇࡢணᚋ࡜┦㛵ࡍࡿ㑇ఏᏊ Golgi phosphoprotein 3
㸦GOLPH3㸧
GOLPH3 ࡀࢩ࢔ࣝ㓟㌿⛣㓝⣲࡜⤖ྜࡍࡿࡇ࡜ࡀຠ⋡ⓗ࡞ N-ᆺ⢾㙐ࡢࢩ࢔ࣜࣝ໬࡟㔜せ࡛࠶ࡿࡇ࡜ࢆሗ࿌ࡋࡓ㸦Isaji, et. al., JBC. 2014㸧ࠋGOLPH3 ࡢୖὶ࡜ࡋ࡚
ᑠ⬊㍺㏦ࡢㄪ⠇ᅉᏊ࡛࠶ࡿ࣍ࢫࣇ࢓ࢳࢪࣝ࢖ࣀࢩࢺ࣮ࣝ㸲࢟ࢼ࣮ࢮ IIǂ㸦PI4KIIǂ㸧ࡀண᝿ࡉࢀࡓࠋࡋ࠿ࡋࠊPI4KIIǂ ࡀࢩ࢔ࣜࣝ໬ࢆ࡝ࡢࡼ࠺࡟ㄪ⠇ࡉࢀࡿࡢ࠿ࡣ୙
࡛᫂࠶ࡗࡓࠋࢩ࢔ࣜࣝ໬ N-ᆺ⢾㙐ࡸ⣽⬊⛣ືࡢㄪ⠇ࢆゎᯒࡍࡿࡓࡵ࡟ࠊPI4KIIǂ
ࡢⓎ⌧ࢆᢚไ࡛ࡁࡿㄏᑟᆺࣀࢵࢡࢲ࢘ࣥ(KD)ࡢங⒴⣽⬊ࢆᶞ❧ࡋࡓࠋண᝿㏻ࡾࠊ
KD ⣽⬊࡛ࡣ PI4P 㔞ࡀⴭࡋࡃᢚไࡉࢀ࡚࠸ࡓࠋࡲࡓࠊKD ⣽⬊ࡢ⣽⬊⾲㠃࡟࠾࠸࡚ࢩ࢔ࣜࣝ໬⢾㙐ࢆㄆ㆑ࡍࡿࣞࢡࢳࣥࡢ཯ᛂᛶࡀపୗࡋࠊࡉࡽ࡟ࠊHPLC ࡛ᐃ㔞ࡍࡿ࡜ࠊ」ྜᆺ⢾㙐࡜ẚ࡭ࠊࢩ࢔ࣜࣝ໬⢾㙐ࡀⴭࡋࡃῶᑡࡋ࡚࠸ࡓࠋࡲࡓࠊ࢖ࣥ
ࢸࢢࣜࣥ ǃ1 ࡢࢩ࢔ࣜࣝ໬⢾㙐ࡶྠᵝ࡟పୗࡋ࡚࠸ࡓࠋAKT ࡢࣜࣥ㓟໬࡜࢖ࣥࢸࢢࣜࣥ ǂ3 ౫Ꮡⓗ࡞⣽⬊⛣ືࡣ KD ⣽⬊࡟࠾࠸࡚పୗࡋࡓࡶࡢࡢࠊ⣽⬊⾲㠃ࡢ࢖ࣥࢸࢢࣜࣥศᏊ✀ࡢⓎ⌧㔞ࡣ control ⣽⬊࡜ኚࢃࡽ࡞࠿ࡗࡓࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊ
࢖ࣥࢸࢢࣜࣥ ǂ3ǃ㸯࠾ࡼࡧ PI4KIIǂ ࡣඹᒁᅾࡋࠊࡉࡽ࡟ࠊ࢖ࣥࢸࢢࣜࣥ ǂ3ǃ1 ࠾
ࡼࡧ PI4KIIǂ ࡢ୧᪉ࡢ㐣๫Ⓨ⌧ࡣࠊ㧗ࢩ࢔ࣜࣝ໬ࢆㄏᑟࡋࡓࠋ୍᪉࡛ࠊ࢖ࣥࢸࢢ
ࣜࣥ ǂ3 㑇ఏᏊࡢḞᦆ࡟ࡼࡗ࡚ࠊ⣽⬊඲యࡢࢩ࢔ࣜࣝ໬ࡀ᭷ព࡟పୗࡋࡓࠋ௨ୖ࠿
ࡽࠊங⒴⣽⬊ࡢ㌿⛣•ᾐ₶࡜࠸ࡗࡓᝏᛶ⾲⌧ᆺࡣࠊPI4KIIǂ ࡜࢖ࣥࢸࢢࣜࣥ ǂ3ǃ㸯࡜ࡢ㛫ࡢ」ྜయ࡟ࡼࡗ࡚ࢩ࢔ࣜࣝ໬ࡀቑຍࡍࡿࡇ࡜࡟ࡼࡗ࡚ࠊಁ㐍ࡉࢀࡿ࡜࠸࠺඲ࡃ᪂つ࡞ศᏊᶵᵓࢆᥦ♧ࡋࡓࠋཧ⪃ᩥ⊩
Isaji T, Im S, Kameyama A, Wang Y, Fukuda T, Gu J.
J Biol Chem. 294, 4425-4436 (2019)
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7


࠙⤒Ṕࠚ
ᖹᡂ 12 ᖺ㸸ᒱ㜧⸆⛉኱Ꮫ⸆Ꮫ㒊༞ᴗ㸪ᖹᡂ 14 ᖺ㸸ᒱ㜧⸆⛉኱Ꮫ኱Ꮫ㝔⸆Ꮫ◊✲⛉༤ኈ๓ᮇㄢ⛬ಟ஢㸪ᖹᡂ 18 ᖺ㸸኱㜰኱Ꮫ኱Ꮫ㝔་Ꮫ⣔◊✲⛉༤ኈᚋᮇㄢ⛬ಟ஢㸦༤ኈ࣭་Ꮫ㸧㸪ྠᖺᮾ໭⸆⛉኱Ꮫ⣽⬊ไᚚᏛຓᡭ㸪ᖹᡂ 20 ᖺ㸸ྠ ຓᩍ,ᖹᡂ 28 ᖺ: ྠ ㅮᖌ㸦ᖹᡂ 28 ᖺ ኱Ꮫྡࡀᮾ໭་⛉⸆⛉኱Ꮫ࡟ᨵ⛠㸧ࠋ⌧ᅾࠊ⢾㙐ࡢไᚚᶵᵓ࡟㛵ࡍࡿ◊✲࡟ᚑ஦ࠋ
࠙ཷ㈹ࡢឤ᝿࡜ᢪ㈇ࠚ
ࡇࡢᗘࠊ௧࿴ඖᖺ⟽Ᏺ௝୍㑻㈹ዡບ㈹ࢆ㡬ࡁࡲࡋࡓࠋ⟽Ᏺඛ⏕ࡣࡌࡵᮾ໭⢾㙐◊✲఍ࡢඛ⏕᪉࡟῝ࡃឤㅰ⏦ࡋୖࡆࡲࡍࠋ⢾㙐◊✲ࢆጞࡵࡓࡁࡗ࠿ࡅࡣࠊ⣽⬊ࡢ◊✲ࡀࡋࡓ࠸࡜ࠊ༤ኈㄢ⛬࡛኱㜰኱Ꮫ࣭㇂ཱྀ┤அඛ⏕ࡢᩍᐊࡢ㛛ࢆ྇࠸ࡓࡇ࡜࡛ࡋ
ࡓࠋಟኈㄢ⛬ࡲ࡛ࡣࠊᒱ㜧⸆⛉኱Ꮫࡢཎ᫂ඛ⏕ࡢࡶ࡜ࠊ⢾௦ㅰ㓝⣲ࡢ㏿ᗘㄽゎᯒࢆࡋ࡚࠸ࡲࡋࡓࠋ㇂ཱྀ◊࡛ࡣ⌧ᅾࡢୖྖ࡛ࡶ࠶ࡿ㢳ᘓᅜඛ⏕ࡢୗࠊ᥋╔ศᏊ࢖ࣥࢸࢢࣜࣥࡢ GnT-III ࡟ࡼࡿಟ㣭ࢆぢ࠸ࡔࡋࡲࡋࡓࠋ⢭〇ࡋࡓ࢖ࣥࢸࢢࣜࣥࡢᛶ㉁ࢆẚ࡭࡚⢾㙐ᵓ㐀ࡢ㐪࠸࡟ࡼࡗ࡚ࣜ࢞ࣥࢻ⤖ྜ⬟ࡀኚ໬ࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡲ
ࡋࡓࠋ⌧ᡤᒓ࡟⛣ࡗ࡚࠿ࡽࡣࠊ࢖ࣥࢸࢢࣜࣥࡢ௜ຍ㒊఩ࡢྠᐃ࡜ᶵ⬟ゎᯒࢆ⾜ࡗ࡚ࡁࡲࡋࡓࠋ⢾㙐ࡢⓎ⌧ᶵᵓ࡟⯆࿡ࡀ࠶ࡗ࡚ࠊGOLPH3 ࡟ࡼࡿࢩ࢔ࣝ㓟㌿⛣㓝⣲ࡢไᚚᶵᵓࡸ PI4KIIǂ ࡟ࡼࡿ⢾㙐ไᚚࡢ◊✲࡟ࢩࣇࢺࡋ࡚ࡁࡲࡋࡓࠋࡲࡔࡲࡔࠊ
⢾㌿⛣㓝⣲࡟ࡣ୙᫂࡞㒊ศࡀከ࠸࡛ࡍࡀࠊࡦ࡜ࡘࡦ࡜ࡘ᫂ࡽ࠿࡟ࡋ࡚࠸ࡁࡓ࠸࡜
⪃࠼࡚࠸ࡲࡍࠋࡇࡢ◊✲ࢆ⾜࠺࡟࠶ࡓࡾࠊ᭱ᚋ࡟Ꮫ⏕ࡢ㡭࠿ࡽࡢᣦᑟᩍဨ࡛࠶ࡾ
⌧ᡤᒓࡢୖྖ࡛ࡶ࠶ࡿ㢳ᘓᅜඛ⏕ࢆࡣࡌࡵࠊࡈᣦᑟ࣭ࡈ༠ຊ࠸ࡓࡔࡁࡲࡋࡓඹⴭ
⪅ࡢඛ⏕᪉࣭኱Ꮫ㝔⏕ࠊࡈᨭ᥼ࡃࡔࡉࡗࡓ◊✲ᐊࡢ࣓ࣥࣂ࣮࡟ᚰࡼࡾឤㅰ⏦ࡋୖࡆࡲࡍࠋ㧗ྡ࡞⟽Ᏺඛ⏕ࡢྡࢆෙࡋࡓ㈹ࡢཷ㈹ࢆບࡳ࡟ࠊ⢾㙐ࡢไᚚ࡟ࡘ࠸࡚ࠊ
ங⒴௨እࡢࡀࢇ࡟࠾࠸࡚ࡶᶵ⬟ࡋ࡚࠸ࡿ࠿᫂ࡽ࠿࡟ࡋ࡚࠸ࡁࡓ࠸࡜⪃࠼࡚࠸ࡲࡍࠋ
⢾㙐⛉Ꮫࡢࡉࡽ࡞ࡿⓎᒎ࡟ᑡࡋ࡛ࡶ㈉⊩࡛ࡁࡿࡼ࠺࡟ᘬࡁ⥆ࡁ⢭㐍ࡋ࡚ࡺࡁࡲࡍࠋ
௒ᚋ࡜ࡶࡈᣦᑟࡈ㠴᧡ࡢ࡯࡝ࡼࢁࡋࡃ࠾㢪࠸⏦ࡋୖࡆࡲࡍࠋ
ఀᕥ἞ ▱ᘺ8ඃ⚽ㄽᩥ㈹Fluorescence quenching-based assay for measuring Golgi endo-ǂmannosidase
Kanae Sano, Taiki Kuribara, Nozomi Ishii, Ayumi Kuroiwa,
Toshitada Yoshihara, Seiji Tobita, Kiichiro Totani, and Ichiro Matsuo
Chemistry an Asian Journal, 2019, 14, 1965-1969.
DOI: 10.1002/asia.201900240
బ㔝 ຍⱑ (⩌㤿኱Ꮫ⌮ᕤᏛᗓ ≀㉁࣭⏕࿨⌮ᕤᏛ㡿ᇦ ༤ኈᚋᮇㄢ⛬஧ᖺ)
Golgi endo-ǂ-mannosidase㸦G-EM㸧ࡣࢩࢫࢦࣝࢪ࡟ᒁᅾࡍࡿ࢚ࣥࢻᆺࡢ⢾ຍỈศゎ㓝⣲࡛ࠊ࢔ࢫࣃࣛࢠࣥ⤖ྜᆺ⢾㙐 A-arm ᵓ㐀୰ࡢ Glc1~3Man1 ṧᇶࢆษ᩿ࡍ
ࡿࠋࡇࡢᇶ㉁≉␗ᛶ࠿ࡽࠊG-EM ࡣṇᖖ࡞⢾㙐ࣉࣟࢭࢩࣥࢢࢆཷࡅ࡚࠸࡞࠸ࡲࡲ
ᑠ⬊య࠿ࡽࢦࣝࢪయ࡬ㄗࡗ࡚㍺㏦ࡉࢀࡓ⢾ࢱࣥࣃࢡ㉁ୖࡢ⢾㙐ࢆṇᖖ࡞⢾㙐ࣉࣟࢭࢩࣥࢢ㐣⛬࡬࡜ᡠࡍᙺ๭ࢆᢸ࠺ࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿࠋᮏ◊✲࡛ࡣ⣽⬊ෆࡢ GEM άᛶࢆࣜ࢔ࣝࢱ࢖᳨࣒࡛ฟࡍࡿࡇ࡜ࢆᚿྥࡋࠊFRET ᾘගࡢゎᾘࢆసືཎ⌮࡜ࡋࡓ⢾㙐ࣉ࣮ࣟࣈࢆࢹࢨ࢖ࣥࡋࡓࠋࡍ࡞ࢃࡕ G-EM ࡀㄆ㆑ࡍࡿ Glc1Man3 ᵓ㐀ࢆྜᡂᚋࠊFRET ࣌࢔࡜ࡋ࡚㠀㑏ඖᮎ➃ഃ࡟⺯ගᛶ⨨᥮ᇶࠊ㑏ඖᮎ➃ഃ࡟ᾘගᛶ
⨨᥮ᇶࢆᑟධࡋࡓࠋᚓࡽࢀࡓࣉ࣮ࣟࣈ࡟ࡼࡾ G-EM άᛶ᳨ฟࢆ⾜ࡗࡓ࡜ࡇࢁࠊ㓝⣲཯ᛂࡢ㐍⾜࡟క࠸⺯ගᙉᗘࡀቑຍࠊG-EM άᛶࢆ ᐃ࡛ࡁࡿࡇ࡜ࢆ☜ㄆࡋࡓࠋࡉࡽ࡟ࠊࣉ࣮ࣟࣈࡢ㠀㑏ඖᮎ➃ഃ࡟⺯ගᛶ⨨᥮ᇶࢆᑟධࡋࡓࡇ࡜࡛ ǂ-glucosidase⪏ᛶࢆ௜୚ࡍࡿࡇ࡜ࡶ࡛ࡁࡓࠋᮏ◊✲ࡣࠊChemistry an Asian Journal ࡢ very
important paper ࡟㑅ฟࡉࢀࡓࠋ
࠙ཷ㈹ឤ᝿࡜ᢪ㈇ࠚ
ࡇࡢᗘࡣ௧࿴ඖᖺᗘ⟽Ᏺ௝୍㑻ඃ⚽ㄽᩥ㈹ࢆ㈷ࡾගᰤ࡟Ꮡࡌࡲࡍࠋ㑅⪃ጤဨࡢඛ⏕᪉࡟ཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ௒ᅇࡢᏛ⾡ㄽᩥࡣࠊ⚾ࡀ➨୍ⴭ⪅࡜ࡋ࡚ฟ∧ࡉࢀࡓே⏕ึࡢㄽᩥ࡛ࠊ⚾࡟࡜ࡗ࡚グᛕࡍ࡭ࡁᮏㄽᩥࢆཷ㈹ᑐ㇟࡜ࡋ࡚㑅ฟࡋ࡚࠸ࡓ
ࡔࡅࡓࡇ࡜ࡣㄔ࡟ᖾ㐠࡞ࡇ࡜࡜ᛮࡗ࡚࠸ࡲࡍࠋ
⚾ࡣࠊ⩌㤿኱Ꮫ ᯇᑿ୍㑻ᩍᤵࡢࡈᣦᑟࡢࡶ࡜ࠊ⢾㙐ࡢ໬Ꮫྜᡂࢆᇶ┙࡜ࡋࠊ⢾ຍỈศゎ㓝⣲ࡢάᛶ ᐃࣉ࣮ࣟࣈࡢྜᡂࡸ࢔ࢫࣃࣛࢠࣥ⤖ྜᆺ⢾㙐ࡢ඲ྜᡂ◊✲ࢆ⾜࡞ࡗ࡚࠸ࡲࡍࠋ⢾㙐ྜᡂ࡛ࡣᚲせ࡞༢⢾ㄏᑟయࢆᩘⓒࢢ࣒ࣛࢫࢣ࣮࡛ࣝ኱⫹࡟ྜᡂࡍࡿ୍᪉࡛ࠊఱẁ㝵࡟ࡶࢃࡓࡗ࡚┠ⓗ≀ࡲ࡛ࡼ࠺ࡸࡃࡓ࡝ࡾ╔ࡁࠊࢃࡎ࠿࡞
㔞ࡢ࢜ࣜࢦ⢾ࢆៅ㔜࡟ᢅ࠺ሙ㠃࡟ࡶ┤㠃ࡋࡲࡍࠋ⢾㙐ㄏᑟయࡢಖㆤᇶࢆ㝖ཤࡍࡿᕤ⛬࡟࠾࠸࡚ࡣࠊ໬ྜ≀ࡢぶỈᛶ࣭␯Ỉᛶࡀ኱ࡁࡃኚ໬ࡍࡿࡓࡵࠊ᫬࡟ࡣㄏᑟయ
ࡢᛶ≧ࡢኚ໬࡟ᑐᛂࡋࡁࢀࡎࠊࡏࡗ࠿ࡃྜᡂࡋࡓㄏᑟయࢆᾘኻࡍࡿ࡜࠸࠺ࡇ࡜ࡶ
㉳ࡁࡲࡍࠋ⚾ࡣࡑࡢࡼ࠺࡞ⱞ࠸ᛮ࠸ࡶ⤒㦂ࡋࡘࡘࠊ⮬ศࡢ᭱⤊┠ⓗ≀࡬࡜ࡓ࡝ࡾ

9╔ࡃࡓࡵ࡟ࡣࠊ཯ᛂࡢ✀㢮࡜ࢫࢣ࣮ࣝ࡞࡝≧ἣ࡟ᛂࡌࡓᰂ㌾ᛶࡀᚲせ࡞ࡢࡔ࡜ᖖࠎឤࡌࠊᴦࡋࡳ࡞ࡀࡽ᪥ࠎዧ㜚ࡋ࡚࠾ࡾࡲࡍࠋ௒ᅇࡢཷ㈹ෆᐜࡢ◊✲ࡣࠊᮇᚅ
ࡋࡓ㏻ࡾ࡟ࣉ࣮ࣟࣈࡀษ᩿ࡉࢀ࡚⺯ගᙉᗘࡀቑຍࡍࡿᵝᏊࢆほᐹࡍࡿࡇ࡜ࡀ࡛ࡁ
ࡲࡋࡓࡀࠊ⣽⬊ෆ࡛ࡢάᛶ᳨ฟ࡟ࡣ⮳ࡗ࡚࠸ࡲࡏࢇࠋ௒ᚋࡶ᪂ࡓ࡞⢾㙐ࣉ࣮ࣟࣈࡢྜᡂ◊✲࡟ᣮᡓࡋ࡞ࡀࡽࠊ⣽⬊ෆ࡛ࡢάᛶ᳨ฟ࡞࡝ࠊ⮬ศ࡟࡜ࡗ࡚᪂ࡓ࡞㡿ᇦࢆ⢭ຊⓗ࡟ᗈࡆ࡚࠸ࡁࡓ࠸࡜ᛮ࠸ࡲࡍࠋ
⚾ࡣࠊ᫖ᖺ 11 ᭶ࡼࡾ⣙㸯ᖺ㛫ࡢணᐃ࡛ᾏእ␃Ꮫࡢᶵ఍ࢆᚓ࡚ࠊࢽ࣮ࣗࢪ࣮ࣛࣥࢻ
࢝ࣥࢱ࣮࣋ࣜ኱Ꮫࡢ Antony Fairbanks ඛ⏕ࡢࡶ࡜࡛᪂ࡋ࠸◊✲ࢸ࣮࣐࡟ྲྀࡾ⤌ࢇ࡛࠸ࡲࡍࠋࢽ࣮ࣗࢪ࣮ࣛࣥࢻࡣࡺࡗࡓࡾ࡜ࡋࡓẼ㢼ࡀ㨩ຊⓗ࡞ᅜ࡛ࠊ⚾ࡀᡤᒓࡍࡿ኱Ꮫࡢ◊✲⎔ቃ࡟࠾࠸࡚ࡶྠᵝࡢ㞺ᅖẼࡀឤࡌࡽࢀࡲࡍࠋᅜእ࡛◊✲⏕άࢆ㐣ࡈࡋ࡚࠸ࡿ⌧ᅾࠊ᪥ᮏ࡟࠾࠸࡚ከࡃࡢඛ⏕᪉ࡸ௰㛫ࠊᜨࡲࢀࡓ⢾㉁⛉Ꮫࡢ⎔ቃ࡟ᨭ࠼ࡽࢀ◊✲ࢆ⾜࡞ࡗ࡚࠸ࡓࡇ࡜ࢆ㌟࡟Ỹࡳ࡚ឤࡌ࡚࠾ࡾࡲࡍࠋᖐᅜᚋࡣࠊࡇ
ࢀࡲ࡛௨ୖ࡟◊✲࡟ᑐࡋ࡚┿ᦸ࡟ྲྀࡾ⤌ࡳࠊ᪥ᮏࡢ⢾㙐⛉Ꮫ◊✲࡟㈉⊩࡛ࡁࡿࡼ࠺ࠊࢽ࣮ࣗࢪ࣮ࣛࣥࢻ࡛ࡢ◊✲␃ᏛࢆᏑศ࡟ᴦࡋࡳ࡞ࡀࡽᡂ㛗ࡋ࡚ཧࡾࡓ࠸࡜ᛮ
ࡗ࡚࠾ࡾࡲࡍࠋ᭱ᚋ࡟ࠊ᪥ࠎ⇕ᚰ࡟ࡈᣦᑟࡃࡔࡉ࠸ࡲࡋࡓᯇᑿ୍㑻ඛ⏕ࡣࡌࡵ◊✲ᐊࡢⓙᵝࠊ㛵ಀ⪅ࡢⓙᵝ࡟῝ࡃឤㅰ⏦ࡋୖࡆࡲࡍࠋ
బ㔝 ຍⱑ

10ඃ⚽ㄽᩥ㈹NPC1L1 ౫Ꮡⓗ࡞⭠⟶࠿ࡽࡢࢥࣞࢫࢸ࣮ࣟࣝ྾཰ࡣ
⭷࣐࢖ࢡࣟࢻ࣓࢖ࣥ ࡢ࢞ࣥࢢࣜ࢜ࢩࢻ GM3 ࢆᚲせ࡜ࡍࡿ
NPC1L1-dependent intestinal cholesterol absorption requires gangliosideGM3 in membrane microdomains
Wataru Nihei, Masakazu Nagafuku, Hirotaka Hayamizu, Yuta Odagiri, Yumi
Tamura, Yui Kikuchi, Lucas Veillon, Hirotaka Kanoh, Kei-ichiro Inamori,
Kenta Arai, Kazuya Kabayama, Koichi Fukase, Jin-ichi Inokuchi
J Lipid Res, 2018, 59:2181-2187.
஧⎼ ΅ (኱㜰኱Ꮫ኱Ꮫ㝔 ⌮Ꮫ◊✲⛉ ໬Ꮫᑓᨷ ≉௵◊✲ဨ)
13 ᅇ⭷㈏㏻ࢱࣥࣃࢡ㉁ NPC1L1(Niemann-pick C1 Like 1)ࡣ⬡㉁ࣛࣇࢺ࡜ࡼࡤ
ࢀࡿ⭷࣐࢖ࢡࣟࢻ࣓࢖ࣥ࡟ᒁᅾࡋࠊࢥࣞࢫࢸ࣮ࣟࣝ࡟ᐩࡴ⣽⬊⭷㡿ᇦ࡜ඹ࡟ྲྀࡾ
㎸ࡲࢀࡿࡇ࡜࡛⭠⟶࠿ࡽࡢࢥࣞࢫࢸ࣮ࣟࣝ྾཰ࢆᢸ࠺ࠋࡋ࠿ࡋࠊNPC1L1 ࡟ࡼࡿࢥࣞࢫࢸ࣮ࣟࣝ㍺㏦ࡢヲ⣽࡞ศᏊᶵᗎ࡟ࡣ୙᫂࡞Ⅼࡀከ࠸ࠋᮏ◊✲࡛ࡣ᪂ࡓ
࡟ࠊNPC1L1 ࡣࡑࡢᶵ⬟࡟㓟ᛶࢫࣇ࢕ࣥࢦ⢾⬡㉁࡛࠶ࡿ࢞ࣥࢢࣜ࢜ࢩࢻ GM3 ࢆᚲせ࡜ࡍࡿ஦ࢆぢฟࡋࡓࠋHEK293T ⣽⬊࡟࠾࠸࡚ GM3 ྜᡂ㓝⣲(GM3S)ࢆḞኻࡉࡏࡓ HEK293T
GM3SKO ⣽⬊࡛ࡣࢥࣥࢺ࣮ࣟࣝ⣽⬊࡜ẚ㍑ࡋ࡚ࠊࢥࣞࢫࢸ࣮ࣟࣝ౫Ꮡⓗ࡞
NPC1L1 ࡢ⣽⬊ෆྲྀࡾ㎸ࡳࡀᢚไࡉࢀࠊࢥࣞࢫࢸ࣮ࣟࣝྲྀࡾ㎸ࡳ⬟ࡀపୗࡋ࡚
࠸ࡓࠋࡉࡽ࡟ࠊ⬡㉁␗ᖖ⑕ࣔࢹ࣐ࣝ࢘ࢫ࡛࠶ࡿ ApoE ኚ␗ (ApoEshl)࣐࢘ࢫࡢ
GM3S ࢆḞኻࡉࡏࡓ ApoEshl/GM3SKO ࣐࢘ࢫ࡛ࡣࠊ㧗ࢥࣞࢫࢸ࣮ࣟࣝ⾑⑕ࡀⴭ᫂࡟ᨵၿࡉࢀ࡚࠸ࡓࠋḟ࡟࣏ࣜࢱࣥࣃࢡ㉁୰ࡢࢥࣞࢫࢸ࣮ࣟࣝྵ㔞࡟ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓ࡜ࡇࢁࠊApoEshl ࣐࢘ࢫ࡜ẚ㍑ࡋ࡚ ApoEshl/GM3SKO ࣐࢘ࢫ࡛ࡣ࢟ࣟ
࣑ࢡࣟࣥ⏬ศ୰ࡢࢥࣞࢫࢸ࣮ࣟࣝྵ㔞ࡀ኱ᖜ࡟పୗࡋ࡚࠸ࡓࠋࡇࢀࡽࡣ GM3S
Ḟᦆ࡟ࡼࡿ⭠⟶࠿ࡽࡢࢥࣞࢫࢸ࣮ࣟࣝ྾཰ࡢపୗࢆ♧၀ࡋࠊ⣽⬊ࢆ⏝࠸ࡓ᳨ウ࡜ྠᵝ࡟ GM3S Ḟᦆ࡟ࡼࡿ NPC1L1 ࡢᶵ⬟పୗࢆᨭᣢࡍࡿ⤖ᯝ࡛࠶ࡿࠋࡑࡇ࡛ࠊ
⭠⟶࠿ࡽࡢࢥࣞࢫࢸ࣮ࣟࣝ྾཰⋡ࢆ᳨ウࡋࡓ࡜ࡇࢁࠊApoEshl ࣐࢘ࢫ࡜ẚ㍑ࡋ࡚
ApoEshl/GM3SKO ࣐࢘ࢫ࡛ࡣ⭠⟶࠿ࡽࡢࢥࣞࢫࢸ࣮ࣟࣝ྾཰⋡ࡀపୗࡋࠊࢥࣞ
ࢫࢸ࣮ࣟࣝࡢ⤒ཱྀᢞ୚࡟క࠺ NPC1L1 ࡢ⤧ẟෆ㒊࡬ࡢྲྀࡾ㎸ࡳࡀᢚไࡉࢀ࡚࠸
ࡓࠋࡇࢀࡽࡢ⤖ᯝࡣ NPC1L1 ࢆ௓ࡋࡓ⭠⟶࠿ࡽࡢࢥࣞࢫࢸ࣮ࣟࣝ྾཰࡟࠾࠸࡚
࢞ࣥࢢࣜ࢜ࢩࢻࡀ㔜せ࡞ᙺ๭ࢆᯝࡓࡍ஦ࢆ♧၀ࡋ࡚࠸ࡿࠋ

11

࠙ཷ㈹ࡢឤ᝿࡜ᢪ㈇ࠚ
 ࡇࡢᗘࡣ௧࿴ඖᖺᗘ➨ 3 ᅇ⟽Ᏺ௝୍㑻㈹ඃ⚽ㄽᩥ㈹ࢆ㈷ࡾ኱ኚගᰤ࡟Ꮡࡌࡲ
ࡍࠋᮾ໭⢾㙐◊✲఍ࡢᑂᰝጤဨࡢඛ⏕᪉࡟ཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ
ᮏ◊✲ࡢ㐙⾜࡟࠶ࡓࡾࠊࡈᣦᑟ࣭ࡈຓゝࢆ㡬ࡁࡲࡋࡓ஭ࣀཱྀඛ⏕ࢆࡣࡌࡵከࡃ
ࡢඛ⏕᪉ࠊ◊✲ᐊࡢⓙᵝ࡟ᚰࡼࡾឤㅰ⏦ࡋୖࡆࡲࡍࠋ௒ᚋ࡜ࡶఱ༞ࡈᣦᑟࡈ㠴᧡ࡢ࡯࡝ࡼࢁࡋࡃ࠾㢪࠸⏦ࡋୖࡆࡲࡍࠋ
஧⎼ ΅


12

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‫ࠄ‪ๅ‬ڂݜ‬
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‫‬
‫‬
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‫‪13‬‬

㸺ᶵ⬟⑓ែศᏊ㒊㛛㸼≉௵ᩍᤵ  ஭ࣀཱྀ ோ୍෸ᩍᤵ   ✄᳃ ၨ୍㑻ㅮᖌ    Ọ⚟ ṇ࿴ຓᩍ    ⊁㔝 ⿱⪃༤ኈ◊✲ဨ ᪂⏣ ᪸኱⢾ᒀ⑓ᛶ⭈⑕࡟࠾ࡅࡿࢢࣟ࣎⣔⢾⬡㉁ࡢ⑓ែ⏕⌮Ꮫⓗᙺ๭⢾ᒀ⑓ᛶ⭈⑕ࡣ, ⢾ᒀ⑓ࡢ୺せ࡞ྜే⑕ࡢ୍ࡘ࡛࠶ࡾ, ㏱ᯒᑟධࡢ୺࡞ཎᅉ⑌
ᝈ࡜ࡋ࡚ၥ㢟࡜࡞ࡗ࡚࠸ࡿ. ⢾ᒀ⑓ᛶ⭈⑕ࡣ, ⭈ᶵ⬟ࡀ୍➃పୗ࣭㐍⾜ࡍࡿ࡜἞⒪ࢆ⾜ࡗ࡚ࡶ᏶඲࡟ࡣᅇ᚟ࡏࡎ, 㐍⾜ࢆ㐜ࡽࡏࡿࡇ࡜ࡋ࠿࡛ࡁ࡞࠸. ࡋࡓࡀࡗ࡚,᪩ᮇⓎぢ࡜᪂つ἞⒪ࡢࡓࡵࡢ⑓ែ⏕⌮Ꮫⓗゎᯒࡀ㔜せ࡜࡞ࡿ. ࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡣ, ࢫࣇ࢕ࣥࢦ⬡㉁࡛࠶ࡿࢭ࣑ࣛࢻ࡜ࢢࣝࢥ࣮ࢫࡸ࢞ࣛࢡࢺ࣮ࢫ, ࢩ࢔ࣝ㓟࡞࡝ࡢ⢾࡛ᵓᡂࡉࢀ࡚࠸ࡿ. ࢭ࣑ࣛࢻࡣ, ࢫࣇ࢕ࣥࢦࢩࣥሷᇶ࡟⬡⫫㓟(࢔ࢩࣝᇶ)ࡀ
࢔࣑ࢻ⤖ྜ࡛⤖ྜࡋ࡚࠾ࡾ, ከࡃࡢศᏊ✀ࡀᏑᅾࡋ࡚࠸ࡿ. ࢭ࣑ࣛࢻศᏊ✀ࡢከᵝᛶࡣ, ࢔ࢩࣝ㙐㛗[㛗㙐⬡⫫㓟(C16-20)࡜ᴟ㛗㙐⬡⫫㓟(C22-26)]ࡸಟ㣭(2-Ỉ㓟໬࣭஧㔜⤖ྜ)ࡀ␗࡞ࡿ⬡⫫㓟࡟ࡼࡗ࡚, ࢭ࣑ࣛࢻࡀᵓᡂࡉࢀࡿࡓࡵ⏕ࡌࡿ. ࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡣ, ࡀࢇ, ⢾ᒀ⑓, ⚄⤒ኚᛶ⑌ᝈ࡞࡝, ࡉࡲࡊࡲ࡞⑌ᝈ࡟࠾࠸࡚⑓
ែ⏕⌮Ꮫⓗᙺ๭ࢆᢸࡗ࡚࠸ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ. ࡋ࠿ࡋ, ⢾ᒀ⑓ᛶ⭈⑕࡟࠾ࡅࡿࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡢ⑓ែ⏕⌮ⓗᙺ๭࡟ࡘ࠸࡚ࡣ, ᮍࡔ୙࡛᫂࠶ࡿ. ᮏ◊✲࡛ࡣ,⢾ᒀ⑓ᛶ⭈⑕࠾ࡅࡿࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡢ⑓ែ⏕⌮Ꮫⓗ࡞ព⩏ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ
࡟, ⭈⮚ࡢࢫࣇ࢕ࣥࢦ⢾⬡㉁ࢆゎᯒࡋࡓ.
⢾ᒀ⑓ࣔࢹ࣐ࣝ࢘ࢫࡸ㧗⬡⫫㣗㈇Ⲵ࣐࢘ࢫ, ࢫࢺࣞࣉࢺࢰࢺࢩࣥㄏᑟᛶ 1 ᆺ⢾ᒀ⑓࣐࢘ࢫࡢ⭈⮚࡛Ⓨ⌧ኚ໬ࡍࡿࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡀ␗࡞ࡿࡇ࡜ࡀุ᫂ࡋࡓ. 㣗ḧᢚไ࡟㛵୚ࡍࡿࣞࣉࢳࣥཷᐜయࡀኚ␗ࡋࡓ db/db ࣐࢘ࢫ࡛ࡣ, ࢢࣝࢥࢩࣝࢭࣛ
࣑ࢻࡸࣛࢡࢺࢩࣝࢭ࣑ࣛࢻࡢⓎ⌧ࡀቑຍࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ, ᮏ◊✲࡛ࢢࣟ࣎⣔ࢫࣇ࢕ࣥࢦ⢾⬡㉁(Gb3Cer)ࡢⓎ⌧ࡀ, ࢥࣥࢺ࣮࣐ࣟࣝ࢘ࢫ࡜ẚ㍑ࡋ࡚ప
࠸ࡇ࡜ࢆぢ࠸ࡔࡋࡓ. ⢾ᒀ⑓ࡣ」ᩘࡢ㑇ఏᅉᏊ࡜⎔ቃᅉᏊࡢ୧᪉ࡀⓎ⑕࡟㛵୚ࡍࡿࡇ࡜࠿ࡽ, ⢾ᒀ⑓Ⓨ⑕࡟」ᩘࡢ⑌ᝈឤཷᛶ㑇ఏᏊࡀ㛵୚ࡍࡿ KK ࣐࢘ࢫ࡟㧗⬡⫫㣗㈇Ⲵࡍࡿࡇ࡜࡛, ࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡢ⑓ែ⏕⌮Ꮫⓗព⩏ࢆ᳨ウࡋࡓ. ⭈⮚ࡢࢫࣇ࢕ࣥࢦ⢾⬡㉁Ⓨ⌧ࢆゎᯒࡋࡓ⤖ᯝ, db/db ࣐࢘ࢫ࡜ࡣᑐ㇟ⓗ࡟ࢢࣟ࣎⣔ࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡀⓎ⌧ቑຍࡋ࡚࠸ࡓ. db/db ࡜ KK ࣐࢘ࢫࡢ⭈⮚࡛, Ⓨ⌧ኚ໬ࡍࡿࢫ
ࣇ࢕ࣥࢦ⢾⬡㉁ࡀᑐ↷ⓗ࡞⌮⏤ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ࡟, ࣞࣉࢳࣥ㑇ఏᏊࡀኚ␗ࡋ
ࡓ ob/ob ཬࡧࢫࢺࣞࣉࢺࢰࢺࢩࣥㄏᑟᛶ 1 ᆺ⢾ᒀ⑓࣐࢘ࢫࡢゎᯒࢆ⾜ࡗࡓ. ob/ob࣐࢘ࢫࡣ, db/db ࣐࢘ࢫ࡜㢮ఝࡢⓎ⌧ኚ໬ࣃࢱ࣮ࣥࢆ♧ࡋ, ob/ob ࣐࢘ࢫ࡬ࡢࣞࣉ
ࢳࣥᢞ୚ࡣ⭈ Gb3Cer ࡢⓎ⌧㔞ࢆ, ࢥࣥࢺ࣮࣐ࣟࣝ࢘ࢫ࡜ྠࣞ࣋ࣝࡲ࡛ᅇ᚟ࡉࡏ

14ࡓ. ⯆࿡῝࠸ࡇ࡜࡟, ࢫࢺࣞࣉࢺࢰࢺࢩࣥㄏᑟᛶ 1 ᆺ⢾ᒀ⑓࣐࢘ࢫࡶ, ob/ob,

db/db ࣐࢘ࢫ࡜ྠᵝ࡟⭈ Gb3Cer ࡢⓎ⌧ࡀపୗࡋ࡚࠸ࡓ. ࢫࢺࣞࣉࢺࢰࢺࢩࣥㄏ
ᑟᛶ⢾ᒀ⑓࣐࢘ࢫࡣ, ࢖ࣥࢫࣜࣥࡢḞஈ࡟ࡼࡾࣞࣉࢳࣥࡀྜᡂ࣭ศἪࡉࢀࡿ⬡⫫⤌⧊ࡢ⃭ῶ, ࡑࢀ࡟క࠺పࣞࣉࢳࣥ⾑⑕ࢆ࿊ࡍࡿ. ࡲࡓ, KK ࣐࢘ࢫࡢሙྜ, 㐣㣗ࡸ㧗⬡⫫㣗㈇Ⲵࡢ⤖ᯝ, ⬡⫫⤌⧊ࡀ⫧኱ࡋ㧗ࣞࣉࢳࣥ⾑⑕ࢆ࿊ࡍࡿ. ࡇࢀࡽࡢ஦
ᐇࢆ㋃ࡲ࠼ࡿ࡜, ୖグࡢ⤖ᯝࡣ, ࣞࣉࢳࣥࡀ⭈⮚ࡢࢢࣟ࣎⣔⢾⬡㉁ࡢⓎ⌧࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿ.
᭱㏆ࡢ◊✲࡛ࡣ, ࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡢᵓᡂ⬡⫫㓟ࡀ␗࡞ࡿศᏊ✀ࡀ,⏕⌮άᛶ࡟኱ࡁ࡞ᙳ㡪ࢆ୚࠼ࡿࡇ࡜ࡀ♧ࡉࢀࡘࡘ࠶ࡿ. ࡋࡓࡀࡗ࡚, ⢾ᒀ⑓ᛶ⭈⑕࡟㛵㐃ࡋ
࡚Ⓨ⌧ኚ໬ࡍࡿࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡢศᏊ✀ࢆゎᯒࡍࡿࡇ࡜ࡣ, ⑓ែ⏕⌮Ꮫⓗ࡟㔜せ࡛࠶ࡿ. ᮏ◊✲࡛ࡣ, 㧗⬡⫫㣗ࢆ 8 㐌㛫㈇Ⲵࡋࡓ KK ࣐࢘ࢫࡢ⭈⮚࡛, ቑຍࡀ☜ㄆࡉࢀࡓࢢࣟ࣎⣔⢾⬡㉁ࡢศᏊ✀ゎᯒࢆ, ᾮయࢡ࣐ࣟࢺࢢࣛࣇ-ࢱࣥࢹ࣒㉁㔞
ศᯒィࢆ⏝࠸࡚⾜ࡗࡓ. ࡑࡢ⤖ᯝ, 㣬࿴ࡢᴟ㛗㙐ศᏊ✀(C22, C23, C24)ࡀ≉࡟ቑຍࡋ࡚࠸ࡓ. ⢾ᒀ⑓᢬ᢠᛶࡢ C3H/HeN ࣐࢘ࢫࡢ⭈⮚࡛ࡣ, KK ࣐࢘ࢫࡢ⭈⮚࡛Ⓨ⌧ࡢప࠸ 2-Ỉ㓟໬⬡⫫㓟ࡀ⤖ྜࡋࡓศᏊ✀ࡀ㧗Ⓨ⌧ࡋ࡚࠸ࡓ.
ୖグࡢ⤖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 ࢢ ࣟ ࣎ ⣔ ⢾ ⬡ ㉁ ࡣ , Toll-like receptor 4 (TLR4) ࣜ ࢞ ࣥ ࢻ (LPS,
HMGB1)ࡢᏑᅾୗ, TLR4 㑅ᢥⓗ࡞࣏ࢪࢸ࢕ࣈࣔࢪ࣮ࣗࣞࢱ࣮࡜ࡋ࡚⅖⑕ಁ㐍άᛶࢆᣢࡘࡇ࡜ࡀ♧၀ࡉࢀࡓ (Fig. 1). 㧗⾑⢾≧ែࡢ៏ᛶ࡞ᣢ⥆࡟ࡼࡾ, ⤊ᮎ⢾໬⏘≀(AGEs)ࡢ⏘⏕ࡸ㓟໬ࢫࢺࣞࢫࡀㄏ㉳ࡉࢀ, ⭈⮚࡬ࡢάᛶ໬࣐ࢡࣟࣇ࢓࣮ࢪ
ࡢᾐ₶ࡸ࣓ࢧࣥࢠ࣒࢘⣽⬊ࡢ TLR4 Ⓨ⌧ࡀୖ᪼ࡍࡿ. ຍ࠼࡚, ⢾ᒀ⑓࡛ࡣ LPS ࡸ
HMGB1 ࡞࡝ࡢ⾑୰⃰ᗘࡶୖ᪼ࡍࡿࡇ࡜࠿ࡽ, ⭈⮚ࡣ⅖⑕ࡀច㉳ࡉࢀࡸࡍ࠸≧ែ࡟㝗ࡗ࡚࠸ࡿ. ࣞࣉࢳࣥࡣどᗋୗ㒊࡟స⏝ࡋ㣗ḧᢚไࡸ࢚ࢿࣝࢠ࣮ᾘ㈝ஹ㐍࡟㛵ࢃࡿࡔࡅ࡛࡞ࡃ, ච␿⣽⬊࡟స⏝ࡍࡿࡇ࡜࡛⅖⑕ᛶࢧ࢖ࢺ࢝࢖ࣥࡢศἪࢆಁ㐍ࡋ,⅖⑕཯ᛂ࡟㛵୚ࡍࡿ. ⅖⑕ᛶࢧ࢖ࢺ࢝࢖ࣥࡣ, ⭈⣒⌫య⣽⬊ࡢࢢࣟ࣎⣔⢾⬡㉁ࡢ
Ⓨ⌧ࢆቑຍࡉࡏࡿ. ࡋࡓࡀࡗ࡚, 㧗⬡⫫㣗㈇Ⲵ࡟ࡼࡾ⫧኱ࡋࡓ⬡⫫⤌⧊࠿ࡽศἪ
ಁ㐍ࡉࢀࡓࣞࣉࢳࣥࡀ, ⭈⮚ࡢࢢࣟ࣎⣔⢾⬡㉁ࡢⓎ⌧ࢆቑຍࡉࡏ, ቑຍࡋࡓࢢࣟ
࣎⣔⢾⬡㉁ࡀ, TLR4 ࣜ࢞ࣥࢻᏑᅾୗ, TLR4 ࡟స⏝ࡍࡿࡇ࡜࡛⅖⑕཯ᛂࢆಁ㐍ࡋ,⢾ᒀ⑓ᛶ⭈⑕ࡢ⑓ែᙧᡂ࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀᮏ◊✲ࡼࡾ♧၀ࡉࢀࡿ. ࣞࣉࢳࣥ࡟ࡼࡿ⭈⮚ࡢࢢࣟ࣎⣔⢾⬡㉁ࡢⓎ⌧ኚ໬࣓࢝ࢽࢬ࣒ࡢࡉࡽ࡞ࡿゎ᫂࡟ࡼࡾ, ⢾ᒀ⑓ᛶ⭈⑕ࡢ᪂ࡓ࡞デ᩿ἲࡸ἞⒪⸆㛤Ⓨࡢ㐍ᒎ࡟ࡘ࡞ࡀࡿࡇ࡜ࡀண᝿ࡉࢀࡿ.

15

Fig. 1. Gb3Cer and Gb4Cer ࡢ TLR4 ࢩࢢࢼࣝ࡟࠾ࡅࡿసᴗ௬ㄝ
A: TLR4 signaling by LPS. Dimerization of TLR4/MD-2 units required, atleast, one molecule of LPS. B: Synergistic activation of TLR4/MD-2 by LPS andGb3Cer/Gb4Cer. Gb3Cer/Gb4Cer associate with TLR4/MD-2 complex as
modulator in presence of LPS, and enhance inflammatory response throughtrans-interaction. C: Gb3Cer/Gb4Cer alone could not induce activation of
TLR4/MD-2 complex.

㸺Ⓨ⾲ㄽᩥ㸼
1. Overexpression of HexCer and LacCer containing 2-hydroxylated fatty acids incholangiocarcinoma and the association of the increase of LacCer (d18:1-h23:0) withshorter survival of the patients. Silsirivanit A, Phoomak C, Teeravirot K, Wattanavises
S, Seubwai W, Saengboonmee C, Zhan Z, Inokuchi J, Suzuki A, Wongkham S (2019)
Glycoconj. J. 36, 103-111.
2. Visual Function in Mice Lacking GM3 Synthase. Hiraoka M, Ohkawa E, Abe A, Murata
M, Go S, Inokuchi J, Ohguro H (2019) Curr. Eye Res. 44, 664-670.
3. Serum GM3(d18:1-16:0) and GM3(d18:1-24:1) levels may be associated withlymphoma: An exploratory study with haematological diseases. Nishikawa M, Kurano
M, Nitta T, Kanoh H, Inokuchi J, Yatomi Y (2019) Sci. Rep. 9, 6308.
4. Globo-series glycosphingolipids enhance lipopolysaccharide-induced inflammationand play a pathophysiological role in diabetic nephropathy. Nitta T, Kanoh H, Inamori
K, Suzuki A, Takahashi T, Inokuchi J (2019) Glycobiology 29, 260-268.
5. ࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡢ⏕ྜᡂ࡜ࡑࡢไᚚ.
஭ࣀཱྀோ୍ࠊᒾῲ࿴ஂ
ࢭ࣑ࣛࢻ◊✲ࡢ᪂ᒎ㛤 ࢭ࣑ࣛࢻ◊✲఍⦅ P44-50 㣗ရ໬Ꮫ᪂⪺♫6. ࢫࣇ࢕ࣥࢦ⢾⬡㉁ࡢ⬡⫫㓟㙐ࢆ௓ࡋࡓ⮬↛ච␿ᛂ⟅ࡢไᚚᶵᵓ.
⊁㔝⿱⪃ࠊ஭ࣀཱྀோ୍
ࢭ࣑ࣛࢻ◊✲ࡢ᪂ᒎ㛤 ࢭ࣑ࣛࢻ◊✲఍⦅ P145-150 㣗ရ໬Ꮫ᪂⪺♫16

㸺Ꮫ఍Ⓨ⾲㸼
࣭⢾⬡㉁࢞ࣥࢢࣜ࢜ࢩࢻࡢḞᦆ࡟ࡼࡿ⫧‶ࣔࢹ࣐ࣝ࢘ࢫࡢ⑓ែᨵၿ࠾ࡼࡧཷᐜయ
ࢩࢢࢼࣝ࡬ࡢᙳ㡪 ✄᳃ၨ୍㑻 ➨ 7 ᅇ AAA (Academy of Aging and CArdiovascular-Diabetes Research)㸪
ᮾி㸪2019 ᖺ 1 ᭶㸪₇㢟␒ྕ 2
࣭Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4
signaling in obesity
Hirotaka Kanoh, Takahiro Nitta, Akemi Suzuki, Jin-ichi Inokuchi
Keystone symposia: Innate Immune Receptors: Roles in Immunology and Beyond,Taipei, Taiwan, Mar/2019, Poster No. 2009
࣭Homeostatic and Pathogenic Roles of Ganglioside GM3 Molecular Species in
TLR4 Signaling
Jin-ichi Inokuchi
FEBS Special Meeting 2019 Sphingolipid Biology: Sphingolipids in Physiology andPathology May 6-10 Cascais, Portugal. Program book page14࣭ᴟ㛗㙐࢞ࣥࢢࣜ࢜ࢩࢻ࡟ࡼࡿ TLR4 ไᚚ࣓࢝ࢽࢬ࣒࡜ Halo-tag ࡟ࡼࡿ࣓࢝ࢽࢬ
࣒ゎᯒࡢヨࡳ
⊁㔝⿱⪃኱㜰኱Ꮫ Halo-tag ࢭ࣑ࢼ࣮, ኱㜰኱Ꮫ࣭⌮Ꮫ◊✲⛉, 2019 ᖺ 5 ᭶࣭⢾ᒀ⑓ᛶ⭈⑕࡟࠾ࡅࡿ⭈ Gb3Cer Ⓨ⌧ㄏᑟᶵᵓ࡟㛵ࡍࡿゎᯒ᪂⏣᪸኱2019 ᖺᗘ㸦௧࿴ඖᖺᗘ㸧᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊୺ദ ➨ 18 ᅇ⏕≀໬Ꮫⱝᡭ◊✲⪅ࢭ࣑ࢼ࣮, ௝ྎ, 2019 ᖺ 7 ᭶࣭⢾㙐࡟ࡼࡿ⏕ά⩦័⑓࣭៏ᛶ⅖⑕ᛶ⑌ᝈ➼ࡢ᪂つデ᩿࣭἞⒪ἲࡢ㛤Ⓨ஭ࣀཱྀோ୍ࠊ⊁㔝⿱⪃➨ 51 ᅇ ᪥ᮏ⮫ᗋ᳨ᰝ་Ꮫ఍/ᮾ໭ᨭ㒊⥲఍ ≉ูㅮ₇, ௝ྎ, 2019 ᖺ 7 ᭶࣭㧗⬡⫫㣗㈇Ⲵ࡟క࠺どᗋୗ㒊⅖⑕࡟࠾ࡅࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢᶵ⬟ⓗᙺ๭ ✄᳃ၨ୍㑻㸪⊁㔝 ⿱⪃㸪᪂⏣ ᫡኱㸪஭ࣀཱྀோ୍
 ➨ 38 ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪ྡྂᒇ㸪2019 ᖺ 8 ᭶㸪₇㢟␒ྕ P-155
࣭⢾ᒀ⑓ᛶ⭈⑕࡟࠾ࡅࡿ⭈ Gb3Cer Ⓨ⌧ㄏᑟᶵᵓ࡟㛵ࡍࡿゎᯒ᪂⏣᪸኱, ⊁㔝⿱⪃, ✄᳃ၨ୍㑻, 㕥ᮌ᫂㌟, ஭ࣀཱྀோ୍
➨ 38 ᅇ ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍, ྡྂᒇ, 2019 ᖺ 8 ᭶, せ᪨㞟 p.105
࣭࢞ࣥࢢࣜ࢜ࢩࢻࡢ࢔ࢩࣝ㙐ᵓ㐀࡟ᇶ࡙࠸ࡓ⮬↛ච␿ᛂ⟅ࡢㄪ⠇ᶵᵓ⊁㔝⿱⪃, ᪂⏣᪸኱, 㕥ᮌ᫂㌟, ஭ࣀཱྀோ୍
➨ 38 ᅇ ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍, ྡྂᒇ, 2019 ᖺ 8 ᭶, せ᪨㞟 p.77
࣭Homeostatic and Pathogenic Roles of GM3 Ganglioside Molecular Species in
TLR4 Signaling in Metabolic Disorders17

Jin-ichi Inokuchi, Hirotaka Kanoh
Glyco25 (25th International symposium on Glycoconjugates),
Milan, Italy, Aug/2019, Program p.26
࣭⢾⢾ᒀ⑓ᛶ⭈⑕࡟࠾ࡅࡿ⭈ Gb3Cer Ⓨ⌧ㄏᑟᶵᵓ࡟㛵ࡍࡿゎᯒ᪂⏣᪸኱, ⊁㔝⿱⪃, ✄᳃ၨ୍㑻, 㕥ᮌ᫂㌟, ஭ࣀཱྀோ୍
➨ 13 ᅇ ᮾ໭⢾㙐◊✲఍, ᪂₲, 2019 ᖺ 9 ᭶, せ᪨㞟 p.51
࣭࢞࢞ࣥࢢࣜ࢜ࢩࢻࡢ࢔ࢩࣝ㙐ᵓ㐀࡟ࡼࡿ TLR4 ไᚚ࣓࢝ࢽࢬ࣒
⊁㔝⿱⪃, ᪂⏣᪸኱,㕥ᮌ᫂㌟, ஭ࣀཱྀோ୍
➨ 13 ᅇ ᮾ໭⢾㙐◊✲఍, ᪂₲, 2019 ᖺ 9 ᭶, せ᪨㞟 p.52
࣭ࣞࣞࣉࢳࣥ࡟ࡼࡿ⭈ࢫࣇ࢕ࣥࢦ⢾⬡㉁ Gb3Cer ࡢⓎ⌧ㄏᑟ᪂⏣᪸኱, ⊁㔝⿱⪃, ✄᳃ၨ୍㑻, 㕥ᮌ᫂㌟, ஭ࣀཱྀோ୍
➨ 92 ᅇ ᪥ᮏ⏕໬Ꮫ఍኱఍, ᶓ὾, 2019 ᖺ 9 ᭶, せ᪨㞟 p.133
࣭⫧‶࡟క࠺どᗋୗ㒊⅖⑕࡟࠾ࡅࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢᙺ๭ ✄᳃ၨ୍㑻㸪⊁㔝 ⿱⪃㸪᪂⏣ ᫡኱㸪஭ࣀཱྀோ୍
 ➨ 92 ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍㸪ᶓ὾㸪2019 ᖺ 9 ᭶㸪₇㢟␒ྕ 1P-371
࣭⢾⢾⬡㉁࡟ࡼࡿ៏ᛶ⅖⑕⑌ᝈࡢ᪂ࡓ࡞デ᩿࣭἞⒪ἲࡢ㛤Ⓨ஭ࣀཱྀோ୍, ⊁㔝⿱⪃➨17ᅇ ⢾㉁⛉Ꮫࢥࣥࢯ࣮ࢩ࢔࣒㸦JCGG㸧ࢩ࣏ࣥࢪ࣒࢘
ࢢࣛ࢖ࢥࢧ࢖࢚ࣥࢫ㸤࢚ࣥࢪࢽ࢔ࣜࣥࢢࡢᮍ᮶ -⢾㙐ࡢከᵝᛶ࡜ᬑ㐢ᛶ㸸⢾㙐ᬯྕࡢゎ᫂࡜ไᚚ࡟ྥࡅ࡚-, ி㒔, 2019ᖺ10᭶, せ᪨㞟 p.24
࣭⢾⢾⬡㉁࡟ࡼࡿ៏ᛶ⅖⑕⑌ᝈࡢ᪂ࡓ࡞デ᩿࣭἞⒪ἲࡢ㛤Ⓨ஭ࣀཱྀோ୍➨㸰㸴㸮ᅇ᪥ᮏἪᒀჾ⛉Ꮫ఍ᮾ໭ᆅ᪉఍ඹദࢭ࣑ࢼ࣮
௧࿴ඖᖺ㸯㸯᭶㸷᪥ ᮾ໭་⛉⸆⛉኱Ꮫࠊ௝ྎ
࣭Glycosphingolipids and leptin signaling Kei-ichiro Inamori, Hirotaka Kanoh, Takahiro Nitta, Jin-ichi Inokuchi
11th Asian Community of Glycoscience and Glycotechnology (ACGG) Conference,
Busan, Korea, Nov/2019, Abstract p.62
࣭Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4
signaling in metabolic disorders
Hirotaka Kanoh, Takahiro Nitta, Akemi Suzuki, Jin-ichi Inokuchi
11th Asian Community of Glycoscience and Glycotechnology (ACGG) Conference,
Busan, Korea, Nov/2019, Abstract p.83
࣭GM3 species in metainflammation
Asia Zonca, Hirotaka Kanoh, Takahiro Nitta, Wataru Nihei, Satomi Kudoh, Maria
Ciampa, Laura Mauri, Sandro Sonnino, Jin-ichi Inokuchi, Alessandro Prinetti
11th Asian Community of Glycoscience and Glycotechnology (ACGG) Conference,
Busan, Korea, Nov/2019, Abstract p.168
࣭㧗⬡⫫㣗ㄏᑟᛶࡢどᗋୗ㒊⅖⑕࡟࠾ࡅࡿ࢞ࣥࢢࣜ࢜ࢩࢻࡢᙺ๭

18 ✄᳃ၨ୍㑻㸪⊁㔝 ⿱⪃㸪᪂⏣ ᫡኱㸪஭ࣀཱྀோ୍
 ➨ 42 ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍㸪⚟ᒸ㸪2019 ᖺ 12 ᭶㸪₇㢟␒ྕ 4LBA-092
࣭Homeostatic and pathogenic roles of GM3 ganglioside molecular species in
TLR4 signaling in obesity
Jin-ichi Inokuchi
The first Academia Sinica-Tohoku Medical and Pharmaceutical University (TMPU)
Joint Symposium. December 19-20, 2019, Academia Sinica Campusm Taipei, Taiwan.

19

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

atrophy, 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).

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

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Immunology

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Globo-series glycosphingolipids and 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.

21

Downloaded from https://academic.oup.com/glycob/article-abstract/29/3/260/5199375 by Tohoku Yakka Daigaku Toshokan user on 05 September 2019diabetic 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.

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.

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Downloaded from https://academic.oup.com/glycob/article-abstract/29/3/260/5199375 by Tohoku Yakka Daigaku Toshokan user on 05 September 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-

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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.

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.

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).

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 gene

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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-

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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

MD-2

MD-2

Monomer

Signaling

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.

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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

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T Nitta et al.
species have ionization efﬁciencies comparable to those of theinternal standards used.

Analysis of GSLs
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.

Puriﬁcation of human monocytes

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.

Differentiation of mouse bone marrow-derived
macrophages (BMDMs)

Mass spectrometric (MS) analysis

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.

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

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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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.

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.

normal diet; PBMC, peripheral blood mononuclear cell; T2DM, type 2
diabetes mellitus; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor α;
VLCFAs, very long chain fatty acids.

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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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Cell stimulation, enzyme-linked immunosorbent assay(ELISA) and luciferase assay

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総論編

スフィンゴ糖脂質の生合成とその制御
井ノ口

＊１

仁一

＊２

、岩渕

和久

成分である。一方、糖が結合したものをスフィン◆ はじめにゴ糖脂質（GSL）と呼ぶ。本稿では、GSL生合成・

ド イ ツ 生 ま れ の 神 経 化 学 者 で あ る J. L .W

代謝とその制御機構、ヒトのGSL合成異常およThudichum（1829 - 1901） は、 ヒ ト の 脳 の 両 親

びGM3合成酵素ノックアウトマウスの表現型に媒性の不思議な脳物質に対して、謎を問いかける

ついて述べる。

スフィンクスに因んで、「スフィンゴシン」と名１ スフィンゴ糖脂質（GSL）の生合成

づけた 1）。長鎖アミノアルコールであるスフィン
ゴシン（スフィンゴイド塩基）に脂肪酸が酸アミ

GSLは、セラミドにグルコース、ガラクトー

ド結合した化合物をセラミド、セラミドに更にス、N-アセチルガラクトサミン、N-アセチルグ

リン酸基を有するものをスフィンゴリン脂質と呼ルコサミン、シアル酸などの糖や硫酸の段階的なび、その代表的な脂質はスフィンゴミエリンであ

酵素反応によって、多様な分子が生合成され、細り、動物細胞膜のスフィンゴ脂質グループの主要

胞膜脂質二重層の外側半葉に局在して非対称構造
分泌

スフィンゴ糖脂質（GSLs）

細胞膜

GSLs

GSLs

リソソーム

セラミド
ガラ系列
GM4
SM4

パルミトイルCoA ＋ L -セリン
3-ケトジヒドロスフィンゴシン
ジヒドロスフィンゴシン

GalCer

GalCer

小胞体

ゴルジ体

セラミド
図１A

LacCerGlcCerGlcCer

ジヒドロセラミド

LacCer分岐
ガングリオ系列
ラクト系列
グロボ系列
ネオラクト系列
イソグロボ系列
スルホ系列

スフィンゴ糖脂質の生合成と代謝（A：細胞内小器官におけるGSLs代謝）＊１ 東北医科薬科大学 分子生体膜研究所長、＊２ 順天堂大学大学院医療看護学研究科 教授

129

を形成している。GSLの生合成は、ラクトシルセ

らは、細胞や組織において特徴的な発現特異性をラミド（LacCer）を分岐点としてガングリオ系示す。GM2/GD2合成酵素（β-1,4 N-acetylgalac

列、ラクト系列、グロボ系列、ネオラクト系列、

tosaminyltransferase：B4GalNAcT1；以下GM2S

イソグロボ系列、スルホ系列の多様なGSLが生合と略）は、主に脊椎動物の脳神経系に発現し複成され、また、ガラ系列はガラクトシルセラミド

合型ガングリオシド生合成の鍵となる酵素で、（GalCer）にシアル酸および硫酸基が付加され、

GM3、GD3、LacCerにN-アセチルガラクトサミ

それぞれGM4とSM4が生成する（図１A, B）。シン（N-acetylgalactosamine：GalNAc） を 転 移

アル酸を含むスフィンゴ糖脂質をガングリオシド

し て、 そ れ ぞ れ か らGM2、GD2、GA2（asialo-と呼び、細胞膜マイクロドメインの構成分子としGM2）が生合成される（図１B）。一方、GalCerにて、様々な細胞膜上におけるシグナル伝達を制御

シアル酸が付加しGM4が生合成される（図１B）。

している。ガングリオシドファミリー生合成の最その生合成に関わるシアル酸転移酵素は近年まで

初の分子は、ラクトシルセラミドにシアル酸が転不明であったが、GM3Sノックアウトマウスでは、移して生成するGM3である。GM3は末梢組織の赤血球やミエリンのGM4が消失することから、主要なガングリオシドである。GM3は、糖脂質研

GM3合成酵素がGM4の生合成も担っていること

究の草分けである山川民夫博士がウマ赤血球からが明らかになった 3、4）。

ヘマトシドとして同定されたものである 1）。ヒト

2 スフィンゴ糖脂質生合成制御機構

GM3合成酵素（GM3S、ST3GAL5；以降GM3Sと略）は、斎藤らにより1998年に遺伝子クロー

細胞内におけるスフィンゴ（糖）脂質の生合成

ニングされた 2）。図１Bに示すように、GM3から制御機構について、現時点での作業仮説も含めては多様なガングリオシドが生合成されるが、それ図２に示した。国立感染研の花田は、CERTとよ

スルファチド
グロボ系列

Cer

S

ラクト/ネオラクト系列

SM4
GalCer

Cer

CST

Cer

GM4

Cer

Gb4

Cer

Gb3

GalCer syn

Cer

GlcCer syn

Gb3 syn (A4galt)

Cer

B4galt5/6

GA2

B3gnT5：グルコース

GD3

Cer

GM3
syn
(St3gal5)

Cer

GD3
syn
(ST3sia1)
GD2

GM3

Cer
GM1/GD1b/GA1 syn

nLc4

b1-3

GM3

Cer

GM2/GD2 syn (B4galnt1)

Cer：セラミド

Cer
Cer

LacCerGlcCer

Lc4

b1-4

Lc3

Cer

Cer

(B3galnt4)
GA1：ガラクトース

GD1b

GM1

Cer

Cer

Cer

：N-アセチルガラクトサミン：N-アセチルグルコサミン：シアル酸
S ：硫酸基

図１B

GM1b

GD1a

GT1b

GD1c

GT1a

GQ1b

O系列

a系列
ガングリオ系列

b系列

ガングリオシド系列、グロボ系列、ラクト・ネオラクト系列及びスルファチドの生合成経路

2

30

総論編

スフィンゴ糖脂質の生合成とその制御ばれる小胞体からゴルジ体へ特異的にセラミドをに変換されることが示された 9、10）。おそらく、運ぶ分子を発見した 5）。CERTに結合したセラミ

LacCer合成酵素はゴルジ体で局在が異なる２種

ドは、CERTのPHドメインとゴルジ膜局所に濃類が存在し、FAPP2と結合したGlcCer はグロ

縮されたホスファチジルイノシトール-４-一リンボ系などの中性糖脂質（Gb3など）の生合成に利酸（PI4P）と複合体を形成し、主としてスフィ用されるのであろう。一方、LacCer合成酵素とンゴミエリン（SM）の生合成に利用される。まGM3合成酵素（GM3S）は、複合体を形成してた、小胞体膜の細胞質側で合成されたセラミドのガングリオシドファミリーの合成を制御している

一部は、小胞体、シス/メディアゴルジ膜の細胞可能性が報告されている 11）。従って、細胞特異的質側でグルコシルセラミド（GlcCer）になる 6）。

なスフィンゴ糖脂質分子種の発現機構の包括的な

また、最近の林らの研究に依れば、GlcCer合成

理解には種々の生合成酵素の細胞内局在および動酵素とSM合成酵素1はゴルジのトランス側で複態を徹底的に解明していくことが不可欠である。

合体を形成することが分かっている 7）。この事実

第２章には、現時点までに報告されているスフィ

は、GlcCer合成酵素は、ゴルジ膜のトランス側

ンゴ糖脂質合成酵素の細胞内局在について詳細にに局在していることを示唆している。ただし、そまとめられているので、参照されたい。

の合成活性に関しては、まだ証拠が不十分であGM3SはII型の膜タンパク質であり複数のアスると考えられる. 生成したGlcCerは非ゴルジ経

パラギン結合型糖鎖が付加される。これらの糖路を通って細胞膜に輸送されるか 8）、糖脂質転移

鎖はGM3Sの酵素活性に必須である 12）。GM3Sに（glycolipid-transter）ドメインを持つFAPP2では、N末端側の細胞質領域の長さが異なる３種類

トランスゴルジに輸送され、ゴルジ体膜の内側にの ア イ ソ フォーム（M1-、M2-、M3-GM3S） がフリップした後、ラクトシルセラミド（LacCer）

存在し、それぞれの細胞内動態が大きく異なって

Plasma membrane
Vesicular transport
Non-vesicular transport
Gb3

GM3

（Globosides）（gangliosides）

SM
TGN

LacCer

CERT
Golgi

GM3 SM
trans

Cer

LacCermedialGlcCer
cis
ERGIC

FAPP2
Cer

ER

GlcCerNuclear

図２ スフィンゴ糖脂質生合成制御機構
スフィンゴミエリン、グロボシド、ガングリオシドの糖脂質生合成の最近の局面（作業仮説を含む）

3

31

いる 13）。シアル酸転移酵素ファミリーのなかで3 GM3S ノックアウトマウスの表現型も最も長い68アミノ酸の細胞外領域を有するM1GM3Sは、N末側に逆行輸送シグナルR-basedモ

種々のスフィンゴ糖脂質合成酵素のノックアウ

チーフを持ち、小胞体に局在している（図３）。

トマウスの解析から、細胞種特異的に発現するス

M1-GM3Sの逆行輸送は不完全であり、一部のフィンゴ糖脂質分子種の機能が見いだされつつあ

M1-GM3Sは逆行輸送を逃れ、ゴルジ体まで運ばる（図４）。詳しくは総説を参照されたい 15）。ここ

れた後、GM3を合成する。つまり、M1-GM3Sはでは、GM3Sノックアウトマウスの解析により見安定して小胞体に局在しながら、ゆっくりとゴ

えて来たガングリオシドの機能について述べる。

ルジ体へGM3Sを供給していることが示唆され

GM3の分子レベルでの作用機序が明らかとなっる。また、M2-GM3SとM3-GM3Sはどちらもゴた代表例としては、インスリン受容体の機能制御

ルジ体に局在しているが、M2-GM3Sは速やかにである。肥満モデル動物のZucker fa/faラットとリソソームへ運ばれて分解されるのに対し、M3-

ob/obマウスの脂肪組織においてGM3S遺伝子のGM3Sは安定してゴルジ体に繋留する。３種類の発現増加に伴いGM3発現量が著しく増加するとアイソフォームの量的バランスと局在のバランいう発見を契機に 16）、GM3S KOマウスでインス

スがGM3合成の制御に重要であると推測される

リン感受性の亢進することが証明された 17）。また、（図３）13、14）。今後、ラクトシルセラミドに作用

グルコシルセラミド合成酵素阻害剤を処理するこする６種の酵素（図１A）の細胞内動態が解明さとで、肥満モデル動物のインスリン抵抗性 18）や多れ、スフィンゴ糖脂質生合成制御機構の全貌が解発 性 嚢 胞 腎（polycystic kidney disease, PKD）

明されることが期待される。

などを改善する 19）などの報告が相次ぎ、GM3と２型糖尿病との関わりが注目されている。現在で
リソソーム
分解

GM3生合成transmedialCOPⅠ

ゴルジ体

maturationcis

R-based motif（RRXXXXR）
逆行輸送シグナル

COPⅡ
［R/K］
（X）
［R/K］motif
小胞体搬出シグナル

N結合型糖鎖

小胞体

M1

M2

M3

小胞体

ゴルジ体
リソソーム
安定性：低

ゴルジ体

細胞内局在

安定性：高

細胞内安定性

安定性：高

M1
M2
M3
MHTEAVGGAARRPQKLRSQAAAPACRAMPSEFTSAKLRSDCSRTSLQWYTRTQHKMRRPSSLLIKDICKC

逆行輸送シグナル

図３

小胞体搬出シグナル

GM3合成酵素アイソフォームの細胞内動態 7 - 9）

4

32

TM

総論編

スフィンゴ糖脂質の生合成とその制御

Embryonic
lethality

Gb3

Ugcg

SM4

GalCer

Cer

Cer

Ugt8a

SO3

Cer
α4

Gala-series
GlcCer

A4galtGlobo-series
Embryonic
lethality

Verotoxin
protection
iGb3

MyelinCer defects

Myelindefects

A4galt5
LacCer

Cer
α3

Cer

Gal3st1

GA2

Cer

Cer
B4galnt1

A3galt2

Isoglobo-series

Asialo-series

Enhancedinsulinsignaling,deafness

No obvious
phenotypeSt3gal5

GM3

GM2

Lc3

Cer

Cer

Cer

B4galnt1B3gnt5

Lacto/neolacto-seriesMultipleaberrant
phenotype

Nerve
regeneration
defects

St8sia1
GD3

GD2

Cer

B4galnt1

Neuronal and myelin
defects, male infertility

図４

Cer

Ganglio-series

種々のスフィンゴ糖脂質合成酵素ノックアウトマウスの表現型（引用文献14より改変）

は、このようなGM3発現量の増加によるインスの二重欠損（GM2S/GM3S DKO）マウスは、生

リンシグナル抑制の効果は、インスリン受容体は後すぐに重篤な中枢神経障害を示し、大多数がカベオラと呼ばれる細胞膜上のくぼみ構造に存在早期に死亡する 22）。従って、GM3S KOマウスですることでインスリンによるシグナル伝達を行う

は、GAIの末端ガラクトースにシアル酸が付加さが、肥満に伴いカベオラとは別のマイクロドメイ

れて生成するGM1b、さらにシアル酸が付加されンを形成しているGM3が膜上に増加することでイ

たGD1cなどの「０系列」ガングリオシドが生命

ンスリン受容体の膜直上のリシン残基とGM3の静維持に重要な「a, b系列」ガングリオシドの機能

電気的相互作用が生じ、インスリン受容体がカベを代償していると考えられる（図１B）。

オラから解離すると考えられている

20）。

4 ヒトにおけるガングリオシド合成異常

GM3S KOマウスはメンデルの法則に従う比率
で産まれ、聴覚異常 21） を呈するもののほぼ正常

ヒトでスフィンゴ糖脂質合成酵素の遺伝子変に成長し、寿命も野生型と比較して大きな差は見

異 は、GM3SとGM2Sで 見 出 さ れ て い る。 現 在られない。GM3S KOマウスのほぼすべての臓器までの報告を図５にまとめた。最初に見出されでは、a, b系列のガングリオシドが消失し、通常たGM3S遺 伝 子 変 異 は、 アーミッシュ家 系（ ７

ほとんど発現が見られない０系列 のガングリオ

世紀に米国ペンシルバニア州に移住した保守派

シドが発現する（図１B）。ガングリオシドを完

アーミッシュ家系）で見出されたナンセンス変異

全に欠損したマウスであるGM3SとGM2S遺伝子（288STOP）である23）。このGM3S欠損は幼児期
533

GM3合成酵素（ST3GAL5）
p.Gly201Arg
p.Cys195Ser
p.Arg288＊

p.Glu355Lys
418aa

1

Sialylmotif S

Sialylmotif L

GM2合成酵素
（B4GALNT1）
p.Pro132Glnfs＊7
p.Leu89Profs＊13
p.Gln120＊

p.Lys284Asn

p.Leu487Thrfs*77

p.Gly280̲Leu326del p.Arg300Cys
p.Arg228＊

p.Phe439delp.Thr307̲Val308dup

p.Asp433Ala

1

p.Arg505His
533aa

Glycosy ltransferase,family 2 domain

図５

Nucleotide-diphospho-sugartransferasessuperfamily domain

GM3及びGM2合成酵素で見出された遺伝子変異

ST3GAL5 and B4GNT1 遺伝子配列は、それぞれ NM̲003896.3 および NM̲001478.4にもとずいている。
遺伝子変異の箇所の記載は、Sequence Variant Nomenclature（http://varnomen.hgvs.org/）に従って記載した。

【引用文献】に発症する重篤なてんかん症状、重度の知的障害、

1）山川民夫（1981） 糖脂質物語 講談社学術文庫.
2）Ishii, A. et al. Expression Cloning and Functional
Characterization of Human cDNA for Ganglioside
GM3 Synthase. J. Biol. Chem. 273, 31652 - 31655（1998）
3）Chisada, S. et al, Zebrafish and mouse alpha2,3sialyltransferases responsible for synthesizing GM4
ganglioside. J.Biol. Chem., 284, 30534 - 30546（2009）4）Uemura S. et al. Expression machinery of GM4:
The excess amounts of GM3/GM4S synthase（ST3GAL5）are necessary for GM4 synthesis in
mammalian cells. Glycoconj. J. 31, 101 - 108（2014）5）Hanada, K. et al. Molecular machinery for nonvesicular trafficking of ceramide. Nature, 426, 803 809,（2003）
6）Halter D, et al.: Pre- and post-Golgi translocation of
glucosylceramide in glycosphingolipid synthesis.
J Cell Biol, 179, 101 - 115（2007）7）Hayashi, Y. et al. Complex formation of
sphingomyelin synthase 1 with glucosylceramide
synthase increases sphingomyelin and decreases
glucosylceramide levels. J. Biol. Chem., 293, 1750517522（2018）8）Marcus D. My career as an immunoglycobiologist.
Proc. Jpn. Acad., Ser. B 89,（2013）doi: 10. 2183/pjab.
89. 257
9）DʼAngelo G. et al. Glycosphingolipid synthesis
requires FAPP2 transfer of glucosylceramide.
Nature, 449, 62 - 67（2007）

舞踏病アテトーゼ（choreoathetosis）、脊柱側弯症、脊椎麻痺、顔面形成異常、視覚異常等の症状を呈し、20歳前後で死亡する。その後同じGM3S
変異では、上記の症状に加えフランスでは難聴
24）、米国では皮膚の色素沈着異常 25）などの多彩な異常が報告されている。また、E355Kのミスセン
ス変異では皮膚の色素沈着異常を呈するSalt-andpepper症候群 26）、C195S、G201Rでは神経系の発達障害を呈するレット症候群（Rett syndrome）
様の症状などが報告されている27）。
GM2Sのミスセンス変異はクウェート、イタリア、
オールド・オーダー・アーミッシュの家系において、複合型遺伝性痙攣性対麻痺（ニューロパチー、小脳失調、脳梁の菲薄化、精神発達遅延、痙攣、難聴、網膜色素変性症、魚鱗癬などをともなう疾患）様の症状がある患者で同定されている28 - 30）。
GM3SノックアウトマウスとヒトのGM3S遺伝
子異常を比較すると、ヒトの表現型がより重篤であることがわかる。ヒトとマウスの表現型の違いを解明するには、ガングリオシド生合成制御機構
や関連遺伝子発現の相違等を比較検討していく必要があり、今後の課題である。
6

34

総論編
10）DʼAngelo G, et al. Vesicular and non-vesiculartransport feed distinct glycosylation pathways in
the Golgi. Nature, 501, 116 - 120（2013）11）Giraudo, C.G., & Maccioni, H.J. Ganglioside
Glycosyltransferases Organize in Distinct
Multienzyme Complexes in CHO-K1 Cells. J. Biol.
Chem., 278, 40262 - 40271（2003）12）Uemura, S. et al. Substitution of the N-glycanfunction in glycosyltransferases by specific aminoacids: ST3Gal-V as a model enzyme. Glycobiology, 16,
258 - 270（2006）13）Uemura, S. et al. The Cytoplasmic Tail of GM3
Synthase Defines Its Subcellular Localization,
Stability, and In Vivo Activity. Mol. Biol. Cell, 20,3088 - 3100（2009）14）Uemura, S. et al. The regulation of ER export and
Golgi retention of ST3Gal5（GM3/GM4 synthase）
a n d B 4 G a l N A c T 1（ G M 2 / G D 2 / G A 2 s y n t h a s e ）by arginine/lysine-based motif adjacent to thetransmembrane domain. Glycobiology, 25, 1410 - 422（2015）
15）Allende, M.L. and Rroia, R.L. Simplifying
complexity: genetically resculpting glycosphingolipid
synthesis pathways in mice to reveal function.
Glycoconj J. 31, 613 - 622（2014）.
16）Tagami, S., Inokuchi, J. et al., Ganglioside GM3
participates in the pathological conditions of insulin
resistance. J. Biol. Chem., 277, 3085 - 3092（2002）17）Yamashita, T. et al. Enhanced insulin sensitivity
in mice lacking ganglioside GM3. Proc Natl Acad Sci
USA, 100, 3445 - 3449（2003）
18）Zhao, H. et al. Inhibiting glycosphingolipid
synthesis improves glycemic control and insulin
sensitivity in animal models of type 2 diabetes.
Diabetes, 56, 1210 - 1218（2007）
19）Natoli, T.A., et al., Inhibition of glucosylceramide
accumulation results in effective blockade ofpolycystic kidney disease in mouse models. NatureMed. 16, 788 - 793（2010）20）Kabayama, K. et al. Dissociation of the insulin
receptor and caveolin-1 complex by ganglioside GM3
in the state of insulin resistance. Proc Natl Acad Sci.
USA, 104, 13678 - 13683（2007）21）Yoshikawa, M. et al. Mice lacking gangliosideGM3 synthase exhibit complete hearing loss due toselective degeneration of the organ of Corti. Proc.
Natl. Acad. Sci. USA, 106, 9483 - 9488（2009）22）Yamashita, T. et al. Interruption of gangliosidesynthesis produces central nervous systemdegeneration and altered axon-glial interactions.
Proc Natl Acad Sci USA, 102, 2725 - 2730（2005）23）Simpson, M.A. et al. Infantile-onset symptomatic
epilepsy syndrome caused by a homozygous loss-offunction mutation of GM3 synthase. Nat. Genet., 36,1225 - 1229（2004）
24）F r a g a k i , K . e t a l . R e f r a c t o r y e p i l e p s y a n d
mitochondrial dysfunction due to GM3 synthase

スフィンゴ糖脂質の生合成とその制御

deficiency. European journal of human genetics. 21,
528 - 534（2013）25）Wang, H. et al. Cutaneous dyspigmentation inpatients with ganglioside GM3 synthase deﬁciency.
Am J Med Genet. A161a, 875 - 879（2013）26） Boccuto, L. et al. A mutation in a gangliosidebiosynthetic enzyme, ST3GAL5, results in salt &pepper syndrome, a neurocutaneous disorder withaltered glycolipid and glycoprotein glycosylation.
Hum Mol Genet. 23, 418 - 433（2014）27）Lee, J.S. et al. GM3 synthase deficiency due to
ST3GAL5 variants in two Korean female siblings:
Masquerading as Rett syndrome-like phenotype. Am
J Med Genet. A161a, 875 - 879（2013）28） Boukhris, A. et al. Alteration of gangliosidebiosynthesis responsible for complex hereditary
spastic paraplegia. Am J Human Genet. 93, 118 - 123（2013）29）Harlalka, G.V. et al. Mutations in B4GALNT1（GM2synthase）underlie a new disorder of gangliosidebiosynthesis. Brain. 136, 3618 - 3624（2013）30）Wakil, S.M. et al. Novel B4GALNT1 mutations in acomplicated form of hereditary spastic paraplegia.
Clinical Genetics. 86, 500 - 501（2014）735㸺⏕య⭷᝟ሗ㒊㛛㸼ᩍᤵ ᮾ ⚽ዲຓᩍ ୰ᕝ ဴேຓᩍ 㯮⏣ ႐ᖾ

Gࢱࣥࣃࢡ㉁ඹᙺཷᐜయ㸦GPCR㸧࡟ࡼࡿ⢾㙐ྜᡂไᚚ࡜⢾㙐࡟ࡼࡿ
GPCRࡢάᛶไᚚ
1. ǃ2 ࢔ࢻࣞࢼࣜࣥཷᐜయࢩࢢࢼࣝ࡟ࡼࡿࣄ࢔ࣝࣟࣥ㓟ྜᡂࡢୖ᪼ ࣄ࢔ࣝࣟࣥ㓟㸦HA㸧ࡣࠊN-࢔ࢭࢳࣝࢢࣝࢥࢧ࣑ࣥ࡜ࢢࣝࢡࣟࣥ㓟ࡢ㸰⢾⧞ࡾ㏉ࡋᵓ㐀࠿ࡽᡂࡿ┤㙐ࡢ㧗ศᏊከ⢾࡛࠶ࡿࠋHA ࡣ⣽⬊እ࣐ࢺࣜࢵࢡࢫࡢᵓᡂศᏊ
࡜ࡋ࡚ࠊ⤌⧊ࡢᙎᛶࡸ≀ᛶ⥔ᣢ࡟㛵ࢃࡿ௚ࠊ๰യ἞⒵ࡸ⣽⬊ቑṪࢆไᚚࡍࡿࠋࡲ
ࡓࠊHA ⏘⏕ࡢቑῶࡸ HA ࡢࢧ࢖ࢬࡀࠊ⅖⑕཯ᛂࡸ⒴ࠊ⾑⟶⑌ᝈ࡞࡝ࡢ⑓ែࢆไ
ᚚࡍࡿࠋHA ࡣ⣽⬊⭷ୖ࡟Ꮡᅾࡋࠊ㸱✀㢮ࡢ࢖ࢯᆺࡀ▱ࡽࢀࡿࣄ࢔ࣝࣟࣥ㓟ྜᡂ㓝⣲㸦HAS1ࠊHAS2ࠊHAS3㸧࡟ࡼࡗ࡚⏘⏕ࡉࢀࡿࠋHAS ࡣከ✀ࡢቑṪᅉᏊࡸࢧ࢖ࢺ࢝࢖ࣥࠊ࣍ࣝࣔࣥ࡞࡝࡛㑇ఏᏊࡢⓎ⌧ࡀไᚚࡉࢀ࡚࠾ࡾࠊ⩻ヂᚋಟ㣭࡟ࡼࡾ
HA ⏘⏕⬟ࡀኚ໬ࡍࡿࠋ㏆ᖺࠊG ࢱࣥࣃࢡ㉁ඹᙺᆺཷᐜయ㸦GPCR㸧࡛࠶ࡿ UDPࢢࣝࢥ࣮ࢫཷᐜయࡢ P2Y14 ࡸ ATP/UTP ཷᐜయࡢ P2Y2 ࡢࢩࢢࢼࣝ࡟ࡼࡾࠊHAS2
㑇ఏᏊⓎ⌧ࡀୖ᪼ࡋ HA ⏘⏕ࡀஹ㐍ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀࠊ⤌⧊ᦆയ㒊఩࡛ࡢ HA⏘⏕ࡢቑຍ࡟ GPCR ࢩࢢࢼࣝࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ (1, 2)ࠋP2Y14 ࡣGǂi ࡜ඹᙺࡋࠊP2Y2 ࡣ Gǂq ࡜ඹᙺࡍࡿࡇ࡜࠿ࡽࠊGPCR ࢩࢢࢼ࡛ࣝࡢ HA ⏘⏕ࡣ༢୍ࢩࢢࢼࣝ࡟ࡼࡿไᚚ࡛ࡣ࡞࠸࡜⪃࠼ࡽࢀࡿࠋ
 ǃ2 ࢔ࢻࣞࢼࣜࣥཷᐜయ㸦ǃ2AR㸧ࡣ࢝ࢸࢥ࣑ࣛࣥࢆࣜ࢞ࣥࢻ࡜ࡍࡿ GPCR ࡛࠶ࡾࠊࡑࡢάᛶ໬ࡣ⾑⟶ࡸẼ⟶ᨭ࡞࡝ࡢᖹ⁥➽ࢆᘱ⦆ࡉࡏࡿస⏝ࢆ⌧ࡍࠋẼ⟶ᨭႍᜥࡢ἞⒪࡛౑⏝ࡉࢀࡿ ǃ సື⸆ࡣࠊǃ2AR ࡜ Gǂs ࡀඹᙺࡍࡿࡇ࡜࡛⏕ࡌࡿࢩࢢࢼ
ࣝࢆ௓ࡋ࡚Ẽ⟶ᨭᖹ⁥➽ࢆᘱ⦆ࡉࡏࡿࡇ࡜ࢆ┠ⓗ࡜ࡋ࡚࠸ࡿࠋࡲࡓࠊẼ⟶ᨭႍᜥ࡛ࡣẼ⟶ᨭࡢ⅖⑕཯ᛂࢆ࡜ࡶ࡞࠸ࠊẼ⟶ᨭႍᜥᝈ⪅ࡢ⫵⬊Ὑίᾮ୰ࡢ HA 㔞ࡀቑຍࡋ࡚࠸ࡿ (3)ࠋࡉࡽ࡟ࠊǃ సື⸆ࡢ㛗ᮇ㐃⏝ࡣࠊǃ2AR ࡜┦஫స⏝ࡋࡓ࢔ࣞࢫࢳ
ࣥ࡟ࡼࡿࢩࢢࢼࣝ࡟ࡼࡾࠊ⅖⑕཯ᛂࡢቑᝏࡸ࣒ࢳࣥࡢ⏘⏕ஹ㐍࡜࠸ࡗࡓ๪స⏝ࡀ⏕ࡌࡿࠋࡇࡢࡇ࡜࠿ࡽᡃࠎࡣࠊǃ2AR ࡟ࡼࡿࢩࢢࢼࣝࡀᖹ⁥➽ࡢ཰⦰ࡔࡅ࡛࡞ࡃࠊ
HA ⏘⏕࡟ࡶᙳ㡪ࡋ࠺ࡿࡢ࡛ࡣ࡞࠸࠿࡜⪃࠼ࡓࠋ ࡑࡇ࡛ࠊHEK293T㸦ࣄࢺ⫾ඣ⭈⮚⏤᮶⣽⬊㸧࡟ ǃ2AR ࢆⓎ⌧ࡉࡏࠊ㠀㑅ᢥⓗ ǃసື⸆࡛࠶ࡿ࢖ࢯࣉࣟࢸࣞࣀ࣮ࣝ㸦ISO㸧ࡸ ǃ2 㑅ᢥⓗసື⸆ࡢࢧࣝࣈࢱ࣮ࣔࣝ
㸦Sal㸧่࡛⃭ࡋࡓࠋᇵ㣴ᾮ୰࡟ศἪࡉࢀࡓ HA 㔞ࢆᐃࡍࡿ࡜ࠊǃ సື⸆่࡛⃭ࡋࡓ⣽⬊࡛ࡣ HA 㔞ࡀቑຍࡍࡿࡇ࡜ࡀศ࠿ࡗࡓࠋḟ࡟ࠊǃ సື⸆่⃭࡟ࡼࡿ HA
㔞ࡢቑຍࡀࠊHAS ࡢⓎ⌧ஹ㐍࡟ࡼࡿࡶࡢ࠿ࢆ᳨ウࡍࡿࡓࡵࠊࣜ࢔ࣝࢱ࢖࣒ PCR
࡟ࡼࡾ HAS 㑇ఏᏊࡢⓎ⌧㔞ࢆㄪ࡭ࡓࠋISO ࡛ 2 ᫬㛫ࠊǃ2AR Ⓨ⌧⣽⬊ࢆ่⃭ࡍ

36

ࡿ࡜ࠊHAS2 㑇ఏᏊࡢⓎ⌧ࡀஹ㐍ࡋࡓࠋHAS2 㑇ఏᏊࡢⓎ⌧ቑຍࡣ Sal ࡛ࡢ่⃭࡛ࡶྠᵝ࡟⏕ࡌࡓࡇ࡜࠿ࡽࠊǃ2AR ࡢάᛶ໬ࡀ HAS2 㑇ఏᏊࡢⓎ⌧ࢆஹ㐍ࡉࡏࡿ࡜⪃࠼ࡽࢀࡓࠋࡉࡽ࡟ࠊ
ǃ2AR ࢩࢢࢼࣝࡀ HAS2
㑇ఏᏊⓎ⌧ࢆஹ㐍ࡉࡏࡿࢩࢢࢼࣝఏ㐩⤒㊰ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊྛ✀㜼ᐖ๣࡛๓ฎ⌮ࢆ⾜࠸ࠊSal ่⃭࡟ࡼࡿ HAS2 㑇ఏᏊⓎ⌧࡬ࡢᙳ㡪ࢆㄪ࡭ࡓࠋࡑ
ࡢ⤖ᯝࠊǃ2AR ࡢάᛶ໬ࡣ
Gǂs - cAMP - PKA - JNK- STAT3 ࢩࢢࢼࣝࢆ௓ࡋ
࡚ HAS2 㑇ఏᏊࡢ㌿෗ࢆஹ㐍ࡋ࡚࠸ࡿࡇ࡜ࡀศ࠿
ࡗࡓࠋ HAS2 㑇ఏᏊࡢࣉ࣮ࣟࣔ ࢱ ࣮ 㡿 ᇦ ࡟ ࡣ STAT3⤖ྜ㒊఩ࡀᏑᅾࡋࠊ
STAT3 ά ᛶ ໬ ࡟ ࡼ ࡗ ࡚
HAS2 㑇ఏᏊⓎ⌧ࡀୖ᪼ࡍࡿ(4)ࠋࡲࡓࠊGǂs ᜏᖖЎ ΠχϪψϨϱण༲ର͹‫׈‬੓Կ͗ +$ ࢊਫ਼Ν ଁՅ͠ⓗάᛶ໬ኚ␗యⓎ⌧⣽⬊ͦΖεήψϩ࡛ࡣ cAMP - PKA ࢩࢢࢼЎ ࡠಊ༂ͶΓΖ Ў$5 ͹‫׈‬੓Կͺɼࢀྖର * νϱϏࣝ࡟ࡼࡗ࡚ STAT3 ࡢάέ࣯ʤ*ЍVʥΝղ͢ͱɼF$033.$ εήψϩΝ‫׈‬੓Կᛶ໬ࡀ⏕ࡌࡿ

ͦ͠Ζɽ3.$ ͺ -1. Ν‫׈‬੓Կͦ͠ɼ 67$7 ͹ 6HUΝϨϱ࢐ԿͤΖɽ͠ΔͶɼ-1. ͗ -$. Ν‫׈‬੓ԿͤΖ͞
ͳͲɼ67$7 ͹ 7\U ΝϨϱ࢐Կ͢ɼ67$7 ͹఺ࣺ‫׈‬੓ԿΝӀًͤ͘͞ɽ‫׈‬੓Կͪ͢ 67$7 ͗ +$6 ͹ϕϫϠʖνʖ྘ҮͶ݃߻͢ɼ+$6 Ҫఽࢢ͹఺ࣺΝထ਒͠(5)ࠋࡇࡢ

ࡇ࡜࠿ࡽࠊǃ2AR ࡟ࡼࡿ
HA ⏘⏕ࡢୖ᪼ࡣࠊǃ2ARࡢࢩࢢࢼࣝࡀ STAT3 ࢆάᛶ໬ࡋࠊHAS2 㑇ఏᏊࡢ㌿෗ࡀஹ㐍ࡍࡿࡇ࡜࡛
⏕ࡌࡿ࡜⪃࠼ࡽࢀࡿࠋ

ͦɼ +$6 νϱϏέ࣯͹൅‫ଁ͗ݳ‬ՅͤΖ͞ͳͶΓΕ +$

HAS2 㑇ఏᏊࡢࣉ࣮ࣟࣔ

ࢊਫ਼͗৏তͤΖͳߡ͓ΔΗΖɽࢱ࣮㡿ᇦ࡟ࡣ CREB ⤖ྜ㒊 ఩ ࡶ Ꮡ ᅾ ࡋ ࠊ cAMP -PKA ࢩࢢࢼࣝࡀ CREB ࢆάᛶ໬ࡍࡿ࡜ࡉࢀࡿࡀࠊᡃࠎࡢᐇ㦂⤖ᯝ࠿ࡽࡣ ǃ2ARࡢࢩࢢࢼࣝࡀ CREB ࡟ࡼࡾ HAS2 㑇ఏᏊࡢⓎ⌧ࢆไᚚࡍࡿࡇ࡜ࡣ☜ㄆ࡛ࡁ࡞࠿

37

ࡗࡓࠋ
 ࡲࡓࠊǃ2AR ࡢࢩࢢࢼࣝ࡟ࡣࠊ࢔ࣞࢫࢳࣥ࡜ࡢ┦஫స⏝ࢆ௓ࡋࡓࢩࢢࢼࣝࡶᏑ
ᅾࡍࡿࠋ࢔ࣞࢫࢳࣥࡢࢩࢢࢼࣝࡣ୺࡟ MAP ࢟ࢼ࣮ࢮ㸦MEK1/2 - ERK1/2㸧ࢆ௓ࡍࡿࡀࠊǃ2AR ࡟ࡼࡿ HAS2 㑇ఏᏊࡢⓎ⌧ቑຍࡣࠊMEK1/2 㜼ᐖ๣࡛ࡣᢚไࡉࢀ
࡞࠿ࡗࡓࠋẼ⟶ᨭႍᜥ࡟࠾࠸࡚ࡣࠊ࢔ࣞࢫࢳࣥࡢࢩࢢࢼࣝࡀ⅖⑕཯ᛂࡢቑᝏ࡞࡝
ࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࡀࠊǃ2AR ࡟ࡼࡿ HA ⏘⏕ࡢቑຍࡣ⅖⑕཯ᛂࡢᢚไࡸ⤌⧊ಟ᚟ࡢಁ㐍࡜࠸ࡗࡓ᭷┈࡞ຠᯝࡢࡳࢆ♧ࡍࡢ࠿ࡶࡋࢀ࡞࠸ࠋǃ2AR ࡢάᛶ໬࡟ࡼࡾ⏘⏕ࡉࢀࡓ HA ࡀ⣽⬊ࡸ⤌⧊࡟࡝ࡢࡼ࠺࡞స⏝ࢆཬࡰࡋ࡚࠸ࡿࡢ࠿
ࡣ௒ᚋࡢ᳨ウㄢ㢟࡛࠶ࡿࠋ
2. ࢞ࣥࢢࣜ࢜ࢩࢻ࡜ࢥࣥࢻࣟ࢖ࢳࣥ◲㓟่⃭࡟ࡼࡿࣈࣛࢪ࢟ࢽࣥ B2 ཷᐜయࡢ୙ᛂ໬࣭ෆᅾ໬ࡢᶵᵓ
 ࣈࣛࢪ࢟ࢽࣥ B2 ཷᐜయ㸦B2R㸧ࡣࠊ⾑⟶ᖹ⁥➽ࡸ⚄⤒⤌⧊ࢆࡣࡌࡵ࡜ࡍࡿከ
ࡃࡢ⤌⧊࡟ᜏᖖⓗ࡟Ⓨ⌧ࡋ࡚࠾ࡾࠊ⅖⑕ࡸ⑊③ࠊ⾑⟶ᣑᙇ࡟㛵୚ࡍࡿ GPCR ࡛࠶ࡿࠋ௨๓ᡃࠎࡣࠊB2R Ⓨ⌧ CHO-K1 ⣽⬊ࡢ࢞ࣥࢢࣜ࢜ࢩࢻ GT1b ࡜ࢥࣥࢻࣟ
࢖ࢳࣥ◲㓟 C㸦CSC㸧࡛ࡢ㡰ḟ่⃭࡟ࡼࡾࠊB2R ࡢ࢔ࢦࢽࢫࢺ࡛࠶ࡿࣈࣛࢪ࢟ࢽ
ࣥ㸦BK㸧࡟ࡼࡿ⣽⬊ෆ Ca2+ୖ᪼ࡀᢚไࡉࢀࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ (6)ࠋGPCR ࢆ࢔ࢦࢽࢫࢺ࡟㛗᫬㛫࠶ࡿ࠸ࡣ⧞ࡾ㏉ࡋᭀ㟢ࡍࡿ࡜ࠊ࢔ࣞࢫࢳࣥࡀ⤖ྜࡋ࡚ G ࢱࣥ
ࣃࢡ㉁࡜ࡢ⤖ྜࡀ㜼ᐖࡉࢀ㸦୙ᛂ໬㸧ࠊࡉࡽ࡟ࡣ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫࡉࢀࡿ㸦ෆᅾ໬㸧ࠋࡇࡢࡇ࡜࠿ࡽࠊGT1b ࡜ CSC ࡟ࡼࡿ㡰ḟ่⃭ࡣ࢔ࢦࢽࢫࢺ㠀౫Ꮡⓗ࡞୙ᛂ໬࣭ෆᅾ໬ࢆㄏᑟࡋࡓ࡜⪃࠼ࡽࢀࡓࡢ࡛ࠊࡇࡢ୙ᛂ໬ᶵᵓࢆゎᯒࡋࡓࠋ ࡲࡎࠊB2R Ⓨ⌧ HEK293 ⣽⬊ࢆ GT1b ࡜ CSC ࡟ࡼࡾ㡰ḟ่⃭ࡋࠊᢳฟࡋࡓ

*3&5 ͹‫׈‬੓Կͳ෈ԢԿʤGHVHQVLWL]DWLRQ ɼ಼ࡑԿ LQWHUQDOL]DWLRQ 

38⭷⏬ศ࡟ྵࡲࢀࡿ B2R ࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢸ࢕ࣥࢢ᳨࡛ฟࡋࡓࠋࡑࡢ⤖ᯝࠊGT1b ࡜ CSC ࡢ㡰ḟ่⃭࡟ࡼࡾ⣽⬊⭷ୖ࠿ࡽศᏊ㉁㔞ࡢᑠࡉ࠸ࠊᮍ⇍࡞ N ᆺ⢾㙐ࢆ᭷ࡍࡿ B2R ࡀῶᑡࡍࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ⣽⬊⭷࠿ࡽࡢ B2R ᾘኻࡣࠊGT1b
࠾ࡼࡧ CSC ࡢ༢⊂่⃭ࡸྠ᫬่⃭࡛ࡣ࡯࡜ࢇ࡝㉳ࡇࡽ࡞࠿ࡗࡓࠋḟ࡟ࠊ⣽⬊ୖࡢ B2R ῶᑡࡀ୙ᛂ໬࡟ࡼࡿࡶࡢ࠿ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊඹච␿ỿ㝆ἲࢆ⏝࠸࡚
B2R ࡜࢔ࣞࢫࢳࣥࡢ┦஫స⏝ࢆㄪ࡭ࡓࠋGT1b ࡜ CSC ࡢ㡰ḟ่⃭ᚋ࡟ࡣࠊ↓่⃭࡜ẚ㍑ࡋ࡚࢔ࣞࢫࢳࣥ࡜┦஫స⏝ࡋ࡚࠸ࡿ B2R ࡀከ࠸ࡇ࡜ࡀศ࠿ࡗࡓࠋࡲࡓࠊ
B2R ࡢ⢾㙐ࡢᡂ⇍ᗘ࡜ࡣ↓㛵ಀ࡟࢔ࣞࢫࢳࣥࡣ┦஫స⏝ࡋ࡚࠸ࡓࠋࡉࡽ࡟ࠊ
GPCR ࡀ࢔ࣞࢫࢳࣥ࡜┦஫స⏝ࡍࡿࡓࡵ࡟㔜せ࡛࠶ࡿ GPCR ࢟ࢼ࣮ࢮ㸦GRK㸧ࢆ㜼ᐖࡍࡿ࡜ࠊGT1b ࡜ CSC ࡢ㡰ḟ่⃭࡟ࡼࡿ B2R ࡢ⣽⬊⭷ୖ࠿ࡽࡢῶᑡࡶ
㜼ᐖࡉࢀࡓࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊGT1b ࡜ CSC ࡢ㡰ḟ่⃭ࡣࠊB2R ࡟ᑐࡋ࡚❧యᵓ㐀ୖࡢኚ໬ࢆᘬࡁ㉳ࡇࡋࠊB2R ࡀ GRK ࡟ࡼࡿࣜࣥ㓟໬ࢆཷࡅࡸࡍࡃ࡞ࡾࠊ
࢔ࢦࢽࢫࢺ㠀౫Ꮡⓗ࡟࢔ࣞࢫࢳࣥ࡜┦஫స⏝ࡍࡿࡇ࡜࡛୙ᛂ໬ࡋࠊෆᅾ໬ࡍࡿࡇ
࡜ࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊB2R ࡢ⢾㙐ࡢᡂ⇍ࡣࠊෆᅾ໬ࡉࢀࡸࡍࡉ࡟ᙳ㡪ࡍࡿࡇ࡜ࡶ♧၀ࡉࢀࡓࠋ

References
1. Jokela, T.A., Karna, R., Makkonen, K.M., Laitinen, J.T., Tammi, R.H., and Tammi,
M.I. (2014) Extracellular UDP-glucose activates P2Y14 Receptor and Induces Signal
Transducer and Activator of Transcription 3 (STAT3) Tyr705 phosphorylation andbinding to hyaluronan synthase 2 (HAS2) promoter, stimulating hyaluronan synthesisof keratinocytes. J Biol Chem. 289, 18569-18581
2. Rauhala, L., Jokela, T., Karna, R., Bart, G., Takabe, P., Oikari, S., Tammi, M. I.,
Pasonen-Seppanen, S., and Tammi, R.H. (2018) Extracellular ATP activates
hyaluronan synthase 2 (HAS2) in epidermal keratinocytes via P2Y2, Ca2+ signaling,and MAPK pathways. Biochem J. 475, 1755-1772
3. Garantziotis, S., Brezina, M., Castelnuovo, P., and Drago, L. (2016) The role ofhyaluronan in the pathobiology and treatment of respiratory disease. Am J Physiol
Lung Cell Mol Physiol. 310, L785-795
4. Tammi, R.H., Passi, A.G., Rilla, K., Karousou, E., Vigetti, D., Makkonen, K., andTammi, M.I. (2011) Transcriptional and post-translational regulation of hyaluronan
synthesis. Febs j. 278, 1419-1428
5. Liu, A.M., Lo, R.K., Wong, C.S., Morris, C., Wise, H., and Wong, Y.H. (2006)
Activation of STAT3 by G Įs distinctively requires protein kinase A, JNK, andphosphatidylinositol 3-kinase. J Biol Chem. 281, 35812-35825
6. Shimazaki, A., Nakagawa, T., Mitoma, J., and Higashi, H. (2012) Gangliosides andchondroitin sulfate desensitize and internalize B2 bradykinin receptors. Biochem.
Biophys. Res. Commun. 420, 193-198.

39

㸺Ꮫ఍Ⓨ⾲㸼
࣭ȕ2 ࢔ࢻࣞࢼࣜࣥཷᐜయࢩࢢࢼࣝ࡟ࡼࡿࣄ࢔ࣝࣟࣥ㓟ྜᡂࡢୖ᪼
㯮⏣႐ᖾࠊ୰ᕝဴேࠊᮾ ⚽ዲ➨ 38 ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪ྡྂᒇ㸪2019 ᖺ 8 ᭶㸪せ᪨㞟 p.154
࣭࢞ࣥࢢࣜ࢜ࢩࢻ࡜ࢥࣥࢻࣟ࢖ࢳࣥ◲㓟่⃭࡟ࡼࡿࣈࣛࢪ࢟ࢽࣥ B2 ཷᐜయࡢ୙
ᛂ໬ไᚚᶵᵓ
▼஭Ꮥ࿴㸪㯮⏣႐ᖾ㸪୰ᕝဴே㸪ᮾ ⚽ዲ
➨ 13 ᅇ ᮾ໭⢾㙐◊✲఍㸪᪂₲㸪2019 ᖺ 9 ᭶㸪せ᪨㞟 p.53

40㸺⣽⬊ไᚚᏛ㒊㛛㸼ᩍᤵ  㢳 ᘓᅜ෸ᩍᤵ ⚟⏣ ཭ᙪㅮᖌ  ఀᕥ἞ ▱ᘺ⢾㙐ಟ㣭ࡢ≉␗ᛶ࡜ཷᐜయࡢᶵ⬟ไᚚ୍⯡ⓗ࡟ࠊ⢾ࢱࣥࣃࢡ㉁ࡣᑠ⬊యࡸࢦࣝࢪయࡢ࢜ࣝ࢞ࢿࣛ࡟࠾࠸࡚⢾㙐࡛ಟ㣭ࡉࢀࠊ㐺ษ࡞ሙᡤ࡟㐠ࡤࢀᶵ⬟ࡍࡿ࡜ࡼࡃ▱ࡽࢀ࡚࠸ࡿࡀࠊࡑࡢヲ⣽࡞ศᏊᶵᗎࡣ
ᚲࡎࡋࡶ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞࠸ࠋ࢖ࣥࢸࢢࣜࣥࡣ ǂ 㙐࡜ ǃ 㙐࠿ࡽᡂࡿ࣊ࢸࣟ஧㔞య࡛ࠊ୺࡟⣽⬊እ࣐ࢺࣜࢵࢡࢫ㸦ECM㸧࡬ࡢ⣽⬊᥋╔ࢆ፹௓ࡍࡿ⭷㈏㏻ᆺ⢾ࢱࣥ
ࣃࢡ㉁࡛ࠊ⣽⬊ෆࢱࣥࣃࢡ㉁ࡢࢳࣟࢩࣥࣜࣥ㓟໬ࡸపศᏊ㔞 GTP ⤖ྜࢱࣥࣃࢡ㉁ࡢάᛶ໬ࢆ㏻ࡌ࡚⣽⬊ෆ㦵᱁⣔ࡢ෌⦅ᡂࡸቑṪ㺃ศ໬ࡢࢩࢢࢼࣝఏ㐩࡟῝ࡃ㛵ࢃࡿࠋࡲࡓࠊ࢖ࣥࢸࢢࣜࣥ࡜⣽⬊ቑṪᅉᏊཷᐜయ࡜ࡢ┦஫స⏝ࡣࠊ⣽⬊ࡢቑṪ࣭⛣ື࣭⏕Ꮡ࡞࡝ࡢ⏕⌮ⓗ࡞㐣⛬ࡢࡳ࡞ࡽࡎࠊࡀࢇ⣽⬊ࡢቑṪ࣭㌿⛣㺃ᾐ₶࡞࡝⑓⌮ⓗ࡞㐣⛬࡟ࡶ㔜せ࡞ᙺ๭ࢆᢸࡗ࡚࠸ࡿࠋࡇࢀࡲ࡛ࠊ⚾㐩ࡣ௦⾲ⓗ࡞⣽⬊᥋╔ศᏊ࡛࠶ࡿ࢖ࣥࢸࢢࣜࣥ ǂ5ǃ1 ࡟௜ຍࡉࢀࡓ N-ᆺ⢾㙐ࡢᶵ⬟ゎᯒࢆ⾜࠸ࠊN-ᆺ⢾㙐ࡀǂ5ǃ1 ࡢ࣊ࢸࣟ஧㔞యࡢᙧᡂࡸ⣽⬊⭷ୖ࡛ࡢⓎ⌧࡟ᚲ㡲࡛࠶ࡾࠊࡲࡓ࢖ࣥࢸࢢࣜࣥ
ࢆ௓ࡍࡿ⣽⬊⛣ື•ቑṪ࡞࡝ࡢᶵ⬟ࢆ≉␗ⓗ࡟ไᚚࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋࡋ
࠿ࡋࠊࡑࡢ≉␗ᛶࢆ⏕ࡌࡿศᏊᶵᵓ࡟㛵ࡋ࡚ࡣࡲࡔ୙࡛᫂࠶ࡿࠋᡃࠎࡣࠊࢩ࢔ࣝ㓟㌿⛣㓝⣲ࡸ ǂ1,6 ࣇࢥ࣮ࢫ(ࢥ࢔ࣇࢥ࣮ࢫ)㌿⛣㓝⣲ (Fut8)࡞࡝ࡢᶵ⬟࡟ὀ┠ࡋࠊ࢖ࣥࢸࢢࣜࣥ ǂ5ǃ1 ࡸࢡࣛࢫ III ཷᐜయࢳࣟࢩࣥ࢟ࢼ࣮࡛࠶ࡿ Fms-Like Tyrosine
Kinase 3㸦FLT3㸧ࡀࣔࢹࣝศᏊ࡜ࡋ࡚ࠊ⢾㙐ಟ㣭ࡢ≉␗ᛶ࠾ࡼࡧ⢾㙐࡟ࡼࡿཷᐜ
యࡢᶵ⬟ไᚚ࡟㛵ࡋ࡚୺࡟௨ୗࡢ㸱Ⅼ࡟ࡘ࠸࡚◊✲ࡋࡓࠋ
I. N-ᆺ⢾㙐ࡢ ǂ2,3 ࢩ࢔ࣜࣝ໬ࡢ≉␗ᛶ࡟㛵ࡍࡿ◊✲ࢩ࢔ࣝ㓟࡞࡝⢾㙐ᮎ➃࡟఩⨨ࡍࡿᵓ㐀ࡣࠊ⢾㙐࡜⢾㙐ㄆ㆑ศᏊ࡜ࡢ┤᥋ⓗ࡞᥋
ゐࡢሙࢆᙧᡂࡍࡿࡓࡵࠊ⑓ែኚ໬ࡸឤᰁ࣭ච␿స⏝࡟࠾࠸࡚ᯝࡓࡍᙺ๭ࡀ኱ࡁ࠸ࠋ
ࢩ࢔ࣝ㓟ࢆ௜ຍࡍࡿࢩ࢔ࣝ㓟㌿⛣㓝⣲ࡣࢫ࣮ࣃ࣮ࣇ࢓࣑࣮ࣜࢆᙧᡂࡋ࡚࠸ࡿࡀࠊ
N ᆺ⢾㙐࡟௜ຍࡉࢀࡿࡢࡣ ǂ2,3 ࡜ ǂ2,6 ⤖ྜ࡛࠶ࡿࠋǂ2,6 ⤖ྜ࡛௜ຍࡍࡿࡢࡣ
ST6GAL1 㑇ఏᏊ 1 ✀㢮ࡔࡀࠊྠࡌ ǂ2,3 ⤖ྜࢆゐ፹ࡍࡿ ǂ2,3 ࢩ࢔ࣝ㓟㌿⛣㓝⣲ࡣ ST3GAL3ࠊST3GAL4 ࠾ࡼࡧ ST3GAL6 ࡢ 3 ✀ࡢ㓝⣲ࡀᏑᅾࡋ࡚࠸ࡿࠋࡇࡢ
ࡓࡵࠊǂ2,3 ࢩ࢔ࣝ㓟ࡀ⏕య࡟㔜せ࡛࠶ࡿࡇ࡜ࡣከࡃࡢሗ࿌࠿ࡽ᫂ࡽ࠿࡛࠶ࡿ࡟ࡶ
࠿࠿ࢃࡽࡎࠊ3 ✀Ꮡᅾࡍࡿࡇ࡜࡟ព࿡ࡸࠊࡑࡢᶵ⬟ゎᯒࡣᮍゎ᫂࡞ࡲࡲṧࡉࢀ࡚࠸ࡿࠋࡇࡢၥ㢟࡟࢔ࣉ࣮ࣟࢳࡍࡿࡓࡵࠊCRISPER/Cas9 ࢩࢫࢸ࣒ࢆ⏝࠸࡚ࠊࡑࢀࡒࢀ
㓝⣲Ḟᦆ㸦KO㸧⣽⬊ᰴࢆసᡂࡋࡓࠋ⣽⬊ቑṪࠊࢥࣟࢽ࣮ᙧᡂ⬟ࡸ ERK࣭AKT ࡞

41࡝ࢩࢢࢼࣝఏ㐩࡟㛵ࢃࡿศᏊࡢࣜࣥ㓟໬ࢆᣦᶆ࡟᳨ウࡋࡓ࡜ࡇࢁࠊ
ST3GAL4-KO ࡣ ST3GAL3-KO ࡸ
ST3GAL6-KO ⣽⬊࡜ࡣ␗࡞ࡿ⾲⌧ᆺࢆ♧ࡋࡓ(1)ࠋࡇࢀࡣࠊ␗࡞ࡿ ǂ2,3 ࢩ࢔ࣝ㓟㌿⛣㓝⣲ࡀྠࡌ⣽⬊ෆ࡟Ꮡᅾࡋ࡚
࠸࡚ࡶࠊ┦⿵ࡋ࡚࠸࡞࠸ࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋǃ1 ࢖ࣥࢸࢢࣜࣥࡸ EGFR ࢆࣔࢹࣝᶆⓗศᏊ࡜ࡋ࡚ࡼࡾヲ⣽࡟᳨ウࡋࡓࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊǃ1 ࢖ࣥࢸࢢࣜࣥࡢ ǂ2,3 ࢩ࢔ࣜࣝ໬ࡣ ST3GAL4-KO ⣽⬊࡛పୗࡍࡿࡢ࡟ᑐࡋ࡚ࠊEGFR ࡢ ǂ2,3 ࢩ࢔ࣜࣝ໬ࡣ ST3GAL6-KO ⣽⬊࡛పୗࡋࡓ (ྑᅗ)ࠋࡉࡽ࡟ࠊST3GAL6-KO ⣽⬊࡟ 3 ࡘࡢ㑇ఏᏊࢆࡑࢀࡒࢀ㐣๫Ⓨ⌧ࡋࡓ࡜ࡇࢁࠊST3GAL6 ࡢ㐣๫Ⓨ⌧ࡢࡳࡀ EGFR ࡢ ǂ2,3 ࢩ࢔ࣜࣝ໬࡜⣽⬊ᙧែࢆᅇ᚟ࡉࡏࡿࡇ࡜ࡀ࡛ࡁࡓࠋྠᵝ࡟ࠊST3GAL4-KO ⣽⬊࡟ ST3GAL4 ࢆ㐣๫Ⓨ⌧ࡉࡏࡓ᫬ࡢࡳ ǃ1 ࡢࢩ࢔ࣜࣝ໬ࡀᅇ᚟ࡋࡓࠋ
ࡇࢀࡽࡢ⤖ᯝࡣࠊ3 ✀ࡢ ǂ2,3 ࢩ࢔ࣝ㓟㌿⛣㓝⣲ࡀࡑࢀࡒࢀᅛ᭷ࡢᙺ๭ࢆࡶࡕࠊ␗
࡞ࡿᶆⓗࢱࣥࣃࢡ㉁ࢆಟ㣭ࡋ࡚࠸ࡿࡇ࡜ࢆ᫂☜࡟♧ࡋ࡚࠸ࡿࠋ့ங㢮ࡢ」㞧࡞⏕యࢩࢫࢸ࣒ไᚚࡢࡓࡵ࡟ࠊከᵝ࡞ࢩ࢔ࣝ㓟㌿⛣㓝⣲ࡀᚲせ࡞⌮⏤ࡢゎ᫂࡟⧅ࡀࡿ
ྍ⬟ᛶࡀ࠶ࡿࠋ
II. EpCAM ࡀ࢖ࣥࢸࢢࣜࣥ ǃ1 ࡜┦஫స⏝ࡋ⒴⣽⬊ࡢ᥋╔࡜⛣ືࢆไᚚࡍࡿ
࢖ࣥࢸࢢࣜࣥࡸ࢝ࢻ࣊ࣜࣥ࡞࡝ࡢ⣽⬊᥋╔ศᏊࡣ⣽⬊ࡢᶵ⬟ࡸ⣽⬊㛫ࢥ࣑ࣗࢽࢣ࣮ࢩࣙࣥ࡟῝ࡃ㛵ࢃࡿࠋୖ⓶≉␗ⓗ࡞⣽⬊㛫᥋╔ศᏊ࡟ EpCAM㸦Epithelial
cell adhesion molecule㸧ࡀ࠶ࡿࠋEpCAM ࡣࠊከࡃࡢࡀࢇ࡟࠾࠸࡚㧗࠸Ⓨ⌧ࡀㄆࡵࡽࢀࡿࡓࡵࠊ἞⒪ᶆⓗ࣭デ᩿࠾ࡼࡧணᚋࡢ࣐࣮࣮࢝࡜ࡋ࡚ὀ┠ࡉࢀ࡚࠸ࡿศᏊ
࡛࠶ࡿࡀࠊࡑࡢᶵ⬟࡟ࡘ࠸࡚୙᫂࡞Ⅼࡀከ࠸ࠋࡀࢇ◊✲ࡢࣔࢹࣝ⣽⬊ᰴ 7 ✀(MiaPaCa-2, A431, CW2, 293T, HeLa, MDA-MB231 ࠾ࡼࡧ HepG2)ࡢ EpCAM Ⓨ⌧ࢆㄪ࡭ࡓ࡜ࡇࢁࠊ኱⭠⒴⣽⬊ᰴ CW-2 ࠾ࡼࡧ㢮ୖ⓶⒴⣽⬊ᰴ A431 ࡛≉࡟㧗࠸Ⓨ⌧ࢆࡳ࠸ࡔࡋࡓ(2)ࠋ㧗Ⓨ⌧ࡋ࡚࠸ࡿ⣽⬊࡟࠾ࡅࡿ EpCAM ࡢᶵ⬟ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊCRISPER/Cas9 ࢩࢫࢸ࣒ࢆ⏝࠸࡚ࠊ
EpCAM Ḟኻ⣽⬊ࢆసᡂࡋࡓࠋCW-2 ⣽⬊ࡸ A431 ⣽⬊࡜ࡶ࡟ࠊEpCAM ࡢⓎ⌧ࡀ↓ࡃ࡞ࡿ࡜ࠊ⣽⬊ࡢᙧែࡀ㛗ࡃఙࡧࡓ⣽⬊࠿ࡽ୸࠸ᙧែ࡟ኚ໬ࡋࡓࠋࡇࡢኚ໬ࡣࠊEpCAM ࡀ⣽⬊᥋╔࣭ఙᒎࢆไᚚࡍࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋࡲࡓࠊ⣽⬊ቑṪ࣭⛣ື࣭ࢥࣟࢽ࣮ᙧᡂ⬟ࡢపୗ࡜ࠊ࢖ࣥࢸࢢࣜࣥࡢୗὶࡢ୰ᚰศᏊ࡛࠶ࡿ focal
adhesion kinase(FAK)ࡸࠊAKTࠊERK ࡞࡝⣽⬊ෆࡢࢩࢢࢼࣝศᏊࡢࣜࣥ㓟໬ࡶపୗࡋࡓࠋ࢖ࣥࢸࢢ࢖ࣥ࡜ EpCAM ࡢ⏕⌮ⓗ┦஫స⏝ࢆㄪ࡭ࡿࡓࡵࠊඹච␿ỿ㝆ἲࢆ⏝࠸᳨࡚ウࡋࡓ࡜ࡇࢁࠊEpCAM ࡣ࢖ࣥࢸࢢࣜࣥ ǃ1 㙐࡜」ྜయࢆᙧᡂࡋ࡚࠸

42ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋࡉࡽ࡟ࠊ࢖ࣥࢸࢢࣜࣥ ǃ1 㙐࡜࣊ࢸࣟ஧㔞యࢆᙧᡂࡋ࡚࠸ࡿ࢖
ࣥࢸࢢࣜࣥ ǂ 㙐ࡢⓎ⌧ࢆ᳨ウࡋࡓ࡜ࡇࢁࠊ࢖ࣥࢸࢢࣜࣥ ǂ5 㙐ࡢⓎ⌧ࡀ EpCAM
Ḟᦆ࡟ࡼࡾపୗࡍࡿࡇ࡜ࡀศ࠿ࡗࡓࠋࡇࡢࡇ࡜ࡣࠊᏳᐃⓗ࡞࢖ࣥࢸࢢࣜࣥ ǂ5ǃ1 ࡢⓎ⌧࡟ EpCAM ࡀ㔜せ࡛࠶ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ௨ୖ࠿ࡽࠊEpCAM ࡢⓎ⌧࡜ṇ࡟┦㛵ࡍࡿࡀࢇࡢ἞⒪᢬ᢠᛶ⋓ᚓࡣࠊEpCAM-࢖ࣥࢸࢢࣜࣥ」ྜయ࡟ࡼࡿ⣽⬊ᶵ⬟ࡢไᚚ࡟㛵㐃ࡍࡿྍ⬟ᛶࡀᙉࡃ♧၀ࡉࢀࡓࠋࡇࢀࡣࠊ௒ᚋ EpCAM-࢖ࣥࢸࢢ
ࣜࣥ」ྜయࢆᶆⓗ࡜ࡋࡓ᪂ࡋ࠸ᢠ⭘⒆ᡓ␎ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࢁ࠺ࠋ
ࡲࡓࠊ᭱㏆ࠊᡃࠎࡣ࢖ࣥࢸࢢࣜࣥ ǂ5 㙐ࡢࡳ࡞ࡽࡎࠊǃ1 㙐ୖࡢ EGFR ࡜⤖ྜࡍࡿ⢾㙐௜ຍ㒊఩ࡢྠᐃ࡟ᡂຌࡋࡓ(3)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊ⭷ୖࡢ࢖ࣥࢸࢢࣜࣥࢆ௓
ࡍࡿ」ྜయࡢᙧᡂᶵᗎࡢゎ᫂࡟ᙺ࡟❧ࡘ࡜⪃࠼ࡽࢀࡿࠋ
III. FLT3 ࢆ௓ࡋࡓࢩࢢࢼࣝఏ㐩⤒㊰άᛶ໬࡟࠾ࡅࡿ Fut8 ࡢᶵ⬟
Fms-Like Tyrosine Kinase 3㸦FLT3㸧ࡣࢡࣛࢫ III ཷᐜయࢳࣟࢩࣥ࢟ࢼ࣮ࢮࣇ
࢓࣑࣮ࣜࡢ࣓ࣥࣂ࣮࡟ᒓࡍࡿ⢾ࢱࣥࣃࢡ㉁࡛ࠊᛴᛶ㦵㧊ᛶⓑ⾑⑓㸦AML㸧ᝈ⪅ࡢ⣙ 3 ศࡢ 1 ࡟άᛶ໬ኚ␗ࡀㄆࡵࡽࢀࡿࠋFLT3 ࡣࣜ࢞ࣥࢻࡢ⤖ྜ࡟ࡼࡾࠊ஧㔞య໬࣭⮬ᕫࣜࣥ㓟໬ࡀ⏕ࡌࡿࡀࠊάᛶ໬ኚ␗࡛ࡣࣜ࢞ࣥࢻ㠀Ꮡᅾୗ࡛ࡶᜏᖖⓗ࡟άᛶ໬ࡉࢀࡿࠋ⣽⬊ࡣ⑓ⓗ≧ែ࡟㝗ࡿ࡜ࠊṇᖖ࡜ࡣ␗࡞ࡗࡓᵓ㐀ࢆᣢࡘ⢾㙐ࡀ⏕ᡂࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࡢ࡛ࠊᮏ◊✲࡛ࡣ FLT3 ࡢ⣽⬊እࢻ࣓࢖ࣥ࡟௜ຍࡉࢀࡓ
N ᆺ⢾㙐࡟↔Ⅼࢆᙜ࡚ࡓゎᯒࢆ⾜ࡗࡓࠋ㔝⏕ᆺ㸦WT㸧ࠊ⭷㈏㏻ࢻ࣓࢖ࣥࡢ㏆ഐ࡟
ぢࡽࢀࡿෆ㒊ࢱࣥࢹ࣒㔜」㸦ITD㸧ࠊ࠾ࡼࡧ FLT3 ࡢࢳࣟࢩࣥ࢟ࢼ࣮ࢮࢻ࣓࢖ࣥ
㸦TKD㸧ࡢάᛶ໬ኚ␗యࡢ N ⤖ྜᆺ⢾㙐ࡢᵓ㐀ࡀ␗࡞ࡿࡇ࡜ࢆぢฟࡋࡓ(4)ࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊIL-3 ౫Ꮡᛶ pro-B ⣽⬊ᰴ Ba/F3 ࡟ WT ࡲࡓࡣኚ␗య FLT3 㑇ఏᏊࢆᑟධࡋࡓ࡜ࡇࢁࠊ⣽⬊ࡢࢥ࢔ࣇࢥࢩࣝ໬ࢆ኱ࡁࡃㄏᑟࡋࡓࠋ FLT3 ࢆ௓ࡋࡓࢩࢢࢼࣝఏ㐩࡟࠾ࡅࡿࢥ࢔ࣇࢥࢩࣝ໬ࡢᶵ⬟ࢆゎ᫂ࡍࡿࡓࡵ࡟ࠊCRISPR / Cas9ࢩࢫࢸ࣒ࢆ౑⏝ࡋ࡚ࠊࢥ࢔ࣇࢥࢩࣝ
໬㓝⣲࡛࠶ࡿ Fut8 ࢆḞᦆࡉࡏࡓࣀ
ࢵࢡ࢔࢘ࢺ㸦KO㸧Ba/F3 ⣽⬊ࢆ☜❧ࡋ FLT3 㑇ఏᏊࢆᑟධࡋࡓ࡜ࡇࢁࠊ
㦫ࡃ࡭ࡁࡇ࡜࡟ࠊFLT3-WT ࢆⓎ⌧ࡉࡏࡓ KO ⣽⬊ࡣ IL-3 㠀౫Ꮡⓗ࡟⣽⬊ቑṪࢆᘬࡁ㉳ࡇࡋࡓ㸦ྑᅗ㸧ࠋIL3 㠀Ꮡᅾୗ࡛ࡢ Ba/F3 ⣽⬊ࡢቑṪࡣ
FLT3 ࡢάᛶ໬࡜᫂☜࡞┦㛵ࢆ♧ࡍࡇ࡜࠿ࡽࠊࡇࡢቑṪࡣⓎ⌧ࡉࡏࡓ㔝⏕ᆺ FLT3 ࡀࢥ࢔ࣇࢥࢩࣝࡉࢀ࡚࠸࡞࠸ࡇ࡜࡟ࡼࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋFut8KO⣽⬊࡟Ⓨ⌧ࡉࡏࡓ㔝⏕ᆺ FLT-3(FLT3-WT)ࡣࠊୗὶ࡛࠶ࡿ STAT5ࠊAKT ࠾ࡼࡧ
ERK ࢩࢢࢼࣝఏ㐩⤒㊰ࢆάᛶ໬ࡉࡏࠊ⣽⬊ࡢࢳࣟࢩࣥࣜࣥ㓟໬ࣞ࣋ࣝࢆ኱ᖜ࡟ቑ

43ຍࡉࡏࡓࠋࡇࢀࡽࡢάᛶ໬ࡣࠊFut8 ࡢⓎ⌧ࢆᅇ᚟ࡉࡏࡿࡇ࡜࡛ᾘኻࡋࡓࠋࡉࡽ࡟ࠊ
ࢳࣟࢩࣥ࢟ࢼ࣮ࢮ㜼ᐖ๣ࢆῧຍࡋࡓ࡜ࡇࢁࠊFut8KO ࡲࡓࡣࣇࢥࢩࣝ໬㜼ᐖ๣࡟ࡼࡗ࡚ㄏᑟࡉࢀࡿ⣽⬊ቑṪࡀ㜼ᐖࡉࢀࡓࠋࢥ࢔ࣇࢥࢩࣝ໬࡟ࡼࡿ FLT3 ࡢάᛶ໬ࡢᶵᵓࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊᢠ FLT3 ᢠయࢆ⏝࠸࡚Ⓨ⌧㒊఩ࢆ᳨ウࡋࡓ࡜ࡇࢁࠊ
FLT3-WT ⣽⬊࡜ྠࡌⓎ⌧ࣃࢱ࣮ࣥࠊࡍ࡞ࢃࡕ୺࡟⣽⬊⾲㠃࡟Ⓨ⌧ࡋ࡚࠸࡚ࠊⓎ⌧㒊఩࡟ኚ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡑࡇ࡛ࠊFLT3 ࡢࣜ࢞ࣥࢻ㠀Ꮡᅾୗ࡛ࡶ FLT3ࡀ஧㔞య໬࣭⮬ᕫࣜࣥ㓟໬࡟࡛ࡁࡿ࠿᳨ウࡋࡓ࡜ࡇࢁࠊFut8KO ⣽⬊࡛ࡣࣜ࢞ࣥ
ࢻ่⃭࡞ࡋ࡛ FLT3 ࡢ஧㔞యᙧᡂࡀㄏᑟࡉࢀ࡚࠸ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ
ᮏ◊✲ࡣࠊࢥ࢔ࣇࢥࢩࣝ໬ࡀ FLT3 ࡢάᛶ໬ࡢㄪ⠇ᶵ⬟࡟῝ࡃ࠿࠿ࢃࡗ࡚࠸ࡿࡇ
࡜ࢆ♧ࡋࡓࠋࡇࡢ▱ぢࡣࢥ࢔ࣇࢥࢩࣝ໬ࡢไᚚ࡟ࡼࡿ AML ࡢ἞⒪࡟᭷ຠ࡞⸆๣
ࡢ㛤Ⓨ࡟౯್࠶ࡿ᪉ྥᛶࢆᥦ౪ࡍࡿࡶࡢ࡛࠶ࢁ࠺ࠋ

㸺Ⓨ⾲ㄽᩥ㸼
1. ST3GAL3, ST3GAL4, and ST3GAL6 differ in their regulation of biological functions
via the specificities for the Į2,3ဨsialylation of target proteins. Qi, F., Isaji, T., Duan,
C., Yang, J., Wang, Y., Fukuda, T. and Gu, J. FASEB J., 34: 881-879. 2020
2. EpCAM associates with integrin and regulates cell adhesion in cancer cells. Yang, J.,
Isaji, T., Zhang, G., Qi, F., Duan, C., Fukuda, T. and Gu, J. Biochem Biophys Res
Commun. doi: 10.1016/j.bbrc.2019.11.152., 2019
3. Identification of the minimal N-glycosylation on integrin Į5ȕ1 required for its inhibitory
effect on EGFR signaling and cell proliferation. Hou, S., Hang, Q., Isaji, T., Fukuda, T.
and Gu, J. Biochem Biophys Res Commun. doi: 10.1016/j.bbrc.2019.11.188., 2019
4. Deficiency of core fucosylation activates cellular signaling dependent on FLT3
expression in a Ba/F3 cell system. Duan, C., Fukuda, T., Isaji, T., Qi, F., Yang, J., Wang,
Y., Takahashi, S. and Gu, J. FASEB J., doi: 10.1096/fj.201902313RR. 2020
5. Core Fucosylation of Maternal Milk N-glycan Evokes B Cell Activation by Selectively
Promoting the L-fucose Metabolism of Gut Bifidobacterium spp. and Lactobacillus spp.
li, M., Bai, Y., Zhou, J., Huang, W., Yan, J., Tao, J., Fan, Q., Liu, Y., Mei, D., Yan, Q.,
Yuan, J., Malard, P., Wang, Z., Gu, J., Taniguchi, N. and Li, W. MBio. doi:
10.1128/mBio.00128-19, 2019
6. Establishment and characterization of a fucosylated protein-specific mAb for fucosylated
D-fetoprotein: a potential application for clinical research. Egashira1, Y., Suganuma, M.,
Kataoka, Y., Higa, Y., Ide, N., Kamada, Y., Gu, J., Fukagawa1, K. and Miyoshi, E. Sci
Rep. 9:12359. doi: 10.1038/s41598-019-48821-x. 2019
7. Ablation of core fucosylation attenuates the signal transduction via T cell receptor tosuppress the T cell development. Liang, W., Mao, S., Li, M., Zhang, N., Sun, S., Fang,
H., Zhang, J., Gu, J., Wang, J. and Li, W. Mol Immunol. 112:312-321. doi:
10.1016/j.molimm.2019.06.011., 201944

㸺Ꮫ఍Ⓨ⾲㸼
࣭ Functions of glycosylation in cell adhesion and EMT of cancer cells㢳ᘓᅜ,୰ᅜ⛉Ꮫ㝔㐣⛬ᕤ⛬◊✲ᡤ࣭⏕໬ᕤ⛬ᅜᐙ㔜Ⅼᐇ㦂ᣐⅬ◊✲ㅮᗙ㸪໭ிᕷ㸪2019 ᖺ 3 ᭶
࣭ PTM of glycosylation in cell adhesion and EMT of cancer cells and diseases
㢳ᘓᅜ,໭ி኱Ꮫ࣭⏕࿨⛉ᏛᏛ㝔Ꮫ⾡ࢭ࣑ࢼ࣮㸪໭ிᕷ㸪2019 ᖺ 3 ᭶
࣭ Glycosylation in cancer cells and diseases
㢳ᘓᅜ,ኳὠ་⛉኱Ꮫ࣭ᇶ♏་Ꮫ㝔Ꮫ⾡ࢭ࣑ࢼ࣮㸪ኳὠᕷ㸪2019 ᖺ 3 ᭶
࣭ Significance of core fucosylation in brain and its relationship with diseases㢳ᘓᅜ2019 International Academic Forum on Neuroinflammation and Psychopharmacology,
༡㏻ᕷ, 2019 ᖺ 5 ᭶, せ᪨㞟 p.6࣭⢾⢾㙐ಟ㣭࡟ࡼࡿ࢖ࣥࢸࢢࣜࣥࡢ㑅ู㍺㏦ࢰ࣮ࣥࡢไᚚ࡜ࡑࡢศᏊᶵᗎࡢゎ᫂ 㢳ᘓᅜ
 ➨ 2 ᅇ࢜ࣝ࢞ࢿࣛࢰ࣮ࣥ ◊✲఍, ᮾி, 2019 ᖺ 5 ᭶
࣭Glycosylation in cell adhesion and EMT of cancer cells and diseases 㢳ᘓᅜInternational Brain Workshop 2019 in Sendai: Molecular Basis and Therapeutics for
Intractable Neuropsychiatry Diseases, ௝ྎᕷ, 2019 ᖺ 8 ᭶㸪せ᪨㞟 p.14
࣭⣽⬊ෆእࡢ⢾㙐Ⓨ⌧࡜࢖ࣥࢸࢢࣜࣥᶵ⬟࡜ࡑࡢไᚚ
㢳ᘓᅜ㸪ఀᕥ἞▱ᘺ㸪⚟⏣཭ᙪ➨ 38 ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪ྡྂᒇ㸪2019 ᖺ 8 ᭶㸪せ᪨㞟 p.131
࣭PI4KIIĮ ࡜࢖ࣥࢸࢢࣜࣥ Į3ȕ1 ࡢ」ྜయᙧᡂ࡟ࡼࡿ N-ᆺ⢾㙐ㄪ⠇ఀᕥ἞▱ᘺ㸪௵⩧ቲ㸪டᒣ᫛ᙪ a㸪⚟⏣཭ᙪ㸪㢳ᘓᅜ(ᅜ❧◊✲㛤Ⓨἲே⏘ᴗᢏ⾡⥲ྜ◊✲ᡤ ⏕࿨ᕤᏛ㡿ᇦ a)➨ 38 ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪ྡྂᒇ㸪2019 ᖺ 8 ᭶㸪せ᪨㞟 p.132
࣭Į1,6 ࣇࢥࢩࣝࢺࣛࣥࢫࣇ࢙࣮ࣛࢮ(Fut8)Ḟᦆ࡟ࡼࡿࢢࣜ࢔⣽⬊ࡢάᛶ໬⚟⏣཭ᙪࠊᗉᏊ㞙ேࠊ㝣㟂ࠊᙇ෤ᱵࠊఀᕥ἞▱ᘺࠊ㢳ᘓᅜ➨ 38 ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪ྡྂᒇ㸪2019 ᖺ 8 ᭶㸪せ᪨㞟 p.210
࣭⒴⒴⣽⬊࡟࠾ࡅࡿ PI4KIIĮ ࡜࢖ࣥࢸࢢࣜࣥ Į3ȕ1 ࡢ」ྜయᙧᡂ࡟ࡼࡿ N-ᆺ⢾㙐ࡢㄪ⠇ఀᕥ἞▱ᘺ㸪௵⩧ቲ㸪டᒣ᫛ᙪ a㸪⚟⏣཭ᙪ㸪㢳ᘓᅜ(ᅜ❧◊✲㛤Ⓨἲே⏘ᴗᢏ⾡⥲ྜ◊✲ᡤ ⏕࿨ᕤᏛ㡿ᇦ a)➨ 13 ᅇᮾ໭⢾㙐◊✲఍, ᪂₲, 2019 ᖺ 9 ᭶, せ᪨㞟 p.20
࣭ ST3GAL3, ST3GAL4, and ST3GAL6 differ in their regulation of biological
functions via the specificities for the Į2,3-sialylation of target proteins
Feng Qi, Tomoya Isaji, Chengwei Duan, Jie Yang, Yuqin Wang,
Tomohiko Fukuda and Jianguo Gu45

➨ 13 ᅇᮾ໭⢾㙐◊✲఍, ᪂₲, 2019 ᖺ 9 ᭶, せ᪨㞟 p.58
࣭PI4KIIĮ ࡜࢖ࣥࢸࢢࣜࣥ Į3ȕ1 ࡢ」ྜయᙧᡂ࡟ࡼࡿ⒴⣽⬊࡟࠾ࡅࡿ N-ᆺ⢾㙐ࡢㄪ⠇ఀᕥ἞▱ᘺ, ௵ ⩧ቲ, டᒣ᫛ᙪ a, ⚟⏣཭ᙪ, 㢳ᘓᅜ㸦ᅜ❧◊✲㛤Ⓨἲே⏘ᴗᢏ⾡⥲ྜ◊✲ᡤ ⏕࿨ᕤᏛ㡿ᇦ a㸧
➨ 92 ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍㸪ᶓ὾㸪2019 ᖺ 9 ᭶㸪₇㢟␒ྕ P-0191㸪T10m-02
࣭ࢥࢥ࢔ࣇࢥ࣮ࢫ࡟ࡼࡿࢢࣜ࢔⣽⬊㐣άᛶ໬ࡢᢚไ
⚟⏣཭ᙪ, ᗉᏊ㞙ே, 㝣㟂, ᙇ෤ᱵ, ఀᕥ἞▱ᘺ, 㢳ᘓᅜ➨ 92 ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍㸪ᶓ὾㸪2019 ᖺ 9 ᭶㸪₇㢟␒ྕ 3P-008
࣭Functional N-glycosylation in cell adhesions 㢳ᘓᅜ The 5th China-Australia conference on Immunity in Tuberculosis & Wuhan University
Famous Medical Scientists Forum㸪Ṋ₎ᕷ㸪2019 ᖺ 11 ᭶㸪せ᪨㞟 p13
࣭A novel regulatory mechanism of N-glycan sialylation by the GOLPH3-PI4KIntegrin complex in cancer cells 㢳ᘓᅜ The 11th International Symposium of Tumor Biological Diagnosis and Therapy inNanjing㸪༡ிᕷ㸪2019 ᖺ 11 ᭶㸪せ᪨㞟 p90
࣭PI4KIIĮ ࡜࢖ࣥࢸࢢࣜࣥ Į3ȕ1 ࡢ」ྜయᙧᡂ࡟ࡼࡿ᪂ࡓ࡞ N-⢾㙐ไᚚ࣓࢝ࢽࢬ
࣒ఀᕥ἞▱ᘺ, ⚟⏣཭ᙪ, 㢳ᘓᅜ➨㸰ᅇ࢜ࣝ࢞ࢿࣛࢰ࣮ࣥ ⱝᡭࡢ఍, ᮾி, 2019 ᖺ 11 ᭶, せ᪨㞟 P17࣭⢾⢾㙐ಟ㣭࡟ࡼࡿ࢖ࣥࢸࢢࣜࣥࡢ㑅ู㍺㏦ࢰ࣮ࣥࡢไᚚ࡟ࡘ࠸࡚㢳ᘓᅜ, ఀᕥ἞▱ᘺ, ⚟⏣཭ᙪ, ᡞᓥᣅ㑻 a, ୰㔝᫂ᙪ a(⌮໬Ꮫ◊✲ᡤ ග㔞ᏊᕤᏛ◊✲ࢭࣥࢱ࣮a)➨ 3 ᅇ࢜ࣝ࢞ࢿࣛࢰ࣮ࣥ ◊✲఍, ᮾி, 2019 ᖺ 11 ᭶, せ᪨㞟 P2
࣭Regulation of integrin functions by N-glycosylation㢳ᘓᅜ➨㸲㸰ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍, ⚟ᒸ, 2019 ᖺ 12 ᭶, せ᪨㞟 P175
࣭A novel regulatory mechanism of N-glycan sialylation by the GOLPH3-PI4KIntegrin complex in cancer cells㢳ᘓᅜ
The first Academia Sinica-Tohoku Medical and Pharmaceutical University (TPMU)
Joint Symposium on Glycoscience, Academia Sinica, Taipei, 2019 ᖺ 12 ᭶

46

Received: 16 July 2019|

Revised: 23 September 2019|

Accepted: 30 September 2019

DOI: 10.1096/fj.201901793R

RESEARCH ARTICLE

ST3GAL3, ST3GAL4, and ST3GAL6 differ in their regulation of
biological functions via the specificities for the α2,3-sialylation oftarget proteins
Feng Qi1,2 | Tomoya Isaji1 | Chengwei Duan1
Tomohiko Fukuda1 | Jianguo Gu1| Jie Yang1 | Yuqin Wang3 |1

Division of Regulatory Glycobiology,
Institute of Molecular Biomembrane
and Glycobiology, Tohoku Medical andPharmaceutical University, Sendai, Japan2

Department of Pharmacy, The Fourth
Affiliated Hospital of Nantong University,
Yancheng, China
3

Department of Pharmacology, Pharmacy
College, Nantong University, Nantong,
China
Correspondence
Jianguo Gu, Division of Regulatory
Glycobiology, Institute of Molecular
Biomembrane and Glycobiology, Tohoku
Medical and Pharmaceutical University,
4-4-1 Komatsushima, Aoba-ku, Sendai,
Miyagi 981-8558, Japan.
Email: jgu@tohoku-mpu.ac.jp
Funding information
MEXT | Japan Society for the Promotionof Science (JSPS), Grant/Award Number:
15H04354, 19H03184 and 19K07049;
National Natural Science Foundation ofChina (NSFC), Grant/Award Number:
31670807; Science and Technology Projectfrom Jiangsu Traditional Chinese Medicine
Bureau, Grant/Award Number: YB2017072;
Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Grant/Award Number: 18H04868

Abstract
The α2,3-sialylation of N-glycans is considered important but complicated because
the functions of the three β-galactoside α2,3-sialyltransferases, ST3GAL3, ST3GAL4,
and ST3GAL6, could be compensating for one another. To distinguish their specificfunctions, we established each individual knockout (KO) cell line. Loss of either the
ST3GAL3 or ST3GAL6 genes decreased cell proliferation and colony formation, as
opposed to the effect in the ST3GAL4 KO cells. The phosphorylation levels of ERK
and AKT were significantly suppressed in the ST3GAL6 KO and ST3GAL3 KO cells,
respectively. The cell aggregations were clearly observed in the KO cells, particularly
the ST3GAL3 KO and ST3GAL6 KO cells, and the expression levels of E-cadherin
and claudin-1 were enhanced in both those cell lines, but were suppressed in the
ST3GAL4 KO cells. Those alterations were reversed with an overexpression of eachcorresponding gene in rescued cells. Of particular interest, the α2,3-sialylation levelsof β1 integrin were clearly suppressed in the ST3GAL4 KO cells, but these were increased in the ST3GAL3 KO and ST3GAL6 KO cells, whereas the α2,3-sialylationlevels of EGFR were significantly decreased in the ST3GAL6 KO cells. The decreasein α2,3-sialylation increased the α2,6-sialylation on β1, but not EGFR. Furthermore,a cross-restoration of each of the three genes in ST3GAL6 KO cells showed thatoverexpression of ST3GAL6 sufficiently rescued the total α2,3-sialylation levels, cellmorphology, and α2,3-sialylation of EGFR, whereas the α2,3-sialylation levels of β1
were greatly enhanced by an overexpression of ST3GAL4. These results clearly demonstrate that the three α2,3-sialyltransferases modify characteristic target proteins andregulate cell biological functions in different ways.
KEYWORDS
EGFR, glycoproteins, N-glycan, integrin β1, α2,3-sialyltransferase

Abbreviations: BSA, bovine serum albumin; ConA, concanavalin A; CRISPR/Cas-9, clustered regularly interspaced short palindromic repeats/caspase-9;
DMEM, Dulbecco's modified Eagle's medium; DOX, doxycycline; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; KO, knockout;
MAM, Maackia amurensis lectin; RES, rescued cell; SSA, Sambucus sieboldiana agglutinin; ST3GAL3, β-galactoside α2,3-sialyltransferase 3; ST3GAL4,β-galactoside α2,3-sialyltransferase 4; ST3GAL6, β-galactoside α2,3-sialyltransferase 6; ST6GAL1, β-galactoside α2,6-sialyltranferase 1; WT, wild type.
FASEB BioAdvances. 2020;34:881–897.

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QI ET AL|IN T RO D U C T ION

expression of ST3GAL3 has been associated with tumor progression, differentiation, and metastasis in extrahepatic cholangiocarcinoma,32 and secondary tumor recurrence in gastriccancer.33 ST3GAL6 is known to promote cell adhesion andmigration of multiple myeloma cells by increasing the synthesis of selectin ligands, and a knockdown of ST3GAL6 has
reduced the ability of multiple myeloma cells to home in thebone marrow, which prolonged the rate of survival in a xenograft mouse model.34 On the other hand, there has been noconsensus on the clinical significance of ST3GAL4. Some
researchers reported a decrease in ST3GAL4 mRNA expression in gastrointestinal and ovarian cancer,35,36 while others
have shown significant increases in cervical intraepithelial
neoplasia, colorectal cancer, and pancreatic adenocarcinoma
tissues.37-39 These different phenomena are yet to be clearly
explained. ST3GAL3, ST3GAL4, and ST3GAL6 show a similar enzymatic substrate specificity in catalyzing the transferof sialic acid on the terminal Gal residue of the disaccharide Galβ1-3/4GlcNAc of glycoproteins,40 which makes it
plausible that the α2,3-sialylation modification of glycoproteins represents a co-involvement and/or a compensation. As
mentioned above, the tissue and tumor-specific expressionsof these enzymes have been extensively reported either bycomparing their contents in cancer tissues, as well as in thetissues adjacent to cancers,41-43 or by investigating their overexpressions or the knockdown of a single gene.34,39,44 Suchapproaches, however, are insufficient to explain the biological functions of α2,3-sialylation, and its underlying molecular mechanisms.
To distinguish the respective functions of ST3GAL3,ST3GAL4, and ST3GAL6, we used the clustered, regularlyinterspaced, short palindromic repeats/caspase-9 (CRISPR/
Cas-9) system45 to establish individual gene knockout (KO)HeLa cell lines, and restored the KO cells with the same ordifferent genes to compare their functions. We found thatthe three enzymes differed in their modification of the α2,3sialylation of target proteins and in the distinct biological
functions each plays in cell adhesion, cell growth, and colony
formation.

N-Glycan biosynthesis is complex and involves many enzymes in the endoplasmic reticulum (ER) and the Golgi apparatus, but small numbers of enzymes found in the Golgi
apparatus are responsible for branching and extending, as
well as for sialylation.1 In mammals, sialic acids usually serveas the terminal monosaccharides of the glycans in glycoproteins and glycolipids.2,3 β-Galactoside α2,3-sialyltransferase1(ST3GAL1) and ST3GAL2-6 and β-galactoside α2,6-sialyltransferase 1 (ST6GAL1) and ST6GAL2 in the Golgi apparatus are responsible for the syntheses of Neu5Ac(α2,3)Galand Neu5Ac(α2,6)Gal.4 ST3GAL1 and ST3GAL2 are almost
exclusively used in the type III disaccharide Galβ1-3GalNAc
that is found in O-glycans and glycolipids.5 ST3GAL5 isalmost exclusively used in lactosyl-ceramide as an acceptor substrate for GM3 production, which is also known as
GM3-synthase.6 In particular, ST3GAL3, ST3GAL4, andST3GAL6 are known to be involved in the synthesis of
N-glycans terminated with Neu5Ac(α2,3)Gal.7 On the other
hand, ST6GAL1 appears to play an important role in thesynthesis of N-glycans terminated with Neu5Ac(α2,6)Galin many kinds of cells and tissues.8-10 There is ample evidence that sialic acids are important bioinformatic molecules,
which play pivotal roles in biological, pathological, and immunological processes through modifying glycoproteins andglycolipids on the cell surface, including nervous systemembryogenesis,11 cell-cell interactions,12,13 signal transduction,14 bacterial and viral infection,15,16 etc
In addition, sialic acids on cell membranes are closely
related to tumor invasion and metastasis.17,18 Many studies
have shown that the expression of ST6GAL1 is increased indiverse carcinomas, which may highly correlate with tumor
progression. For example, ST6GAL1 regulates macrophage
apoptosis by controlling the α2,6-sialylation of tumor necrosis factor receptor-1.19 ST6GAL1 is up-regulated in colon
carcinoma, and its metastasis and poor prognosis is ascribedto sialylation of the Fas death receptor by ST6GAL1 that provides protection against Fas-mediated apoptosis.20 Moreover,differentiation of human dendritic cells is accompanied byan increased expression of sialylated glycans, mainly throughthe activities of ST3GAL1 and ST6GAL1.21 However,among the six enzymes of α2,3-sialyltransferases, ST3GAL3,ST3GAL4, and ST3GAL6 participate mainly in the generation of NeuAcα2,3Galβ3/4GlcNAc, which is the precursor ofsLea or sLex,22,23 two important tumor-associated sialylated
glycoconjugates.24-26 In fact, the increased sialylation andexpressions of these sialyltransferases genes in cervical cancer were also reported.27-31 Therefore, study of the expression and biological functions of ST3GAL3, ST3GAL4, andST3GAL6 in tumors is of great importance.
Until now, several reports have chronicled the functionsof these cells in various types of tumors. For example, the

2
2.1

|

M AT E R I A L S A N D M E T H O D S|

Antibodies and reagentsThe experiments were performed with the following antibodies: rabbit mAbs against Claudin-1 (#13255T), epidermal growth factor receptor (EGFR) (#3777), ERK1/2(#4695S), phospho-ERK1/2 (#4370S), AKT (#9272), andphospho-AKT (#4060) were obtained from Cell Signaling
Technology; mouse mAbs against E-cadherin (#610182),
N-cadherin (#610920) and β1 integrin (#610468) were fromBD Biosciences; mouse mAbs against ɑ-tubulin (#T6199)48

| 883

QI ET ALand FLAG (#F1804) was from MilliporSigma; rabbit mAbs
against GAPDH (FL-335, #SC-25778) were from Santa Cruz
Biotechnology, Inc Alexa Fluor 647 goat anti-mouse IgG and
streptavidin were obtained from Thermo Fisher Scientific;
Alexa Fluor 488 conjugated anti-mouse (#A11029) was from
Invitrogen; Alexa Fluor 568 phalloidin (#A12380) and TOPRO-3 (#T3605) was from Molecular Probes. The peroxidase-conjugated secondary antibody against rabbit (#7074S)
was obtained from Cell Signaling Technology; peroxidaseconjugated secondary antibodies against mouse (#AP124P)
were from MilliporeSigma; Maackia amurensis (MAM)
lectin (#BA-s7801-2) was from EY Laboratories, Inc;
Concanavalin A (ConA) lectin (#B-1005) from Seikagaku
Corporation; MAM-Agarose (J310) and SSA-Agarose (J318)
were from J-OIL MILLS, Inc An ABC kit was acquired from
Vector Laboratories; doxycycline hyclate (DOX) (#D9891)
was from Sigma-Aldrich; PrimeScript RT reagent Kit with
gDNA Eraser (Perfect Real Time) (#RR047A) was fromTakara, Japan; Quick Taq HS DyeMix (DTM-101) was fromTOYOBO, Japan.

2.2|(FACS) to isolate low MAM-binding transfectants. Stable
KO cells were cloned by a limiting dilution. For isolation of
ST3GAL4 KO cells, 3 days post-transfection, the cells were
subjected to FACS to isolate cells that highly expressed GFP.
Stable clones were isolated by a limiting dilution. The α2,3sialylation levels were confirmed by flow cytometry andWestern blotting using MAM lectin.

2.4 | ST3GAL3, ST3GAL6, and GFP-tagged
E-cadherin expression vectors
We used the Gateway cloning system from Invitrogen for
all overexpression experiments. The cDNAs of human
ST3GAL3 and ST3GAL6 were kindly provided by Dr H.
Narimatsu (National Institute of Advanced Industrial Scienceand Technology, Japan). The Gateway entry vectors wereconstructed as described previously.47 In brief, using LR
clonase (Invitrogen), the sub-cloned cDNAs in entry vectors were transferred into CSIV-TRE-RfA-CMV-KT for the
doxycycline hyclate inducible overexpression. To express
GFP-tagged E-cadherin, expression vector pEGFP-N1-Ecadherin-GFP was transfected using PEI MAX (molecularmass, 40 kDa; Polysciences Inc) as previously described.48

Cell lines and cell culture

The HeLa and 293T cell lines (RIKEN Cell Bank, Tsukuba,
Japan) were cultured at 37°C in Dulbecco's modified Eagle's
medium (DMEM) with 10% fetal bovine serum (FBS), in ahumidified atmosphere containing 5% CO2, except for thevirus production.

2.5|

Virus production and infection

The virus production and infection was performed as described previously.47,49,50 Briefly, ST3GAL4 overexpression
viruses were prepared as previously described.47 To prepare
the ST3GAL3 and ST3GAL6 overexpression viruses, thelentivirus vectors (CSIV-TRE-RfA-CMV-KT) were transfected into 293T cells with packaging plasmids via PEIMAX. The target cells were cultured for 24 hours to obtain
virus media for infection. After infection for 72 hours, cells
were selected using FACSAria II (BD bioscience) to obtain
Kusabira Orange-positive cells (CSIV-TRE-RfA-CMV-KT).
Stable clones were also isolated using a limiting dilution. The
stable cells that expressed Kusabira Orange-positive in fluorescence microscopy were chosen and used in subsequentstudies.

2.3 | Genomic deletion of ST3GAL3,ST3GAL4, or ST3GAL6 in HeLa cells
The pSpCas9(BB)-2A-GFP (PX458) plasmid andpSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid were purchased from Addgene (PX458:Addgene plasmid #48138,
PX459:Addgene plasmid #62988) and were deposited by
Dr Feng Zhang.45 The target site on human ST3GAL4 exon
8 (TTGAACAATGCCCCAGTGGC), as previously described,46 was cloned into PX458. The target site on human
ST3GAL3 exon 8 (TGCCGCCGCTGCATCATCGT) andST3GAL6 exon 5 (CTATGGGATGAGAACATCAG),
which were designed by guide design tool (https://zlab.bio/
guide-design-resources/), were cloned into PX459. The threekinds of KO cell lines were created by electroporating cellswith the vectors containing the target sequences, according to the manufacturer's recommendations (Amaxa cellline Nucleofector kit; Lonza, Basel, Switzerland). One day
post-transfection, the cells transfected with ST3GAL3 KO
or ST3GAL6 KO plasmid and were selected with 1 μg/mL
puromycin for 48 hours. After further incubation for 5 days,
the cells were subjected to fluorescence-activated cell sorting

2.6|

Western blot and immunoprecipitation

Western blot and immunoprecipitation were performed as
described previously with minor modifications.47,48 In brief,
the indicated cells were rinsed twice with ice-cold PBS andthen lysed in a cell lysate buffer (20 mM Tris-HCl, pH 7.4,
150 mM NaCl, 1% Triton X-100) that included proteaseand phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan)
for 30 min. The protein concentrations of lysates were

49

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QI ET AL

2.9

determined via BCA assay (Pierce). Equal amounts of celllysates from each sample were resolved by SDS-PAGE.
Then, the proteins were transferred to PVDF membranes(MilliporeSigma) and detected with primary and secondary antibodies, and visualized by the Immobilon Western
Chemiluminescent HRP Substrate (MilliporeSigma), according to the manufacturer's instructions. For immunoprecipitates, equal amounts of protein lysates from eachsample were immunoprecipitated with MAM-agarose or
Sambucus sieboldiana Agglutinin (SSA)-agarose, which
specifically recognizes ɑ2,3 or ɑ2,6 sialylation, respectively, for 1 hour at 4°C with rotation, and then the immunoprecipitates were washed twice with lysis buffer andsubjected to SDS-PAGE. The precipitated glycoproteins
were detected using different antibodies.

The cell growth curves and colony formation assays were
performed as described previously with minor modifications.51,52 In brief, to assay cell growth, the cells (5 × 104)
were seeded in six-well culture dishes overnight and thenserum starved for 24 hours. After starvation, the cells were
supplied with DMEM containing 10% FBS. Cells in the same
areas on the cultured dishes were photographed and counted
at the indicated times (0, 24, 48, and 72 hours), the folds ofchange in cell numbers were normalized to those at 0 hour andstatistically analyzed. To assay the colony formation, cells(500/per well) were seeded in 6-cm dishes in DMEM containing 10% FBS, incubated at 37°C and the cultured media
were changed twice weekly. After incubation for 14 days, theformed colonies were stained with 0.25% crystal violet, andimages were taken. Colonies containing more than 50 cells
were counted and analyzed statistically from three independent wells of each sample.

2.10 | RT-PCR for detection of mRNA
expression levels
Total RNAs of all cell lines listed in Figure 1A were prepared with TRI reagent (Invitrogen), and 1 μg of total RNA
was reverse-transcribed using a PrimeScript RT reagent
kit with gDNA Eraser according to the manufacturer's instructions. The primers for KO confirmation were designed
around the KO regions. The sequences of those primers areshown in Table 1. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control in PCRruns, and the reaction products obtained were subjected toelectrophoresis using 2% agarose gels containing ethidium
bromide.

2.11|

Immunofluorescence

Immunofluorescence staining was performed as described
previously with minor modifications.51,52 In brief, cells
were cultured on glass-bottom dishes, rinsed twice with
PBS and fixed with 4% paraformaldehyde for 20 min.
A nonspecific blocking solution was applied (PBS,
0.1% Triton X-100, 10% BSA) at room temperature for
1 hour. N-cadherin antibodies were used and followed
by incubation with anti-mouse Alexa Fluor 488 secondary antibodies (Invitrogen) and Alexa Fluor 568 phalloidin (Invitrogen), and then incubated with TO-PRO-3 for
20 min in the dark. After transfection for 48 hours, thosecells overexpressed GFP-tagged E-cadherin were rinsed
twice with PBS and fixed with 4% paraformaldehyde for
20 min, and incubated with TO-PRO-3 for 20 min in thedark. Fluorescence images were observed via confocal
microscopy using a FluoView FV1000 (Olympus, Tokyo,
Japan).

2.7 | Cell growth and colony
formation analysis

2.8|

Flow cytometry analysis|

Cell aggregation assay

Cell aggregation assay was performed as described
previously.55 Briefly, cells (2 × 105 cells/mL) were resuspended in DMEM containing 1% BSA with or without 2 mM EDTA. For each condition, 1 mL of the cellsuspension was added to a 15-mL tube, which was
followed by incubation in a rotator (MACSmixTube
Rotator; Miltenyi Biotec, Bergisch-Gladbach, Germany)at 12 rpm for 6 hours at 37°C. The cell suspensions wereplaced in six-well plates and the field of vision was randomly selected for photography. Cell aggregation assays
were performed using three independent wells for eachsample.

Flow cytometric analysis was performed as described
previously with minor modifications.53,54 Briefly, the indicated semi-confluent cells were detached from the culture dishes using trypsin containing 1 mM EDTA and were
subsequently stained with or without the primary biotinylated MAM, followed by incubation with Alexa Fluor
647-conjugated streptavidin. After washing three times
with PBS, flow cytometric analysis was performed using a
FACSCalibur flow cytometer and Cell Quest Pro software(BD Biosciences).

50

| 885

QI ET AL

(E)

(A)

(C)

(B)

(D)

F I G U R E 1 Abatement of α2,3-sialylation in ST3GAL3, ST3GAL4, or ST3GAL6 KO cells. A, The mRNA expression levels of α2,3sialyltransferases (1-6) and α2,6-sialyltransferase (ST6GAL1) in nine cell lines were detected by RT-PCR. GAPDH was used as a control. B,
WT and three kinds of KO HeLa cells were collected and incubated with (bold line) or without (grey shadow) MAM lectin, which specificallyrecognizes sialic acid ɑ2,3 galactose, followed by incubation with Alexa Fluor 647 streptavidin subjected to flow cytometric analysis. The verticaldashed line indicates the peak reacted with MAM lectin in WT cells. Equal amounts of cell lysates from WT and the three types of KO cells were
immunoblotted with MAM lectin (C) and ConA lectin (D), which preferentially recognizes high-mannose N-glycans. ɑ-Tubulin was used as aloading control. E, Comparison of the mRNA expression levels using RT-PCR between WT and KO cells. The results obtained from using thenormal primers and GAPDH were used as controls

2.12

|

Statistical analysis

test or one-way analysis of variance (ANOVA) by GraphPad
Prism version 5 (GraphPad Software, La Jolla, CA, USA).
Statistical significance was defined as P < .05 (*P < .05;**P < .01; ***P < .001).

All data are expressed as the mean ± standard derivation.
Statistical analyses were performed using either a Student's t

51

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QI ET ALTABLE 1
Gene names

Primer sequences and annealing temperatures for RT-PCR

Sense primer (5′–3′)

Antisense primer (5′–3′)

Annealing temperature (°C)

I. Primers for detection of expression levels
ST3GAL1GACTTGGAGTGGGTGGTGAGACAAGTCCACCTCATCGCAG

57

ST3GAL2

CTCTCGGGCAAGAGCTGTG

CTGCAGCATCATCCACCACC

59

ST3GAL3

CTATGACATTGTGGTGAGACTGACTCTCTCCTTGTAGACGATGTATTT51

ST3GAL4TGAGGGTGGCCCGAGG

CCGGGAGTAGTTGCCAAAGA59

ST3GAL5

CCAATGCCAAGTGAGTACACCTGAGCTCTCTTTACATGGTCAGG

57

ST3GAL6

AGGTGGCTGTTGGAATTGT

GTTGTTGGGTGTTTAGGTTTCTG

51

ST6GAL1GAAAAATGGGCCTTGGCCTG

GAGCAGGAAAACAAGCCTGC

57

E-cadherin

CGTAGCAGTGACGAATGTGGTAC

AACTGGAGAACCATTGTCTGTAGC

59

GAPDH

CGGAGTCAACGGATTTGGTCGTA

AGCCTTCTCCATGGTGGTGAAGAC

57

II. Primers for KO confirmation
ST3GAL3

CCGCCGCTGCATCATCGTTGCACTCACTCTCTCCTTGTAGAC

68

ST3GAL4GAACAATGCCCCAGTGGC

GGGTTGAGAATCCGAATCTGTTTAG

66

ST3GAL6

CCCTATGGGATGAGAACATCAG

TAGGGTCATTGTGAATAGGATCTG

66

3

|

RESULTS

the KO results, we designed these primers for KO confirmation in the KO regions (Table 1). As shown in Figure
1E, compared with using normal primers or GAPDH, the
RT-PCR results obtained from using the primers for KO
confirmation were clearly lower in three KO cells thanthose in each of the corresponding WT cells, suggestingthat the base sequences in the KO regions of three enzymes were changed. These results suggested that the KOsof the ST3GAL3, ST3GAL4, and ST3GAL6 genes weresuccessful.

3.1 | KOs of the ST3GAL3, ST3GAL4, or
ST3GAL6 genes reduced α2,3-sialylation
To investigate the roles of α2,3-sialylation on N-glycans,
first we screened nine cell lines that included HeLa,
MDA-MB-231, MiaPaCa2, PANC-1, HuH-7, 293T, CW-2,
PMF-ko14, and Hep G2 cells based on RT-PCR (Figure1A), to determine a suitable cell line highly expresses α2,3sialyltransferases (ST3GAL3, ST3GAL4, and ST3GAL6),
and marginally expresses ST6GAL1. Using primer sequences (Table 1), we found that the expression levels
of mRNA for those three α2,3-sialyltransferases in HeLa
cells were relatively higher, whereas α2,6-sialyltransferase(ST6GAL1) was relatively lower than those in the othereight cell lines (Figure 1A). So, we chose the HeLa cell line
as a suitable cell model to study. And then to distinguishthe specific functions of the three α2,3-sialyltransferasesof ST3GAL3, ST3GAL4, and ST3GAL6, we establishedeach single KO cell line using the CRISPR/Cas9 system.
As expected, flow cytometric analysis using MAM lectin,
which specifically recognizes ɑ2,3-sialylated galactose,
showed that the ɑ2,3-sialylation levels were decreased in
the ST3GAL3 KO, ST3GAL4 KO, and ST3GAL6 KO
cells, compared with that in the wild type (WT) cells. The
degree of decrease in ɑ2,3-sialylation seemed to be as follows: ST3GAL4 KO > ST3GAL3 KO > ST3GAL6 KO(Figure 1B). The MAM lectin blot showed a similar tendency (Figure 1C). There were no significant differences in
the reactive abilities of ConA lectin, which preferentiallyrecognizes high-mannose types of N-glycans, for either thethree KO cells or WT cells (Figure 1D). To further confirm

3.2 | Comparing cell proliferation andcellular signaling among the ST3GAL3 KO,
ST3GAL4 KO, and ST3GAL6 KO cells
We used these KO cell lines to compare cell biological functions. First, we investigated the effects of α2,3sialyltransferase KO on cell proliferation and found thatcell growth was significantly suppressed in the ST3GAL3
KO and ST3GAL6 KO cells, but not in the ST3GAL4 KO
cells, compared with the WT cells (Figure 2A). It is notable that the cell growth for ST3GAL4 KO cells seemed todemonstrate a cell density-dependent manner, that is, whencells were cultured at a much lower density, the growthrate of ST3GAL4 KO cells was lower than that of the WT
cells, but once cells reached a certain density the growthrate of ST3GAL4 KO cells was significantly increased
at a rate that was even faster than that of the WT cells(Figure 2A). To confirm those phenomena, a clonogenic
assay was conducted to test the single-cell potential for survival and “unlimited” cell division.52,56 The ST3GAL3 andST3GAL6 KO cells consistently formed smaller and fewer52

| 887

QI ET AL

(A)

(D)

(B)

(C)

(F)

(E)

F I G U R E 2 Effects of ST3GAL3 KO, ST3GAL4 KO, and ST3GAL6 KO on cell proliferation and cellular signaling. A, WT and the threekinds of KO cells were seeded in six-well culture dishes overnight and then serum starved for 24 hours. After starvation, the cells were suppliedwith DMEM containing 10% FBS, which was taken as the 0 point (0 hour), and further incubated for 72 hours. The cells in the same areas on
the cultured dishes were photographed and counted at the indicated times (0, 24, 48, and 72 hours), the fold changes in the cell numbers werenormalized to those at 0 hour. Data are represented as the mean ± standard derivation (n = 3 individual experiments). B, C, WT and the threekinds of cells (500/per well, suspended in a single cell) were grown for 14 days, and then stained with crystal violet and the foci in each well
were counted. B, Representative images for colony formation. C, The stained colonies were counted, and quantitative data are presented as themeans ± standard derivation from three independent experiments. D, The expression levels of phosphorylated and total ERK and AKT in the fourcells were examined by Western blotting with the indicated antibodies. GAPDH was used as a loading control. E, Relative ratios of pERK/ERK
in these four cells were quantified (n = 3 independent experiments). F, Relative ratios of pAKT/AKT in these four cells were quantified (n = 3
independent experiments). All values are the means ± standard derivation, one-way ANOVA; *P < .05; **P < .01; ***P < .001

foci, compared with those in the WT cells (Figure 2B,C).
Interestingly, the numbers of foci in ST3GAL4 KO cells
were similar to those in the WT cells, but the foci seemedsmaller than those in the WT cells (Figure 2B,C), which
may support the notion again that cell growth exists in acell density-dependent manner in the ST3GAL4 KO cells.
Western blotting was used to detect the activation of ERK
and AKT that is related to cell proliferation and survival,
we found that the phosphorylation levels of ERK were decreased significantly in ST3GAL6 KO cells (Figure 2D,E),
whereas the phosphorylation levels of AKT were significantly decreased in ST3GAL3 KO cells (Figure 2D,F),
compared with WT or ST3GAL4 KO cells. Together, theseresults clearly indicated that ST3GAL3, ST3GAL4, andST3GAL6 differ in their effect on cell functions via distinct mechanisms.

3.3 | Deletion of ST3GAL3, ST3GAL4, or
ST3GAL6 genes altered cell morphology andinduced cell aggregation
Next, we noticed cell morphological changes after deletion of the three genes. The KO cells all tended to form islands, which were never observed in the WT cells. Amongthem, ST3GAL3 KO cells were the most obvious, followed
by ST3GAL6 KO cells, while the effect in ST3GAL4 KO
cells was much weaker. Immunofluorescent staining of

53

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QI ET AL

(A)

(C)

(D)

(B)

(E)

F I G U R E 3 Knockout of ST3GAL3, ST3GAL4, or ST3GAL6 in HeLa cells, altered cell morphologies, and induced cell aggregation. A,
WT and the three kinds of KO cells were stained with anti-N-cadherin antibody, followed by incubation with fluorescent secondary antibody.
Localization of F-actin was examined by staining with Alexa Fluor 568 phalloidin, and the nucleus by TO-PRO-3. B, The four kinds of cells weredetached from culture dishes and suspended at 2 × 105 cells/mL in 10% FBS culture medium with or without EDTA, followed by constant rotation(12 rpm) for 6 hours at 37°C. An aliquot of each of these cell suspensions was photographed with a phase-contrast microscope. C, The sameamounts of cell lysates from the four cells were examined by Western blotting with the indicated antibodies, and ɑ-tubulin was used as a loading
control. D, Fluorescence images of WT and the three kinds of KO cells overexpressed with E-cadherin-GFP, and the nucleus stained by TO-PRO-3.
E, The mRNA levels of E-cadherin in WT and three KO cells were detected by RT-PCR. GAPDH was used as a control

F-actin showed that the ST3GAL3 KO cells and ST3GAL6
KO cells exhibited cortical actin around the cell surface,whereas ST3GAL4 KO cells did not (Figure 3A). We attempted to perform immunostaining with E-cadherin antibody, but failed. Instead of E-cadherin, immunostaining with
N-cadherin was carried out. N-cadherin was highly expressedin the cell-cell contacts in ST3GAL3 KO and ST3GAL6 KO
cells, which existed in a diffuse state in the cytoplasm andin the cell surface in ST3GAL4 KO and WT cells (Figure
3A). Furthermore, we performed a cell aggregation assay inthe suspension. The three KO cells all tended to form many
aggregates, whereas the WT cells remained as either single
cells or as much smaller aggregates (Figure 3B). Those cellaggregates disappeared in the presence of EDTA, suggesting that the cell-cell adhesion had proceeded in a calciumdependent manner. To better understand the underlying
molecular mechanisms, we compared the expression levelsof some adhesive molecules, which affect cell-cell adhesion, such as claudin-1, E-cadherin, and N-cadherin. It wasinteresting that the expressions of claudin-1 and E-cadherin,
which promote cell-cell adhesion, were greatly increased in
ST3GAL3 KO and ST3GAL6 KO cells, but were decreased
in ST3GAL4 KO cells (Figure 3C). On the other hand, the expression levels of N-cadherin were similar in all cells (Figure
3C). To further determine the changes of E-cadherin expression in four groups of cells, we performed an overexpression54

| 889QI ET ALsystem with GFP-tagged human E-cadherin. The ST3GAL3
KO and ST3GAL6 KO cells expressed more E-cadherin on
the cell surface, compared to the WT cells and ST3GAL4 KO
cells (Figure 3D). In addition, E-cadherin proteins seemed
to be internalized in the ST3GAL4 KO cells. We also performed a RT-PCR to examine E-cadherin expression levelsin those cells, and found that the pattern of E-cadherin expression at mRNA levels (Figure 3E) was similar to that at
the protein levels (Figure 3C). These results again indicatedthat ST3GAL3, ST3GAL4, and ST3GAL6 differ in their effect on cell morphology via different pathways.

would decrease a competitor for a common donor substrate,
CMP-NANA, which could relatively increase the amounts of
CMP-NANA available for ST3GAL4 to catalyze the α2,3sialylation of β1 integrin. However, the α2,3-sialylation levels of
EGFR were only decreased in the ST3GAL6 KO cells, but notin the other two KO cells (Figure 5B,E). Curiously, the changes
in the α2,3-sialylation of N-cadherin were similar among thethree KO cells (Figure 5C,F). On the other hand, the changes inthe α2,6-sialylation of the target proteins, with the exception of
EGFR in the ST3GAL6 KO cells, were the opposite (Figure 5),suggesting that these α2,3-sialyltransferases may compete withST6GAL1 for the same substrates. These results strongly suggest that ST3GAL3, ST3GAL4, and ST3GAL6 differ in theirregulation of biological functions via the modification of different target glycoproteins, that is, each enzyme has preferential
target proteins.

3.4 | Restoration of ST3GAL3, ST3GAL4, or
ST3GAL6 genes in each of the corresponding
KO cells rescued ɑ2,3-sialylation, cellmorphology, and expression of E-cadherin

3.6 | Cross-restoration using ST3GAL3,ST3GAL4, or ST3GAL6 genes in ST3GAL6
KO cells differed in the rescued ɑ2,3sialylation and cell morphology

Given the observations described in Figures 2 and 3, we attempted to determine whether the phenotypes could be rescuedby the restoration of each gene in the corresponding KO cells(RES). The ɑ2,3-sialylation levels were compared between the
KO and RES cells using flow cytometry and Western blotting.
As expected, increased ɑ2,3-sialylation was observed in eachof the RES cells by either flow cytometric analysis (Figure 4A)
or Western blotting with MAM lectin (Figure 4B). The cellmorphologies that tended to form aggregations in the KO cells
were reversed to separated or small aggregation morphologies
in each of the RES cells, particularly in the ST3GAL3 RES
or ST3GAL6 RES cells (Figure 4C). Although the restorationof ST3GAL4 did not signal a significant change in E-cadherin
expression, the enhanced E-cadherin expression levels observed in the ST3GAL3 KO and ST3GAL6 KO cells shown in
Figure 3C were greatly suppressed in the ST3GAL3 RES and
ST3GAL6 RES cells (Figure 4D). These results further supported the notion that ST3GAL3, ST3GAL4, and ST3GAL6
differ in their effect on cell morphology.

To further confirm the specificities of the three α2,3sialyltransferases, we performed a cross-restoration withST3GAL3, ST3GAL4, or ST3GAL6 genes in the ST3GAL6
KO cells. The efficiencies for rescuing the α2,3-sialylationlevels in the ST3GAL6 RES cells, examined using flow cytometry, were clearly higher than those in the cells expressedwith ST3GAL3 or ST3GAL4 (Figure 6A), which was further confirmed by lectin blotting with MAM lectin (Figure
6B). The expression levels of each α2,3-sialyltransferase
were similar among these cells, as confirmed by Western
blotting with anti-Flag antibody (Figure 6B). In addition,
the cell aggregations induced by ST3GAL6 deficiency were
efficiently rescued to normal cell morphology, as with WT
cells, by overexpression of ST3GAL6, but neither ST3GAL3
nor ST3GAL4 overexpression had the same effect (Figure
6C). These results clearly indicated that the overexpressionsof ST3GAL3 or ST3GAL4 could not completely rescue thedecreased α2,3-sialylation levels and functions caused by
ST3GAL6 deletion, further suggesting that these three enzymes have preferential target proteins.

3.5 | ST3GAL3, ST3GAL4, or ST3GAL6
modified target glycoproteins differently
Sialylation is well known to play an important role in cancer cell
invasion and metastasis, as well as in cell growth. Here, we chose
three representative transmembrane glycoproteins, β1 integrin,
EGFR, and N-cadherin to detect the changes in ɑ2,3-sialylationand ɑ2,6-sialylation levels in the KO cells. Of particular interest,
the α2,3-sialylation levels of β1 integrin were only decreased in
the ST3GAL4 KO cells, compared with those in the WT cells.
Conversely, the α2,3-sialylation levels were increased in the
ST3GAL3 KO and ST3GAL6 KO cells (Figure 5A,D). The underlying mechanisms for the enhancement remain unclear, but
it is reasonable that the KOs of ST3GAL3 and ST3GAL6 genes

3.7 | Cross-restoration with ST3GAL3,ST3GAL4, or ST3GAL6 genes in the ST3GAL6
KO cells differ in the rescue levels of ɑ2,3sialylation and ɑ2,6-sialylation on target
glycoproteins
Again, the α2,3-sialylation and α2,6-sialylation levels of
β1 integrin, EGFR, and N-cadherin were tested following

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(A)

(B)

(C)

(D)

F I G U R E 4 Forced expression of ST3GAL3, ST3GAL4, or ST3GAL6 KO genes in each of the corresponding KO cells, and the rescued
cell morphologies and E-cadherin expressions. Each KO cells and restoration of each gene in the corresponding KO (RES) cells were cultured in
the presence of DOX at 1 μg/mL for 72 hours before the following experiments were performed. A, The KO and RES cells were collected andincubated with (bold line) or without (gray shadow) MAM lectin, followed by incubation with Alexa Fluor 647 streptavidin and subjected to flow
cytometric analysis. The vertical dashed lines indicate the peaks reacted with MAM lectin in KO cells. B, Equal amounts of cell lysates fromthese KO and RES cells were examined by immunoblot with MAM lectin and anti-Flag antibodies. GAPDH was used as a loading control. C,
Representative cell morphologies of each KO and RES cells were taken with a phase-contrast microscope. Arrows indicated cell aggregation. D,
The same amounts of cell lysates from each KO and RES cells were analyzed by Western blotting with the E-cadherin antibody, and ɑ-tubulin wasused as a loading control

4cross-restoration with ST3GAL3, ST3GAL4, or ST3GAL6
genes in ST3GAL6 KO cells. The increases in ɑ2,3sialylation and decreases in ɑ2,6-sialylation of β1 integrin were much more obvious in the cells restored with the
ST3GAL4 gene compared with those restored using the otherversions (Figure 7A,D) while both the α2,3-sialylation and
α2,6-sialylation of EGFR that were down-regulated in the
KO of the ST3GAL6 gene were preferentially rescued byan overexpression of ST3GAL6, but not by overexpressionsof either ST3GAL3 or ST3GAL4 (Figure 7B,E). Curiously,
consistent with the data shown in Figure 5B, the expressionof ST3GAL6 seemed correlated with both α2,3- and ɑ2,6sialylation of EGFR. Compared with the other two genes, restoration with the ST3GAL6 gene in the ST3GAL6 KO cells
efficiently increased ɑ2,3-sialylation levels and decreasedthe ɑ2,6-sialylation levels of N-cadherin (Figure 7C). These
results further clearly demonstrated that the three enzymes
have individual specific target proteins, and that ST3GAL4 is
the most critical for the ɑ2,3-sialylation of β1 integrin while
ST3GAL6 is the most important for the ɑ2,3-sialylation of
EGFR.

|

DISCUSSION

In the present study, we used the same HeLa cell line toinvestigate the functions of three α2,3-sialyltransferases,
ST3GAL3, ST3GAL4, and ST3GAL6, which are
the most important sialyltransferases for the α2,3sialylation of N-glycans. We found that (i) the three
α2,3-sialyltransferases had different functions in cell proliferation and cell adhesion, as well as in cellular signaling; (ii) the three α2,3-sialyltransferases individuallypromote specific modifications for different target proteins(Figure 8), for example, ST3GAL4 modifies β1 integrin,
while ST3GAL6 modifies EGFR; and (iii) the three α2,3sialyltransferases show negligible levels of compensationfor ɑ2,3-sialylation, and may compete with ST6GAL1
for sialylation of the same target protein. Our studies are
the first to characterize the ɑ2,3-sialylation of the three
enzymes in the same cell line, which may help us to understand their functional expressions, and explain someprevious results or interpret some controversial observations obtained from different cells or tumor tissues.35,38,57

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(A)

(B)

(C)

(D)

(E)

(F)

F I G U R E 5 Knockout of ST3GAL3, ST3GAL4, or ST3GAL6 in HeLa cells differed in the alteration of the levels of ɑ2,3-sialylation andɑ2,6-sialylation of specific glycoproteins. Equal amounts of cell lysates were immunoprecipitated with MAM-agarose or Sambucus sieboldiana
Agglutinin (SSA)-agarose which specifically recognizes ɑ2,6-sialylation, for 1 hour at 4°C with rotation, and then the immunoprecipitates were
washed twice with lysis buffer and subjected to SDS-PAGE. The precipitated glycoproteins and cell lysates were immunoblotted for β1 integrin(A), EGFR antibody (B), and N-cadherin (C) antibodies. The relative levels of ɑ2,3 and ɑ2,6 sialylation on β1 integrin (D), EGFR (E), andN-cadherin (F) in these four kinds of cells were quantified (n = 3 independent experiments). All values are the means ± standard derivation, oneway ANOVA; *P < .05; **P < .01; ***P < .001

Glycan structures expressed on glycoproteins and glycolipids of cells are essential for regulating normal cell functions.58 Altered tumor-associated carbohydrate antigens havebeen identified in many solid tumors, and the changes in terminal sialylation are very important in malignant transformation and cancer progression.59,60 Until now, most studies
have focused on either the effects of three kinds of sialylation
linkages (α2,3, α2,6, and α2,8) in tumors or on the expression levels of certain sialyltransferases in tumors. Examplesof these studies include the following: α2,3-sialylation of α2
integrin has been related to the bone metastatic behavior of
prostate cancer cells60; malignant transformation of oral epithelium was found to be accompanied by α2,3-sialylation,
wherein α2,6-sialylation was related to disease progression
and metastatic potentials61; melanoma progression has been
associated with a significant increase in α2,3-sialylation on
the surface57; several sialyltransferase genes were found tobe highly expressed in human colon and gastric tumor tissues62,63; and important functions of a single sialyltransferase
have been observed by manipulation of the gene expression
in a cell line.39,44,64 However, very little is known about whichsialyltransferase(s) is/are specifically responsible for thesynthesis of these altered glycans on defined glycoproteins.
The main function of these three enzymes is to modify the
α2,3-sialylation of glycoproteins. As far as we could ascertain, few studies have compared the functions of these genes
in the same cell line. The present study clearly demonstrated
that ST3GAL4 in HeLa cells significantly contributes to total
α2,3-sialylation levels, compared with the effect from either
ST3GAL3 or ST3GAL6, but each sialyltransferase has its
own specific target glycoproteins, which subsequently affects
cellular signaling. ST3GAL4 KO significantly decreases
the α2,3-sialylation of integrin β1, but not that of EGFR;ST3GAL6 KO, however, greatly decreases the α2,3-sialylation of EGFR, but not that of integrin β1. Consistently, thelevels of p-ERK and cell proliferation were down-regulated
in ST3GAL6 KO cells, not in ST3GAL4 KO cells, compared with WT cells. Curiously, the levels of p-AKT were
only suppressed in the ST3GAL3 KO cells, suggesting thatST3GAL3 might specifically modify some glycoproteins that
promote AKT activation to regulate cell proliferation. Thus,
we speculated that the ranges for the α2,3-sialylation of glycoproteins affected by ST3GAL3, ST3GAL4, and ST3GAL6
are different. In fact, the present results were supported by a

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(A)

(B)

(C)

F I G U R E 6 Restoration of either ST3GAL3 or ST3GAL4 genes could not completely rescue the ɑ2,3-sialylation levels and cell morphologies
of impaired ST3GAL6 KO cells. The ST3GAL6 KO cells transfected with or without ST3GAL3, ST3GAL4, or ST3GAL6 genes were cultured
in the presence of DOX at 1 μg/mL for 72 hours before the following experiments were performed. A, These four kinds of cells were collected
and incubated with (bold line) or without (gray shadow) MAM lectin, followed by incubation with Alexa Fluor 647 streptavidin and subjected toflow cytometric analysis. The vertical dashed lines indicate the peaks of the reaction abilities with MAM lectin in the ST3GAL6 KO cells rescued
with or without ST3GAL6 genes. B, Equal amounts of cell lysates were examined by immunoblotting with MAM lectin and anti-flag antibody.
α-Tubulin was used as a loading control. C, Representative cell morphologies of these four kinds of cells were taken with a phase-contrast
microscope. Arrows indicated cell aggregation

previous study, which found that the sialylation of a recombinant glycoprotein, erythropoietin, was specifically modified
by ST3GAL4, and not by either ST3GAL3 or ST3GAL6 in
Chinese Hamster Ovary cells.7 An observation made in thepresent study whereby the α2,3-sialylation of integrin β1 wasconversely increased in both the ST3GAL3 and ST3GAL6
KO cells could also be very important (Figure 5A). Although
the exact underlying mechanism in this novel result will require further studies, it would be plausible to speculate thata KO of either the ST3GAL3 or ST3GAL6 gene may relatively increase the amounts of a common donor substrate,
CMP-NANA, which would allow ST3GAL4 to modify integrin β1, which further suggests that the KO effect of a glycogene may be expressed as both a loss-of-function and again-of-function.
The specificities of the three enzymes for their own target proteins were also proved by the cross-restoration experiments. Overexpression of either ST3GAL3 or ST3GAL4
in the ST3GAL6 KO cells increased the levels of α2,3-sialylation to some extent, but the recovery level observedin ST3GAL6 RES cells was never reached (Figure 6),

suggesting ST3GAL6 has its own intrinsic target proteins, as
described above. In fact, the restorative ability with respect
to the α2,3-sialylation of integrin β1 was the highest in the
ST3GAL4 RES cells, while that of the α2,3-sialylation ofboth EGFR and N-cadherin was the highest in the ST3GAL6
RES cells (Figure 7). Furthermore, cell morphology differedamong the three cross-restoration cells, and only the overexpression of ST3GAL6 could rescue it to the level of WT cells.
When the important functions of integrin β1,65,66 EGFR,67
and N-cadherin68 are considered, there can be little doubtthat the three sialyltransferases differentially regulate cell biological functions, cancer progression, and tumor resistance.
Of course, the specificities of the three enzymes give thema relative approach rather than an all-or-nothing effect. The
overexpression of ST3GAL4 in the ST3GAL6 KO cells alsoslightly increased the α2,3-sialylation levels of EGFR, as
ST3GAL6 did.
Changes in the α2,6-sialylation levels of these KO cells
merit recognition, since α2,3- and α2,6-sialyltranfserases
may compete with common substrates to a certain extent,
and this competition is manifested in specific glycoproteins58

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QI ET AL

(A)

(B)

(C)

(D)

(E)

(F)

F I G U R E 7 The cross-restoration of ST3GAL3, ST3GAL4, or ST3GAL6 in ST3GAL6 KO cells differed in the alteration of sialylation intarget proteins. As described in Figure 6, the ST3GAL6 KO cells transfected with or without ST3GAL3, ST3GAL4, or ST3GAL6 genes, were
cultured in complete medium with 1 μg/mL DOX for 72 hours before the following experiments were performed. Equal amounts of cell lysatesas indicated were immunoprecipitated with MAM-agarose or SSA-agarose for 1 hour at 4°C with rotation, and then the immunoprecipitates were
washed twice with lysis buffer and subjected to SDS-PAGE. The precipitated glycoproteins and cell lysates were immunoblotted for β1 integrin(A), EGFR (B), or N-cadherin (C) antibodies. The relative levels of ɑ2,3- and ɑ2,6-sialylation on β1 integrin (D), EGFR (E), and N-cadherin (F) inthese four kinds of cells were quantified (n = 3 independent experiments). All values are reported the means ± standard derivation (error bars), oneway ANOVA; *P < .05; **P < .01; ***P < .001

such as β1 integrin. Interestingly, among all three genes,
ST3GAL4 deficiency alone reciprocally increased the levelsof α2,6-sialylation (Figure 5), while the overexpression of
ST3GAL4 conversely decreased the α2,6-sialylation levels of
β1 integrin (Figure 7). Therefore, when evaluating the effectof α2,3-sialylation, the influences of α2,6-sialylation could
not be neglected, since the α2,6-sialylation of β1 plays important roles in cell adhesion and cell proliferation.52,69 These
results could also partly explain the observation of different
phenotypes in these three KO cells. Although the decrease in
the α2,3-sialylation of β1 integrin was more obvious in the
ST3GAL4 KO cells, the phenomena of cell-cell aggregationand cell proliferation were much weaker than that observedin the ST3GAL3 KO cells, which might have been due to thesignificant increase in the α2,6-sialylation of β1 integrin in the
ST3GAL4 KO cells. In fact, the expression of ST6GAL1 isimportant for not only β1-mediated cell adhesion but also forcellular signaling such as that seen in the PI3K-AKT signaling pathway.52,70 Our previous results showed that the up-regulation of α2,6-sialylation inhibited E-cadherin expression,71

which might partly explain why the expression levels ofE-cadherin were increased in ST3GAL3 or ST3GAL6 KO
cells, whereas it was decreased in the ST3GAL4 KO cells
in the present study, although we could not identify whether
the levels of sialylated E-cadherin changed after a KO of thethree enzymes. Nevertheless, we did not exclude other possibilities that could affect E-cadherin expression.
It is noteworthy that the down-regulation of α2,3-sialylation decreased the α2,6-sialylation of EGFR in the ST3GAL6
KO cells (Figure 5) while up-regulation of α2,3-sialylation
increased it (Figure 7). Thus far, the details of this molecular mechanism remain unclear, but α2,3-sialylation could
affect EGFR conformation changes, which could result in the
promotion of ST6GAL1 accessibility to some glycosylationsites for α2,6-sialylation. In fact, EGFR contains 12 potential
N-glycosylation sites, and the N-glycans on EGFR have beenreported to play important roles in receptor functions that include ligand binding and tyrosine kinase activity.72 For example, the Asn-420-Gln mutant EGFR did not bind to the EGF
ligand, but spontaneously induced oligomerization, resulting

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QI ET ALF I G U R E 8 The proposed mechanisms for the modification of ɑ2,3-sialylation by ST3GAL3, ST3GAL4, and ST3GAL6 in glycoproteins
expressed on the cell surface in different domains. All membrane glycoproteins such as β1 integrin, EGFR, and N-cadherin were synthesized inribosomes and then transferred from the ER to the Golgi apparatus for oligosaccharide assembly, and then finally transported to the cell surface indifferent domains. The present study clearly supported the notion that there are distinct functional units (80), referred to as “zones,” in the Golgi
apparatus (78, 79), that is, the ɑ2,3-sialylation of β1 integrin was mainly catalyzed in zones containing ST3GAL4, whereas the ɑ2,3-sialylationof EGFR occurred in zones containing ST3GAL6. On the other hand, the ɑ2,3-sialylation of N-cadherin was accomplished in zones containing
ST3GAL3, ST3GAL4, and ST3GAL6. Furthermore, it is worth noting that ST6GAL1 could be ubiquitously distributed in all of those zones. These
results clearly demonstrate that the three α2,3-sialyltransferases of ST3GAL3, ST3GAL4, and ST3GAL6 differed in their modification of target
proteins and in their regulation of cell biological functions

in phosphorylation of the receptor in the absence of a ligand,further supporting the notion that glycans may be involvedin maintaining the proper structure of target proteins.73 Of
course, we could not exclude the effects from glycolipids or/
and O-glycosylated proteins, since the sialyltransferases inthis study were also involved in the modification of glycolipids and/or O-glycoproteins.23,34,74-76
Most sialytransferases modify proteins in the Golgi apparatus. Based on our results, we speculated that three
α2,3-sialytransferases modify glycoproteins (β1, EGFR, andN-cadherin) through different pathways or functional distinct
Golgi units (Figure 8), which can also be referred to as the“zones” of mitochondria77 or Golgi apparatus.78,79 Hiroyuki
et al agreed with our observations and reported that functional distinct Golgi units exist in Drosophila cells80 to selectively modify target proteins such as Notch and GAG coreproteins. Therefore, it is reasonable to postulate that distinct
Golgi zones containing different kinds of sialyltransferases
exist in HeLa cells, and that the majority of β1 integrin or
EGFR might be modified with α2,3-sialylation in the zones
containing ST3GAL4 or ST3GAL6, respectively. ST6GAL1
might ubiquitously exist in those zones together withST3GAL3, ST3GAL4, or ST3GAL6. In addition, the specificities for target protein modification might also depend on
the acceptor structures, it is known that ST3GAL3 seems toprefer the Galβ1-3GlcNAc disaccharide, whereas ST3GAL4and ST3GAL6 appear to prefer the Galβ1-4GlcNAc linkage.22,81,82 The detailed mechanisms will require further
study.
In conclusion, using KO and cross-restoration techniques,
we clearly demonstrated that ST3GAL3, ST3GAL4, andST3GAL6 differ in their modification of target proteins andin their regulation of cell biological functions. Clarifying thespecificities of the three sialyltransferases and the underlying molecular mechanisms will shed light on the biological
functions of ST3GAL3, ST3GAL4, and ST3GAL6 in various
tissues and cells, and could also provide insight into drug developments for cancer and infection therapies.
ACKNOWLEDGMENTS
This work was supported in part by a Grant-in-Aid for
Scientific Research (15H04354 and 19H03184 to JG and
19K07049 to TI) from the Japan Society for the Promotionof Science, grant from National Natural Science Foundationof China (No. 31670807) and Science and Technology
Project from Jiangsu Traditional Chinese Medicine Bureau(YB2017072); and by a Grant-in-Aid for Scientific Researchon Innovative Areas (18H04868 to JG) from the Ministryof Education, Culture, Sports, Science, and Technology ofJapan.

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CONFLICT OF INTEREST
The authors declare no conflicts of interest and no competing
financial interests.
AUTHOR CONTRIBUTIONS
F. Qi, T. Isaji, and J. Gu designed the research; F. Qi performed all experiments; F. Qi and T. Isaji performed flow cytometric analysis; C. Duan, J. Yang, Y. Wang, and T. Fukuda
assisted with experiments; T. Isaji, Y. Wang, T. Fukuda, and
J. Gu analyzed and interpreted the data; F. Qi, T. Isaji, and
J. Gu wrote and revised the manuscript; and all authors approved the final version of the manuscript.
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multidrug resistance in human leukemia by regulating the PI3K/
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79. Sasaki K, Yoshida H. Organelle zones. Cell Struct Funct.
2019;44:85-94.
80. Yano H, Yamamoto-Hino M, Abe M, et al. Distinct functional unitsof the Golgi complex in Drosophila cells. Proc Natl Acad Sci U S
A. 2005;102:13467-13472.
81. Kitagawa H, Paulson JC. Cloning and expression of human Gal
beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase. Biochem Biophys
Res Commun. 1993;194:375-382.
82. Sasaki K, Watanabe E, Kawashima K, et al. Expression cloning of anovel Gal beta (1–3/1-4) GlcNAc alpha 2,3-sialyltransferase using
lectin resistance selection. J Biol Chem. 1993;268:22782-22787.

How to cite this article: Qi F, Isaji T, Duan C, et al.
ST3GAL3, ST3GAL4, and ST3GAL6 differ in theirregulation of biological functions via the specificities
for the α2,3-sialylation of target proteins. FASEB
BioAdvances. 2020;34:881–897. https://doi.
org/10.1096/fj.201901793R

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Received: 10 September 2019|

Revised: 16 December 2019|

Accepted: 23 December 2019

DOI: 10.1096/fj.201902313RR

RESEARCH ARTICLE

Deficiency of core fucosylation activates cellular signaling
dependent on FLT3 expression in a Ba/F3 cell system
Chengwei Duan1 | Tomohiko Fukuda1
Yuqin Wang2 | Shinichiro Takahashi3| Tomoya Isaji1 | Feng Qi1 | Jie Yang1 || Jianguo Gu1

1

Division of Regulatory Glycobiology,
Institute of Molecular Biomembrane
and Glycobiology, Tohoku Medical andPharmaceutical University, Sendai, Japan2

Department of Pharmacology, Pharmacy
College, Nantong University, Nantong,
China
3

Division of Laboratory Medicine,
Faculty of Medicine, Tohoku Medical andPharmaceutical University, Sendai, Japan
Correspondence
Jianguo Gu, Division of Regulatory
Glycobiology, Institute of Molecular
Biomembrane and Glycobiology, Tohoku
Medical and Pharmaceutical University,
4-4-1 Komatsushima, Aoba-ku, Sendai,
Miyagi 981-8558, Japan.
Email: jgu@tohoku-mpu.ac.jp
Shinichiro Takahashi, Division ofLaboratory Medicine, Faculty of Medicine,
Tohoku Medical and Pharmaceutical
University, 1-15-1 Fukumuro, Miyagino-ku,
Sendai, Miyagi 983-8536, Japan.
Email: shintakahashi@tohoku-mpu.ac.jp
Funding information
Japan Society for the Promotion of
Science, Grant/Award Number: 19H03184,
17K09019 and 17K08284; National Natural
Science Foundation of China, Grant/Award
Number: 31670807; Ministry of Education,
Culture, Sports, Science, and Technology ofJapan, Grant/Award Number: 18H04868

Abstract
Fms-like tyrosine kinase 3 (FLT3) is a glycoprotein, that is a member of the class
III receptor tyrosine kinase family. Approximately one-third of acute myeloid leukemia (AML) patients have mutations of this gene, and activation of the FLT3 downstream pathway plays an important role in both normal and malignant hematopoiesis.
However, the role of N-glycosylation for FLT3 activation remains unclear. In thisstudy, we showed that the N-glycan structures on wild type (WT), internal tandem
duplication (ITD), and tyrosine kinase domain (TKD) mutants of FLT3 were different. Interestingly, expression of either WT or mutant FLT3 in Ba/F3 cells, aninterleukin-3 (IL-3)-dependent hematopoietic progenitor cell, greatly induced corefucosylation. To elucidate the function of core fucosylation in FLT3-mediated signaling, we used a CRISPR/Cas9 system to establish α1,6-fucosyltransferase (Fut8)
knockout (KO) cells. Surprisingly, the Fut8KO resulted in cell proliferation in an
IL-3-independent manner in FLT3-WT cells, which was not observed in the parental
cells, and suggested that this proliferation is dependent on FLT3 expression. Fut8KO
greatly increased cellular tyrosine phosphorylation levels, together with an activation of STAT5, AKT, and ERK signaling, which could be completely neutralized
by restoration with Fut8 in the KO cells. Consistently, a tyrosine kinase inhibitor
efficiently inhibited cell proliferation induced by Fut8KO or specific fucosylation
inhibitor. Additionally, immunostaining with FLT3 showed that the proteins weremainly expressed on the cell surface in the KO cells, which is similar to FLT3-WT
cells, but different from the ITD mutant. Finally, we found that Fut8KO could induce
dimer-formation in FLT3 without ligand-stimulation. Taken together, the present
study clearly defines the regulatory function of core fucosylation in FLT3, which
could provide a valuable direction for development of drugs could be effective in thetreatment of AML.
KEYWORDS
acute myeloid leukemia, cellular signaling, core fucosylation, FLT3, Fut8

Abbreviations: AAL, aleuria aurantia lectin; AML, acute myeloid leukemia; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; ER, endoplasmic
reticulum; FBS, fetal bovine serum; FLT3, Fms-like tyrosine kinase 3; Fut8, α1,6-fucosyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;IL-3, interleukin-3; ITD, internal tandem duplication; KO, knockout; PBS, phosphate-buffered saline; PhosL, pholiota squarrosa lectin; PNGase F,
peptide-N-glycosidase F; TKD, tyrosine kinase domain; WGA, wheat germ agglutinin; WT, wild type.
The FASEB Journal. 2020;00:1–14.

wileyonlinelibrary.com/journal/fsb2

64© 2020 Federation of American Societies forExperimental Biology

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IN T RO D U C T ION

their downstream signaling. Conversely, a deficiency in corefucose leads to a marked enhancement of complex formationsuch as activin receptor40 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR),35 which constitutively activated intracellular signaling. Depletion of thecore fucosylation of IgG1 is reported to be the most criticalrole in the enhancement of antibody-dependent cellular cytotoxicity.41,42 Recently, the blocking of core fucosylation inprogrammed cell death protein 1 (PD-1) was shown to reduce the cell-surface expression of PD-1 and enhance T cell
activation, which led to more efficient tumor eradication.43
Those studies clearly suggest that core fucosylation plays important roles in cell-signal transduction.
In the present study, to clarify the impact of core fucosylation on FLT3, we employed a CRISPR/Cas9 system toknockout (KO) Fut8 gene in the Ba/F3 cell line, an interleukin-3 (IL-3) dependent hematopoietic progenitor cell.44 We
found that a deficiency of Fut8 increased the FLT3 dimericformation and intracellular signaling, which led to IL-3 independent cell growth of the FLT3- wild type (WT) cells,
but greatly chemosensitized the cells to PKC412, a tyrosine
kinase inhibitor. These novel findings suggest that core fucosylation plays a pivotal role in both normal and malignant
FLT3 signaling.

Acute myeloid leukemia (AML) is a heterogeneous aggressive disease that accounts for approximately 12% of allhematologic malignancies.1 AML is characterized by the expansion of an abnormal stem cell clone and accumulation of
immature blasts in the bone marrow.2 Over the past decade,accumulating evidence has suggested that leukemogenesis
is a process in which multiple events involving independent
genetic alterations in proto-oncogene or suppressor genes, together with epigenetic or environmental factors, contribute to
the development of a full malignant phenotype.3-7
Fms-like tyrosine kinase 3 (FLT3) is a proto-oncogene expressed in both normal hematopoietic cells and AML cells.
FLT3 plays a pivotal role in hematopoiesis through regulating
the hematopoietic cell proliferation, survival, and differentiation.8-12 Internal tandem duplication (ITD) of the juxtamembrane domain of FLT3 is the most frequent kinase mutationin human AML (affecting 20%-30% of adult AML), and issignificantly associated with leukocytosis and a poor prognosis.11,13,14 The predominant point mutation within the tyrosine
kinase domain (TKD) is D835Y mutation, which is found in
5%-10% of AML patients.15,16 FLT3-ITD and FLT3-TKD
are known to induce ligand-independent cell proliferation incytokine-dependent Ba/F3 and 32D cells via the display ofdifferent signaling properties.17-20 The most significant difference in these signaling properties is STAT5, which FLT3ITD strongly and constitutively activates.17,19 FLT3-ITD
mutations lead to an endoplasmic reticulum (ER)-retained intracellular localization, and a change in the maturation of surface glycosylation and autophosphorylation of the receptor
results in efficient activation of STAT5.21,22 Mass spectrometry analysis exhibited nine potential N-glycosylation sites inthe extracellular domain of FLT3.23 These facts strongly suggest that surface glycosylation changes in the FLT3 receptor
may affect the function and downstream signaling of FLT3.
N-Glycosylation plays critical roles in folding, stability,
and a vast degree of biological functions of glycoproteins.24-26
These different effects on glycoproteins mainly result fromthe different N-linked glycan structures determined by various glycosyltransferases. Among these, α1,6-fucosyltransferase (Fut8) is the only enzyme that catalyzes the transfer of afucose from GDP-fucose to the innermost GlcNAc residue via
α1,6-linkage to form α1,6-fucosylation in mammals.27 The
resulting core fucosylated N-glycans are widely distributed
in a variety of glycoproteins.28 Accumulating data suggeststhat Fut8 and its products may play important roles in various physiological and pathological processes, such as tumor
formation,29,30 inflammation and immune response,31-33 as
well as in central nervous system diseases.34-36 Core fucosylation is known to be crucial for the ligand-binding affinityof transforming growth factor (TGF)-β1 receptor,37 epidermal growth factor (EGF) receptor,38 and integrin α3β139 for

2
2.1

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M AT E R I A L S A N D M E T H O D S|

Antibodies and reagents

Experiments were performed with the following antibodies:
p-STAT5 (#4322S), ERK1/2 (#9102), p-ERK1/2 (#4370),
AKT (#9272), p-AKT (#4060), and FLT3 (#3462S), allof which were acquired from Cell Signaling Technology(Danvers, MA, USA); Total STAT5 (#13-3600) was from
Thermo Fisher, and monoclonal antibody against α-tubulin
was acquired from Sigma (St. Louis, MO, USA). Rabbit
polyclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#sc-25778) and Mouse monoclonal
antibodies against FLT3 (#sc-19635), p-Tyr (sc-7020), andp-Tyr agarose (sc-508 AC), all were from Santa Cruz (Santa
Cruz, CA, USA). Biotinylated aleuria aurantia lectin (AAL)
and wheat germ agglutinin (WGA) were obtained from the
Seikagaku Corp (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). TO-PRO-3 (#T3605)
was obtained from Molecular Probes, Inc (Thermo Fisher,
Waltham, MA, USA); The peroxidase-conjugated goat antibodies against mouse and rabbit IgG were from Cell Signaling
Technology; Goat anti-mouse/rabbit IgG Alexa Fluor 568,
goat anti-rabbit/mouse IgG Alexa Fluor 488, streptavidinconjugate Alexa Fluor 647, and 488 were purchased from

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2.4 | Plasmid constructions andestablishment of Fut8KO cell

Invitrogen (Carlsbad, CA, USA). Recombinant murine IL-3
was obtained from PeproTech (London, United Kingdom).
Neomycin (G418) was purchased from Nacalai Tesque(Kyto, Japan). BS3 linker (A39266, 2 mg × 10) was obtained from Thermo scientific; PKC412 (#M1323) and dimethyl sulfoxide (DMSO) were (#276855) from Sigma; and2-fluoro-L-fucose (2FF) was from Synchem, Inc, IL, USA.

2.2|

The pSpCas9 (BB)-2A-GFP plasmid was purchased from
Addgene (PX458: Addgene #48138), which was depositedby Dr Feng Zhang.49 The target vector of mouse Fut8 gene
was constructed as previously described.50 The plasmid
was transfected into cells according to the manufacturer's
instructions (Amaxa Cell Line Nucleofector kit V). After
2 days of transfection, GFP-positive cells were sorted using a
FACSAria II (BD Bioscience). Following 3 weeks of culture,
Fut8KO cells were sorted by PhosL lectin, and confirmed by
FACS analysis and lectin blotting as described above. Fut8
expression was rescued by electroporating the Fut8KO cellswith the pcDNA3.1 vector containing Fut8 gene as previously described.40
To establish a stable rescue cell line, the CSIV-TRE-RfACMV-KT-Fut8-lentivirus production and infection were
performed as described previously.51 In brief, the CSIV-TRERfA-CMV-KT-Fut8 vectors were co-transfected with pCAGHIVgp and pCMV-VSV-G-RSV-Rev into 293T cells throughcalcium phosphate transfection method. After transfection
for 48 hours, the lentivirus supernatants were collected. The
Fut8KO FLT3-WT Ba/F3 cells were infected with the CSIVTRE-RfA-CMV-KT-Fut8-lentivirus. The infected cells werefirst selected by the Kusabira Orange marker and then were
sorted two times with PhosL lectin using FACS Aria II. The
stable cell lines were used in subsequent studies.

Cell line and cell culture

Parental Ba/F3 cells (CTR) were maintained in RPMI with
10% heat-inactivated fetal bovine serum (FBS), 1 ng/mL recombinant murine IL-3, and 50 μmol/L of 2-mercaptoethanol.
The stably transduced Ba/F3-FLT3-WT cell lines were maintained in RPMI containing 10% FBS, 1 ng/mL recombinantmurine IL-3, 400 μg/mL G418, and 50 μmol/L 2-mercaptoethanol.45 Ba/F3-FLT3-ITD and Ba/F3-FLT3-TKD cells weregrown with 400 μg/mL G418 in RPMI containing 10% heatinactivated FBS and 50 μmol/L 2-mercaptoethanol.45
The 293T (Human embryonic kidney cells) cell line wasobtained from the RIKEN cell bank (Tsukuba, Japan). The
293T cells were grown in Dulbecco modified Eagle medium
containing 10% FBS. Cells were cultured at 37°C in a humidified 5% CO2 atmosphere.

2.3|

Expression plasmids and transfection

To confirm the glycosylation status of FLT3 proteins, wetransfected them with expression pcDNA3.1 vectors containing human FLT3-WT,46 FLT3-ITD,47 or FLT3-TKD45
in 293T cells using PEI MAX (molecular weight, 40 kDa;
Polysciences Inc, PA) and followed the dictates of the US
patent document (US20110020927A1) with minor modifications. Briefly, 12 hours prior to transfection, 5 × 106 cells
were seeded on a 10 cm dish. Each expression vector (12 μg
dissolved in 1mL of dilution buffer (20 mM CH3COONa,
pH = 4.0, 150 mM NaCl) and PEI MAX (36 μg dissolved in
36 μL of 0.2 M HCl and then diluted with the dilution buffer
to 1 mL) were mixed and incubated at RT for 20 min, andthen 2 mL of the mixture was gently transferred to the cell
cultured dish for transfection. After incubation for 6 hours,
the conditioned medium was replaced with a normal culture
medium for further incubation for 48 hours.48 At 48 hours
post transfection, the cell lysates were treated with or without
peptide-N-glycosidase F (PNGase F) (New England BioLabs,
Ipswich, MA, USA) according to the manufacturer's instructions. Then, the digested cell lysates were analyzed byWestern blot. For immunoprecipitation, cell lysates (1 mg)
were immunoprecipitated with anti-FLT3 antibody (1.25 μg)
and 5 μL of Ab-Capcher Mag beads (P-050-1, Protenova).
The immunoprecipitates were analyzed by Western blot.

2.5|

Flow cytometry analysis of cells

Flow cytometric analysis was performed as described before.50 Briefly, cells that indicated a semi-confluency werecollected from the 10 cm culture dishes and centrifuged at
90 g, for 10 minutes. Subsequently, collected cells were
washed with chilled phosphate-buffered saline (PBS) andstained with biotinylated PhosL (1:1000) for 1 hour on ice,followed by incubation with streptavidin-conjugate Alexa
Fluor 647 for 1 hour. During incubation, the cells were mixed
gently every 10 minutes by flicking. After incubation, cells
were washed three times with ice-cold PBS and then analyzed
using a FACS Calibur flow cytometer (BD Biosciences, San
Jose, CA, USA).

2.6 | RT-PCR for detection of mRNA
expression levels
Total RNAs of Ba/F3 cell lines were extracted by Trizol
reagent (Invitrogen), and then reverse-transcribed using a
Prime Script RT Reagent kit with gDNA Eraser (Takara,
Japan) according to the manufacturer's instructions. The

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DUAN ET AL.

specific primers used for the PCR amplification wereshown in Table 1. The GAPDH mRNA was served as acontrol. The obtained reaction products were then subjected to 1.2% agarose gels containing ethidium bromide
for electrophoresis.

2.7|

cells with a hemacytometer. Experiments were performed
in biological triplicates.

2.9|

Western blotting and lectin blotting

2.10

|

MTT assay

To evaluate the cytotoxic effects of PKC412, an MTT(#341-01823, Dojindo) assay was performed. The WT Ba/
F3 cells expressing FLT3 were pretreated with or without
2FF (100 μM) for 3 days. After 3 days, the WT Ba/F3 cells
were plated in quintuplicate at 2 × 104 cells per well in
96-well plates under normal media containing 2FF and
PKC412 at indicated concentrations for another 48 hours.
Then, a 10 μL of MTT solution (5 mg/mL in PBS) was
added to each well and let stand for 4 hours at 37°C. The
plate was then centrifuged at 2,000 rpm, for 10 minutes.
Subsequently, the supernatant was removed and 100 μL of
DMSO was added to each well at 37°C, which was let standfor 10 minutes. Finally, absorption at 570 nm was measured using a microplate reader (Infinite M1000, TECAN,
Japan).

Cell proliferation assay

After washing three times with prewarmed PBS, Ba/F3
parent cells then resuspended with the RPMI 1640 medium
containing 10% FBS, 1 ng/mL recombinant murine IL-3,
and 50 μmol/L of 2-mercaptoethanol. Ba/F3-FLT3-WT
cells then resuspended with the RPMI 1640 medium containing 10% FBS, 1 ng/mL recombinant murine IL-3,
400 μg/mL G418, and 50 μmol/L of 2-mercaptoethanol.
Ba/F3-FLT3-ITD and Ba/F3-FLT3-TKD cells were maintained with RPMI 1640 medium containing 10% FBS,
400 μg/mL G418, and 50 μmol/L of 2-mercaptoethanol. At
daily intervals, following staining with trypan blue, the viable cell number was calculated by counting the unstained
TABLE 1

Confocal microscopy analysis

Cells were processed for immunofluorescence microscopy as
previously described.52 Briefly, cells were washed and suspended in cold PBS containing WGA lectin for 30 minutes
at 4°C to stain the cell membrane. For intracellular staining,cells were fixed with 4% paraformaldehyde for 20 minutes atroom temperature (RT), and washed twice with wash buffer(0.3% BSA in PBS). Cells were plated on poly-L-lysine
coated slides for 20 minutes at RT. To block nonspecificstaining, 400 μL of blocking buffer (10% normal donkey
serum, 0.3% TritonX-100) was added with incubation for
45 minutes at RT. Cells were then stained with FLT3 antibody in dilution buffer (PBS, 1% bovine serum albumin,
1% normal donkey serum, 0.3% TritonX-100, and 0.01%
sodium azide) overnight at 4°C followed by washing withwash buffer and incubation in secondary antibody and with
TO-PRO-3. Slides were rinsed with PBS and mounted using
coverslips, and fluorescence was detected via sequential excitation using an Olympus FV1000 laser-scanning confocal
microscope with an UPlanSApo × 60/1.35 oil objective andhigh-sensitivity gallium arsenide phosphide detector units
operated by F10-ASW version 4.02 software.

Western blot analysis was performed as described previously.50 To prepare the cell lysate, cells were washed withchilled PBS three times and then lysed with cold lysis
buffer (20 mM Tris-HCl (pH = 7.4), 150 mM NaCl, 1%
TritonX-100) containing proteases and phosphatase inhibitor cocktail (Nacalai Tesque). After being subjected to arotor shaker for 40 minutes at 4°C, cell lysates were clearedby centrifugation at 15 000 rpm, for 15 minutes at 4°C,
and the supernatant was transferred to new marked tubes.
The protein concentration was determined using a Pierce
BCA protein assay kit (Thermo Fisher Scientific, Munich,Germany). Equal amounts of protein lysate were resolved
by reducing SDS-PAGE. After electrophoresis, separated
proteins were transferred to PVDF membranes (Millipore,
Billerica, MA, USA) and incubated with indicated primaryand secondary antibodies or with biotinylated lectins as
indicated, and immunoreactive bands were detected using
either an Immobilon Western Chemiluminescent HRP
Substrate (Millipore) or a Vectastain ABC kit (Vector
Laboratories, Burlingame, CA, USA), according to the
manufacturer's instructions.

2.8|

Primer sequences and annealing temperatures (Tm) for RT-PCR

Gene names

Sense primer (5′-3′)

Antisense primer (5′-3′)

Tm (°C)

FLT3

CCCAGTCAATCAGCTTTGGT

CCTGGCTGGTGCTTATGATT

55

Fut8

AGATCTGACAGAGCTGGTCCAG

TCTGTGCGTCTGACATGGACTC

56

GAPDH

ACCCAGAAGACTGTGGATGGCACATTGGGGGTAGGAACAC

56

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DUAN ET AL.

2.11

|

Chemical cross-linking of FLT3

FLT3 on glycosylation. The expression levels of core fucosylation confirmed by AAL lectin, which preferentiallyrecognizes core fucosylated N-glycans,40,50 were enhancedby expression of FLT3 either WT or mutants (Figure 1B),
whereas the reactive abilities with WGA lectin, which recognizes GlcNAc-containing total glycans were similar among
those cells (Figure 1C). To know whether or FLT3 was modified by core fucosylation, we performed an immunoprecipitation with anti-FLT3 antibody in Ba/F3 cells. Unfortunately,
the experiment for detection of FLT3 protein expression was
failed in the Ba/F3 cell. So, we used the 293T cell instead of
Ba/F3 cell to improve protein expression levels, and found
FLT3 was core fucosylation confirmed by AAL lectin blot
as shown in Figure 1D, which was also confirmed in endogenous FLT3 expressed in THP-1 cells, a human acute monocytic leukemia cell line (data not shown). To explore whether
the increased core fucosylation was due to a promotion of
Fut8 expression by FLT3, semi-quantitative RT-PCR was
performed and found that FLT3 did not express in CTR cell
(Figure 1E). Overexpression of either WT- or mutant-FLT3
greatly induced Fut8 mRNA expression (Figure 1F). On thecontrary, the increased levels of core fucosylation in those
FLT3 expressing cells were inhibited by PKC412 (Figure1G), a FLT3 kinase inhibitor, suggesting the FLT3-mediatedsignaling may induce Fut8 expression. That result suggested
that core fucosylation is specifically increased by the expression of either WT or mutant FLT3.

To assay the dimerization of FLT3, a cross-linking experiment was performed as previously described.53 Briefly,
12 hours prior to transfections 1 × 106 cells were seeded ona 6 cm dish, then, transfected with the FLT3-WT expression vector (1 μg) in 293T or Fut8KO 293T cells40 using
PEI MAX as described above. The 293T cells were serumstarved for 8 hours before incubation with or without human
FLT3 ligand (FL) at 100 ng/mL for 30 minutes in 4°C. Crosslinking was performed by an incubation with 1 mM BS3 for
15 minutes at 4°C. The reaction was stopped by adding glycine-HCl (pH = 7.5) to a final concentration at 150 mM andincubating for 5 minutes. After wash three times with chilled
PBS, these cells were harvested with RIPA buffer (Tris-HCl(pH = 7.5), 150 mM NaCl, 1% TritonX-100, 0.1% SDS). The
cell lysates were subjected to 7.5% SDS-PAGE, and analyzed
by Western blot with anti-FLT3 antibody.

2.12

|

Statistical analysis

Results are reported as the means ± SEM. Statistical analyses were performed using an unpaired Student's t test with
Welch's correction (one-tailed) using GraphPad Prism 5.0
software (GraphPad Software Inc). P < .05 was regarded asstatistically significant.

3|

3.2 | Establishment of FLT3-expressed
Fut8KO Ba/F3 cells

RESULTS

3.1 | Comparing of the glycosylation status
of WT with that of FLT3 mutants

To understand the mechanisms of induction for core fucosylation by FLT3, we established Fut8KO cell lines using a murine Ba/F3 cell line, an IL-3-dependent cell line that exhibits
IL-3 independency in the presence of oncogenic signals suchas FLT3-ITD and -TKD.17 The pSpCas9 (BB)-2A-GFP vector containing target sequences were transduced into those
Ba/F3 cell lines as indicated by electroporation, and stable
cell lines were established by positive and negative sortingwith GFP and PhosL lectin as described in the “Methods
and Materials” section. Finally, we successfully confirmed
stable Fut8KO cell lines by flow cytometric analysis using
biotinylated PhosL lectin (Figure 2A) and lectin blotting withAAL (Figure 2B), which specifically recognizes core fucosylated N-glycans.50,54

Glycosylation of proteins often results in a heterogeneous
pattern on Western blots. It is known that there are two formsof human FLT3. One is a mature form at around 150 kDa,
which is thought to be completely glycosylated at the N-linked
glycosylation sites of the extracellular domain of the Golgi
apparatus to form complex types of N-glycans, and is thenexpressed on the cell surface. The other is an immature formaround 130 kDa, which is incompletely glycosylated to forma high mannose type of N-glycans, and is mainly localized in
the ER.22 To confirm the differences in the glycosylation status of FLT3 WT and mutants, we transfected 293T cells withexpression plasmids encoded with FLT3-WT,46 FLT3-ITD,47
or FLT3-TKD,45 and extracted cell lysates after 48 hours forWestern blotting. The bands of FLT3-WT that migrated on
SDS-PAGE were totally different from the other two mutants(Figure 1A), which could be neutralized by the removal of
N-glycans via treatment with PNGase F. All bands of FLT3
were shifted to around 120 kDa upon PNGase F treatment.
On the contrary, we also were curious about the effects of

3.3 | IL-3 independent proliferation in
Fut8KO- FLT3-WT Ba/F3 cells
Fms-like tyrosine kinase 3 plays a crucial role in normal hematopoietic processes such as proliferation, differentiation,

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(A)

(D)

(B)

(C)

(E)

(G)

(F)

F I G U R E 1 Glycosylation patterns of FLT3 proteins and the effects on fucosylation expression. A, The 293T cells were cultured at 70%
confluency, and then transfected with plasmids containing human FLT3-WT, FLT3-ITD, or FLT3-TKD. At 48 hours post transfection, the celllysates were treated with or without PNGase F, and analyzed by Western blot with anti-FLT3 antibody. The effects of FLT3 WT or mutants oncore fucosylation in Ba/F3 cells were examined by AAL lectin blot (B) or WGA lectin blot (C). The same amounts of cell lysates were separated
on 7.5% SDS-PAGE, and the membranes were probed with AAL (top panel), and then re-probed with anti-GAPDH antibody (bottom panel), which
was used as a loading control. D, The same amounts of cell lysates from those indicated transfected 293T cells were immunoprecipitated with
anti-FLT3 antibody, and then the immunoprecipitates were probed with AAL lectin (upper panel) and re-probed with anti-FLT3 antibody (lower
panel) as a loading control. E, RT-PCR using total RNA extracted from different Ba/F3 cells were carried out to examine the mRNA levels of
FLT3. GAPDH was used as control. F, RT-PCR was carried out to examine the mRNA levels of Fut8 in different Ba/F3 cells as indicated. GAPDH
was used as control. G, The same amounts of cell lysates from the different Ba/F3 cells treated with or without PKC412 (1 nM) for 48 hours, wereseparated on 7.5% SDS-PAGE, and the membranes were probed with AAL (top panel), and then re-probed with anti-GAPDH antibody (bottom
panel), which was used as a loading control

and survival.8,12 To test whether Fut8KO regulates cell proliferation, we compared the viable cell number in different
Ba/F3 cell lines. Fut8KO slightly increased cell proliferationin the presence of IL-3 in the CTR (Figure 3A). Quite interestingly, Fut8KO induced cell proliferation in FLT3-WT
cells even in the absence of IL-3 (Figure 3B), whereas the
Fut8KO CTR have no IL-3-independent proliferative activity (Figure 3A). What is more, the restoration with Fut8 genein the Fut8KO WT cells (Rescue cell) resulted in an IL3dependent cell growth (Figure 3B). The cell proliferation of
the rescue cells was completely blocked in the absence of
IL-3. These results suggest that FLT3 expression is necessary
for Fut8KO-induced IL-3-independent cell proliferation. In
addition, ablation of Fut8 partially blocked ITD (Figure 3C)
and TKD (Figure 3D) cell proliferation. These results suggest that the lack of core fucosylation dramatically activate

FLT3-WT. To clarify FLT3 receptor activation mechanisms
related to core fucosylation depletion, we mainly focused on
the FLT3-WT in subsequent studies.

3.4 | Effects of Fut8KO on cellular signaling
in the FLT3-WT Ba/F3 cells
Next, we performed Western blotting to analyze the activation status of STAT5, AKT, and ERK signaling, which areimportant pathways for FLT3 WT and mutant downstream
signaling.17,19,55 Consistently, FLT3-WT could not induce
phosphorylation of STAT5 in the absence of IL-3. However,Fut8KO greatly induced phosphorylation of STAT5 even
without IL-3 (Figure 4A). The activation of AKT and
ERK was also observed in the Fut8KO cells (Figure 4A).

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(A)

(B)

F I G U R E 2 Established Fut8 deficient Ba/F3 cells. A, The parent (CTR), FLT3 WT and mutants of Ba/F3 cells, and Fut8KO cells werecollected and incubated with (bold line) or without (gray shadow) the PhosL lectin, which preferentially recognizes core fucosylated N-glycans,followed by incubation with Biotinylated Alexa Fluor 647 streptavidin and flow cytometric analysis. The vertical dashed lines indicate the peakreacted with PhosL lectin expression in CTR cells. B, Equal amounts of cell lysates from CTR and stably expressed FLT3-WT or mutants wereanalyzed by immunoblot with AAL lectin; GAPDH served as a loading control. Asterisks represent nonspecific bands

3.6 | Deficiency of core fucosylation
increased cellular tyrosine phosphorylationlevels and dimerization of FLT3

Importantly, these inductions were blocked by reexpressionof Fut8 in the KO cells (Figure 4A). The core fucosylation
was confirmed by AAL lectin blotting (Figure 4B).

As we revealed that Fut8KO resulted in the activation of
FLT3-WT downstream signaling, we next examined thetyrosine phosphorylation levels of FLT3. The intracellulardomain of FLT3 has 10 potential tyrosine phosphorylationsites.59,60 The cell lysates and immunoprecipitates with p-Tyr
agarose (PY20) from indicated cells were examined. As a result, ablation of core fucosylation potently increased total tyrosine phosphorylation levels (Figure 6A). There is evidence
that the dimerization of FLT3-WT is an initial and essential
event in ligand-induced signal transduction.61 Therefore, we
compared FLT3 dimerization in 293T and Fut8KO 293T
cells with or without FL. As shown in Figure 6B, the dimer
formation of FLT3 in WT or Fut8KO 293T cells could be
induced by FL. Interestingly, the dimerization was observedin the Fut8KO 293T cells even without FL, but not in the
WT 293T cells. In addition, the upper band of FLT3 (around
150 kDa, so called mature form) rather than lower band(around 130 kDa, so called immature form) participated in
the dimer formation. Collectively, these data suggest that the
deficiency of core fucosylation promotes the dimerizationof FLT3 and results in the aberrant activation of FLT3-WTmediated signaling such as p-STAT5, p-ERK, and p-AKT,

3.5 | Deficiency of core fucosylation did notaffect FLT3 intracellular localization
Changes in FLT3 localization are known to influence downstream signaling. Mutations of FLT3 receptor result in localization in the ER, which affects surface glycoprotein
maturation and activates STAT5, while ligand-activated
WT FLT3 is mainly localized on the cell surface and activates MAP kinase.21,56,57 Therefore, we next performed immunostaining with FLT3 antibody to examine the effectsof core fucosylation on FLT3 intracellular localization. The
FLT3-WT proteins were mainly localized on the cell surface,and co-localized with WGA staining (Figure 5, upper panel).
In Fut8KO cells, the FLT3-WT proteins were also localizedon the cell surface (Figure 5, middle panel). The localization
pattern was quite different that of FLT3-ITD, in which FLT3ITD proteins were mainly localized in the intracellular domain(Figure 5, lower panel), presumably in the ER, as previously
described.21,58 These results suggest that the underlying molecular mechanism for the induction of FLT3-WT by Fut8KO
may be different from activation in the FLT3-ITD mutation.

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(A)

(B)

(C)

(D)

F I G U R E 3 Influences of core fucosylation on Ba/F3 cell proliferation. A, The parent Ba/F3 cells (CTR) and CTR Fut8KO cells were
cultured under normal culture media with or without IL-3 at 1 ng/mL final concentration, and the cell numbers of living cells were measured bytrypan blue exclusion assay at indicated times. CTR KO with IL-3 versus CTR KO without IL-3. B, The parent cells expressing FLT3-WT (WT)or Fut8KO cells expressing FLT3-WT (WT KO), and the WT KO cells restored with Fut8 gene (WT Rescue) were cultured. Cell numbers werecounted as described above. WT KO and WT Rescue with IL-3 versus WT KO and WT Rescue without IL-3, respectively. The parent or Fut8KO
cells expressing FLT3-ITD (C) or FLT3-TKD (D) were also cultured under normal culture media without IL-3 for 24, 48, and 72 hours. The
numbers of living cells were measured by trypan blue exclusion assay. Data represent the average of three independent experiments. All values are
Means ± SEM (n = 3). *P < .05. **P < .01

which induces an IL-3-independent cell proliferation pathway in the Ba/F3 cell system.

proliferation even at 1 nM, which did not affect cell proliferation of the WT cells (Figure 7A). A fluorinated analog of
fucose, 2FF, functions as a metabolic fucosylation inhibitor, which is taken up by cells and converted to GDP-2FF
through endogenous salvage pathways.63,64 Therefore, 2FF
could be considered a specific inhibitor for fucosylation. In
fact, a treatment with 2FF at 100 μM could suppressed corefucosylation in HepG2 cells.30 We also confirmed the inhibitory effect on Ba/F3 cells. Similar to the Fut8KO cells, thesensitivities for the inhibitory effects of PKC412 on cell proliferation were also increased in the cells pretreated with 2FF(Figure 7B). These results suggested that a deficiency of corefucosylation induces a novel signal pathway, but not like theIL-3-dependent signal pathway, which is more sensitive totreatment with a tyrosine kinase inhibitor.

3.7 | PKC412 efficiently inhibited cellproliferation of Fut8KO cells in a relatively
lower dose
Since Fut8KO greatly induced tyrosine phosphorylation
as described above, we examined the effects of PKC412,
a tyrosine kinase inhibitor, on cell proliferation. In fact, it
was reported that the inhibitory effects of PKC412 on cellproliferation could be observed with usage at 500 nM, buteven at 100 nM in Ba/F3 cells expressed FLT3.62 However,it was interesting, that PKC412 significantly inhibited cell

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(A)

(B)

F I G U R E 4 Effects of core fucosylation on intracellular signaling. The wild type (WT), Fut8KO (KO), or restoration of Fut8 in the KO cells(Res) of Ba/F3 cells expressing FLT3 were cultured under normal culture media with (+) or without IL-3 (−) for 24 hours. A, The same amountsof cell lysates were separated on 7.5% SDS-PAGE, and Western blotting with the indicated antibodies including both total and phospho-STAT5,
ERK, and AKT. B, The expression levels of core fucosylation in these cells were blotted with AAL (top panel) and re-probed with anti-GAPDH(low panel). GAPDH served as a loading control

F I G U R E 5 Effects of core fucosylation on FLT3 intracellular localization. The indicated cells were stained for plasma membrane using
WGA lectin (Green), fixation and then adhered to slides coated with poly-L-lysine for further staining with FLT3 (Red) and TO-PRO-3 (Blue).
Slides were visualized by immunofluorescence microscopy using an Olympus FV1000 laser-scanning confocal microscope as described in Methods
and Materials. Bar shows 30 μm

4|

D IS C U SS ION

proliferation in an IL-3 independent manner in FLT3expressing cells, but not in FLT3-negative cells, whichsuggests that this cell proliferation is dependent on FLT3
expression (Figure 8). Of course, we could not exclude influences of Fut8KO from other receptors including IL-3 receptor. We revealed a novel effect for a deficiency of corefucosylation for FLT3 homodimerization and activation ofseveral downstream signaling pathways in the absence ofFms-like tyrosine kinase 3 signaling is important for normal and oncogenic hematopoiesis, but the downstream effect from the modification of FLT3 surface glycoprotein
remains to be elucidated. In the current study, we used
the CRISPR/Cas9 system to establish Fut8KO Ba/F3 celllines, and found that a deficiency of Fut8 resulted in cell

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(A)

(B)

F I G U R E 6 Deficiency of core fucosylation increased cellular tyrosine phosphorylation levels and dimerization of FLT3. The wild type (WT)and Fut8KO (KO) of Ba/F3 cells expressing FLT3 were cultured under normal culture media with or without IL-3 for 48 hours, respectively. A,
The same amounts of cell lysates and immunoprecipitates with p-Tyr agarose (PY20) were separated on 7.5% SDS-PAGE, and the membranes
were blotted with anti-p-Tyr (PY99) and re-probed with anti-α-tubulin, as a loading control. B, The FLT3-WT plasmid was transfected into 293T
or Fut8KO 293T cells. After 48 hours, cells were treated for chemical cross-linking as described under “MATERIALS AND METHODS.” Cell
lysates were separated on 7.5% SDS-PAGE, and blotted with anti-FLT3 antibody to detect FLT3 monomer and dimer. α-Tubulin was used as aloading control

(A)

(B)

F I G U R E 7 Loss of core fucosylation chemosensitized the cells to PKC412, a tyrosine kinase inhibitor. A, The wild type (WT) and the
Fut8KO (KO) of Ba/F3 cells expressing FLT3-WT were cultured in the presence of PKC412 at indicated concentrations, and then cell numbers
were examined after 48 hours. The WT and KO cells were cultured with or without IL-3, respectively. The inhibitory ratio was normalized to thatof each group without the inhibitor as 1. Data are Means ± SEM (n = 3). *P < .05. B, The WT Ba/F3 cells expressing FLT3 were pretreated withor without 2FF (100 μM) for 3 days, and then cultured under normal media containing 2FF and PKC412 at indicated concentrations for another48 hours. The cell numbers were measured using MTT assay. The inhibitory ratio was normalized to that of each group without the inhibitor as 1.
Data are Means ± SEM (n = 3). *P < .05

a ligand. Furthermore, blockage of core fucosylation by
Fut8KO or 2FF, a fucosylation inhibitor, greatly increased
sensitivities for the suppression of cell proliferation byusing PKC412, a tyrosine kinase inhibitor. Therefore, themanipulation of the core fucosylation could provide valuable direction for the development of drugs that could be
effective in treating AML.
As mentioned above, core fucosylation is an important
regulator for receptor-mediated signaling. Our group recently

reported that Fut8 deficiency increases sensitivities to inflammatory stimulators such as IFN-γ or IL-6 in glial celllines,50 and sensitivities for postsynaptic depolarization byenhancing the heteromerization of AMPARs,35 suggestingthat without core fucosylation these receptors might exist inan active state. In contrast, core fucosylation represses several receptor functions as well. Deletion of core fucosylation
down-regulated the EGF-induced phosphorylation of EGF
receptor38 and TGF-β1 receptor-mediated Smad activation.37

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F I G U R E 8 A working model for the role of core fucosylation in regulating FLT3 activity in Ba/F3 cells. As described previously, FLT3 isa member of the class III receptor tyrosine kinase family, which exists in the extracellular (EC) domain that contains 9 potential N-glycosylationsites, a transmembrane (TM), a juxtamembrane (JM), and tyrosine kinase (TK) domains.23 Ba/F3 cells are an IL-3-dependent cell line, and in thepresence of IL-3, STAT5 is potently activated78 (shown in green arrow and green letters). The FLT3-WT signal mainly activates the ERK andAKT pathway in the absence of FLT3 ligand79 (red arrow and red letters). In the present study, the deficiency of core fucosylation induced an
IL-3-independent cell proliferation pathway in the Ba/F3 cell system, in which the expression of FLT3 is essential. A lack of core fucosylation
triggers ligand-independent FLT3 dimerization on the cell surface, resulting in aberrant activation of FLT3-mediated signaling such as p-STAT5,
p-ERK, and p-AKT (yellow arrow), which are different from the observations in the FLT3-ITD and -TKD mutants. Those two mutants are mainly
expressed in the ER, and participate in activation of the STAT5 signaling pathway, rather than the ERK and AKT signaling pathways.21 Thus, theunderlying molecular mechanism for the induction of FLT3-WT by Fut8KO could be different from the activation in the FLT3-mutants, which
could provide valuable direction for the development of drugs that could be effective in the treatment of AML

Sialylation and fucosylation of EGF receptor suppress its
function and activation in lung cancer cells.65 These datasuggest that core fucosylation either positively or negatively
affects receptor functions. In the present study, we also foundthat the effect of core fucosylation differs between WT and
FLT3 mutant receptors. Considering the FLT3-TKD or-ITD mutant could activate downstream signaling pathways
through different mechanisms such as conformation change,
intracellular localization and/or modification status, whichmight be quite different from WT FLT3. Therefore, we could
speculate that the disruption of Fut8 affects other glycoproteins, which interfere with those FLT3 signaling to down-regulate cell proliferation. Further clarifying the mechanisms of
how fucosylation inhibition activates/suppresses FLT3 receptors may be helpful in developing novel targeted therapies forhematological malignancies.
Several important residues in FLT3 activation have beenreported which includes tyrosine residues 589 and 591 forligand-dependent activation of FLT3-WT Ba/F3 cells.66
Tyrosine 589 and 591 are also reported to play importantroles in STAT5 activation and transformation by FLT3ITD.67 Masson et al59 reported that tyrosines 768, 955,
and 969 of FLT3, as phosphorylation sites, and mediatorsof growth factor receptor binding protein (Grb2) interactions lead to the association of Grb2-associated binder 2(Gab2), which contributes to proliferation and survival.
They revealed that these residues are important for the activation of STAT5 and AKT.59 STAT5 is mainly activatedby FLT3-ITD signaling via aberrant localization in the ER
of FLT3-ITD, but barely activated by FLT3-WT signaling.21
We observed that the depletion of core fucosylation did
not change the transmembrane localization of FLT3-WT.
However, FLT3-WT activates not only ERK and AKT, but
STAT5 in the Fut8KO cells (Figure 8). We postulate thatthe activation of STAT5 via the depletion of core fucosylation happens not by an intracellular localization change in
FLT3, but by phosphorylation, or some modifications of theimportant residues of FLT3. The activation mechanisms of
FLT3 receptor via the depletion of core fucosylation should
be clarified in the future.

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FLT3 is reported to undergo glycosylation in the ER,22
and several glycosylation inhibitors were examined for theireffect on the functions of FLT3. One of these compounds isfluvastatin, already approved by the FDA and clinically applied inhibitor of mevalonate synthesis, which apart fromblocking cholesterol synthesis, also inhibits N-glycosylation
by depleting the cells of dolichol phosphate, thus leading toa loss of surface expression and the induction of cell deathin Ba/F3 cell.52 The other compound is 2-deoxy-D-glucose which not only depletes cells of ATP but also impairs
N-glycosylation.68 A possible reason for the selective inhibition of FLT3-ITD-positive cells by compounds affecting
glycosylation may be a further shift of FLT3-ITD toward intracellular localization, thereby abrogating signaling from thecell surface and in turn cell transformation. Tunicamycin isa bacterial antibiotic, which specifically inhibits the transferof activated sugars to dolichol phosphate, an essential step inN-glycosylation of proteins in the ER.69,70 These effects are
partly mediated by arresting FLT3-ITD in an under-glycosylated state and thereby attenuating FLT3-ITD-driven AKT
and ERK signaling.71 Because FLT3 plays a very importantrole in the pathogenesis of AML, various FLT3 inhibitors havebeen developed.72 However, their duration of clinical responseis short because of the rapid development of resistance.73We
found that depletion of fucosylation by Fut8KO in combination with PKC412 efficiently decreases the factor-independent
growth of FLT3-WT cells. Inhibition of N-glycosylation may
be a possible approach for cancer therapy. Theoretically, however, inhibition of core fucosylation inhibitors is less harmful
and should have fewer side effects. To date, several fucosylation inhibitors have been developed,63,64,74 and there are many
good examples of combination therapy with FLT3 inhibitors
for AML.75-77 Therefore, the modulation of FLT3 glycosylation may provide a hint for development of a new therapy for
different types of FLT3-mediated hematological malignancy.

Duan and T. Isaji performed flow cytometric analysis; T.
Isaji, F. Qi, J. Yang, and T. Fukuda assisted with experiments; T. Isaji, Y. Wang, T. Fukuda, S. Takahashi, and J.
Gu analyzed and interpreted the data; C. Duan, S. Takahashi,
and J. Gu wrote and revised the manuscript; and all authors
approved the final version of the manuscript.
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ACKNOWLEDGMENTS
This work was supported in part by a Grant-in-Aid for
Scientific Research (19H03184 to JG, 17K09019 to ST and
17K08284 to TF) from the Japan Society for the Promotionof Science, by a grant from National Natural Science
Foundation of China (No. 31670807), and by a Grant-in-Aid
for Scientific Research on Innovative Areas (18H04868 toJG) from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan.
CONFLICT OF INTEREST
The authors declare no conflicts of interest and no competing
financial interests.
AUTHOR CONTRIBUTIONS
C. Duan, Y. Wang, T. Fukuda, S. Takahashi, and J. Gu designed the research; C. Duan performed all experiments; C.

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How to cite this article: Duan C, Fukuda T, Isaji T,
et al. Deficiency of core fucosylation activates cellularsignaling dependent on FLT3 expression in a Ba/F3cell system. The FASEB Journal. 2020;00:1–14.
https://doi.org/10.1096/fj.201902313RR

77㸺ศᏊㄆ㆑㒊㛛㸼ᩍᤵ ⣽㔝 㞞♸ㅮᖌ Ⳣཎ ᰤ⣖ㅮᖌ ❧⏣ ᓅ⏕ࣞࢡࢳࣥࡢᢠ⭘⒆࣓࢝ࢽࢬ࣒ࡢゎ᫂࡜ࡀࢇ⸆≀἞⒪࡬ࡢᛂ⏝㸯㸧HeLa ⣽⬊࡟࠾ࡅࡿ SAL ྲྀࡾ㎸ࡳᶵᵓࡢゎ᫂ ᙜ◊✲ᐊ࡛ࡣ㸪ࢼ࣐ࢬ༸ࣞࢡࢳࣥ㸦SAL㸧ࡣ㸪HeLa ⣽⬊⭷ୖ࡟Ⓨ⌧ࡋ࡚࠸ࡿࢢ
ࣟ࣎ࢺࣜ࢔࢜ࢩࣝࢭ࣑ࣛࢻ㸦Gb3㸧ࡢ⢾㙐࡟⤖ྜࡍࡿࡇ࡜࡟ࡼࡾࢫࢽࢳࢽࣈ㸦SU㸧
ࡢ⣽⬊ෆ࡬ࡢྲྀࡾ㎸ࡳቑຍཬࡧ⣽⬊እ࡬ࡢ᤼ฟᢚไࢆᘬࡁ㉳ࡇࡋ㸪SU ࡢస⏝ࢆቑᙉࡍࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ㸬ࡋ࠿ࡋ㸪Gb3 ࡟⤖ྜࡋࡓ SAL ⮬㌟ࡣ⣽⬊⭷࡟␃ࡲࡿࡢ࠿㸪࠶ࡿ࠸ࡣ⣽⬊ෆ࡬⛣⾜ࡍࡿ࠿࡟ࡘ࠸࡚ࡣ᫂ࡽ࠿࡟ࡋ࡚࠸࡞࠿ࡗࡓ㸬ᮏᖺᗘࡣ⺯ගᶆ㆑ SAL㸦HL-SAL㸧ࢆస〇ࡋ㸪HeLa ⣽⬊࡬ࡢྲྀࡾ㎸ࡳཬࡧ⣽⬊ෆ࡟࠾ࡅࡿᣲື࡟ࡘ࠸᳨࡚ウࡋࡓ㸬HL-SAL㸦50 μg/mL㸧24 ᫬㛫ฎ⌮࡛㸪HeLa ⣽⬊⭷࡬ࡢ⤖ྜ࠾ࡼࡧ⣽⬊ෆ࡬ࡢ⛣⾜ࡀほᐹࡉࢀࡓ㸬⣽⬊ෆࡢ⺯ගࡣ㸪⭷࡟⤖ྜࡋࡓ HL-SAL ࢆࣁࣉࢸࣥ⢾࡟ࡼࡾ㝖ཤࡋ࡚ࡶࡇࡢ⺯ගࡣṧᏑࡋ࡚࠸ࡓ㸬SAL ࡀ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿ᫬㛫ࢆ᳨ウࡋࡓ࡜ࡇࢁ㸪ฎ⌮ᚋ 0.5 ᫬㛫௨ෆ࡛⭷⤖ྜࡋ㸪⣙
1 ᫬㛫ᚋ࠿ࡽ⃰ᗘ౫Ꮡⓗ࡟ྲྀࡾ㎸ࡲࢀጞࡵ㸪3 ᫬㛫ᚋ࡟ࡣ᰾㏆ഐ࡬ࡢ㞟✚ࡀㄆࡵࡽࢀࡓ㸬ࡲࡓ㸪⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿ SAL ࡀฎ⌮⃰ᗘ࡟౫Ꮡࡍࡿ࠿ྰ࠿ࢆㄪ࡭ࡓ
࡜ࡇࢁ㸪3.125 μg/mL ࡛ࡣ SAL ࡢྲྀࡾ㎸ࡳࡣぢࡽࢀ࡞࠿ࡗࡓࡶࡢࡢ㸪ࡑࢀࡼࡾ㧗࠸⃰ᗘ࡛ࡣ⃰ᗘ౫Ꮡⓗ࡟ྲྀࡾ㎸ࡲࢀࡿࡇ࡜ࡶศ࠿ࡗࡓ㸬ࡇࡢྲྀࡾ㎸ࡳࡣ㸪CRIPR/Cas9 ࢩࢫࢸ࣒࡟ࡼࡾ Gb3 ྜᡂ㓝⣲㑇ఏᏊࢆḞᦆࡉࡏࡓ Gb3 㝜ᛶ HeLa㸦ƦGb3-HeLa㸧࡟࠾࠸࡚ῶᑡࡍࡿࡇ࡜ࡀ♧ࡉࢀࡓࡇ࡜࠿ࡽ㸪SAL ࡢྲྀࡾ㎸ࡳࡣ㸪
Gb3 ࢆ௓ࡍࡿࡶࡢ࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓ㸬Gb3 ࡣ⬡㉁ࣛࣇࢺ㸦GEM㸧࡟Ꮡᅾࡍࡿ⢾⬡㉁࡛࠶ࡿࡇ࡜࠿ࡽ㸪SAL ࡢྲྀࡾ㎸ࡳࡣ⬡㉁ࣛࣇࢺ௓ᅾᆺ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ
࡟ࡼࡿࡢ࡛ࡣ࡞࠸࠿࡜⪃࠼㸪ࡇࡢᶵᵓࡢ㜼ᐖ๣࡛࠶ࡿ࣓ࢳࣝ-ǃ-ࢩࢡࣟࢹ࢟ࢫࢺࣜ
ࣥ㸦MǃCD㸧ࢆ⏝࠸᳨࡚ウࡋࡓ࡜ࡇࢁ㸪⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿ SAL ࡢ㔞ࡀῶᑡࡍࡿᵝᏊࡀㄆࡵࡽࢀࡓ㸬ࡇࢀࡽࡢ⤖ᯝ࠿ࡽ㸪SAL ࡣ HeLa ⣽⬊⭷࡟Ꮡᅾࡍࡿ Gb3 ࡟⤖ྜࡋ㸪⬡㉁ࣛࣇࢺࢆ௓ࡋࡓ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡟ࡼࡾ HeLa ෆ࡟ྲྀࡾ㎸ࡲࢀ࡚࠸ࡿྍ⬟ᛶࡀ♧ࡉࢀࡓ㸬ࡋ࠿ࡋ㸪MǃCD ࡛ࡣ SAL ࡢྲྀࡾ㎸ࡳࡀ᏶඲࡟㜼ᐖࡉࢀ࡞࠿ࡗࡓࡇ࡜࠿ࡽ㸪࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ௨እࡢྲྀࡾ㎸ࡳᶵᵓࡶ㛵୚ࡋ࡚࠸ࡿࡇ࡜
ࡀ⪃࠼ࡽࢀ㸪ูࡢྲྀࡾ㎸ࡳᶵᵓࢆྵࡵ࡚௒ᚋヲ⣽࡟᳨ウࡍࡿᚲせࡀ࠶ࡿ㸬㸰㸧ᑠ㯏⬇ⱆࣞࢡࢳࣥฎ⌮ HeLa ⣽⬊ෆ࡟ぢࡽࢀࡿᾮ⬊ᵝᵓ㐀≀࡟ࡘ࠸࡚ ࡇࢀࡲ࡛࡟ SAL ࡜ࢫࢽࢳࢽࣈ㸦SU㸧ࡢే⏝ฎ⌮࡟ࡼࡾ HeLa ⣽⬊ࡢ⏕Ꮡ⋡ࢆ᭷ព࡟పୗࡉࡏࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ㸬ࡉࡽ࡟㸪ࡇࡢ࣓࢝ࢽࢬ࡟㛵ࡋ࡚ࡣ㸪㸯㸧

78

SAL ฎ⌮࡟ࡼࡾ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿ SU ࡀቑຍࡍࡿ㸪㸰㸧SAL ฎ⌮࡟ࡼࡾෆ㒊ࡀ୰ᛶࡢࣜࢯࢯ࣮࣒ᵝᾮ⬊㸦lysosome like vesicle, LLV㸧ࡀᙧᡂࡉࢀ㸪ࡇࡢᵓ㐀≀࡟ SU ࡀࢺࣛࢵࣉࡉࢀࡿࡇ࡜࡟ࡼࡾ⸆๣ࡢ᤼ἥࡀᢚไࡉࢀࡿ࡜࠸࠺㸰ࡘࡢᶵᵓ࡟ࡼࡾᘬࡁ㉳ࡇࡉࢀࡿ࡜⪃࠼࡚࠸ࡿ㸬௨๓ࡼࡾᑠ㯏⬇ⱆࣞࢡࢳࣥ㸦WGA㸧ࡣ㸪⣽⬊࡟ᑐࡋ࡚⣽⬊Ṛࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ㸬ࡇࡢ⣽⬊Ṛࡣ㸪㏆ᖺࡢ◊✲࡛⣽⬊Ṛࡢ୍✀࡛࠶ࡿࣃࣛࣉࢺ࣮ࢩࢫ࡟ࡼࡿࡇ࡜ࡀ♧ࡉࢀ㸪SAL ฎ⌮ࡢሙྜ࡜㢮ఝࡋࡓᾮ⬊ᵝᵓ㐀≀ࡀᙧᡂࡉࢀࡿࡢࡀ≉ᚩ࡛࠶ࡿ㸬ࡋ࠿ࡋ㸪ࡇࡢᾮ⬊ࡢ≉ᚩ࠾ࡼࡧᙧᡂᶵᵓ࡟㛵ࡍࡿヲ⣽ࡣሗ࿌ࡉࢀ࡚࠸࡞࠸㸬ᮏᖺᗘࡣ㸪ࡇࡢⅬ࡟╔┠ࡋ᳨࡚ウࡋࡓ㸬WGA ࡣ㸪HeLa ⣽⬊࡟ᑐࡋ࡚⣽⬊Ṛࢆㄏᑟࡍࡿࡇ࡜࠿ࡽ㸪ᾮ⬊ᙧᡂᐇ㦂࡟ࡣ⏕Ꮡ⋡ࡀ࡯ࡰኚ໬ࡋ࡞࠸ 6.25 ࠶ࡿ࠸ࡣ 12.5 μg/mL ࡢ⃰ᗘࢆ฼⏝ࡋࡓ㸬WGA
㸦6.25 μg/mL㸧ฎ⌮࡛ࡣ㸪SAL ࡛ぢࡽࢀࡿ LLV ࡜ᙧែⓗ࡟ྠᵝࡢᵓ㐀≀ࡀ⣽⬊ෆ࡟ㄆࡵࡽࢀࡓ㸬ࡇࡢᵓ㐀≀ࡣ㸪௚ࡢ᳜≀ࣞࢡࢳࣥ㸦PNA ࠾ࡼࡧ ConA㸧ฎ⌮࡛ࡣぢࡽࢀࡎ㸪GlcNAc ฎ⌮࡟ࡼࡾ᭷ព࡟ᙧᡂࡀ㜼ᐖࡉࢀࡓ㸬ᾮ⬊ෆ㒊ࡢ pH ࢆ
neutral red ࢆ⏝࠸࡚ㄪ࡭ࡓ࡜ࡇࢁ㸪LLV ࡜ྠᵝ࡟୰ᛶ࡛࠶ࡿࡇ࡜ࡀศ࠿ࡗࡓ㸬ࡲࡓ㸪ᾮ⬊ࡢᙧᡂࡣ M CD ฎ⌮࡟ࡼࡾ୍㒊ᢚไࡉࢀࡓࡇ࡜࠿ࡽ㸪⬡㉁ࣛࣇࢺ௓
ᅾᛶ࡟㉳ࡇࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࡀ㸪ࡇࡢⅬ࡟ࡘ࠸࡚ࡣࡲࡔ୙᫂࡞Ⅼࡀከ࠸㸬ᡃࠎࡣ㸪SAL ฎ⌮࡟ࡼࡾㄏᑟࡉࢀࡿ LLV ࡣ㸪Brefeldin A㸦BFA㸧࡟ࡼࡿ㜼ᐖᐇ㦂ࡢ⤖ᯝ࠿ࡽ㸪ࢦࣝࢪయ࠿ࡽฟⱆࡋࡓࣜࢯࢯ࣮࣒࡟⏤᮶ࡍࡿࡢ࡛ࡣ࡞࠸࠿࡜⪃࠼࡚࠸ࡿ㸬WGA ࡟ࡼࡾᙧᡂࡉࢀࡿᾮ⬊࡟ࡘ࠸࡚ࡶ BFA ࢆ⏝࠸᳨࡚ウࡋࡓࡀ㸪⣽⬊Ṛࢆㄏᑟࡋ࡞࠸⃰ᗘࡢ BFA ࡜ WGA ࢆࡑࢀࡒࢀ⏝࠸࡚ే⏝ฎ⌮ࡋ࡚ࡶ⣽⬊Ṛࡀ㉳ࡇࡿࡓࡵ㸪ࡇࡢⅬ࡟㛵ࡋ࡚ࡶࡉࡽ࡞ࡿ᳨ウࡀᚲせ࡛࠶ࡿ㸬୍᪉㸪HeLa ⣽⬊࡛ࡣ㸪
WGA ฎ⌮࡟ࡼࡾ࣮࢜ࢺࣇ࢓ࢪ࣮࣐࣮࣮࡛࢝࠶ࡿ LC3 ࡢⓎ⌧㔞ࡀቑຍࡍࡿࡇ࡜࠿
ࡽ㸪ࡇࡢᾮ⬊ࡣ࣮࢜ࢺࣇ࢓ࢦࢯ࣮࣒⏤᮶࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓ㸬ࡇࢀࡽࡢࡇ
࡜࠿ࡽ㸪WGA ࡣ㸪GlcNAc ౫Ꮡⓗ࡟⬡㉁ࣛࣇࢺ࡟ᒁᅾࡍࡿ⢾ࢱࣥࣃࢡ㉁࠶ࡿ࠸ࡣ
⢾⬡㉁ࢆ௓ࡋ࡚ LLV ࡜≉ᚩࡀ㢮ఝࡋࡓᾮ⬊ࡢᙧᡂࢆㄏᑟࡋ㸪ࡇࡢ⣽⬊ෆኚ໬ࡀ⣽⬊⏕Ꮡ⋡ࡢపୗ࡟㛵୚ࡋ࡚࠸ࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࡀ㸪୍᪉࡛ SAL ࡣ HeLa ⣽⬊࡟ᑐࡋ࡚⣽⬊Ṛࢆㄏᑟࡋ࡞࠸ࡇ࡜࠿ࡽ㸪ࡇࡢ┦㐪Ⅼ࡟ࡘ࠸࡚⌧ᅾࡉࡽ࡟᳨ウࢆ⾜ࡗ࡚࠸ࡿ㸬㸱㸧࢘ࢩ࢚࢞ࣝ༸⏤᮶ࢩ࢔ࣝ㓟⤖ྜᛶࣞࢡࢳࣥ (cSBL) ࡣ㸪ࣞࢡࢳࣥάᛶ࡜ࣜ࣎
ࢾࢡࣞ࢔࣮ࢮάᛶࢆేࡏᣢࡘከᶵ⬟ࢱࣥࣃࢡ㉁࡛࠶ࡿ㸬cSBL ࡣ㸪⣽⬊ෆ RNAࡢศゎࢆᇶ┙࡜ࡋࡓ㸪᪂つᢠࡀࢇ๣ೃ⿵࡜ࡋ࡚ᮇᚅࡉࢀ࡚࠸ࡿࡀ㸪ࡑࡢᢠ⭘⒆స
⏝ᶵᗎࡣ୙᫂࡞Ⅼࡶከࡃ㸪≉࡟ cSBL ࡟ࡼࡾⓎ⌧ኚ໬ࡍࡿ㑇ఏᏊ࡟ࡘ࠸࡚ࡢሗ࿌
ࡣ࡯࡜ࢇ࡝࡞࠸㸬cSBL ࢆྵࡴᢠ⭘⒆ᛶࣜ࣎ࢾࢡࣞ࢔࣮ࢮࡢ㑇ఏᏊⓎ⌧ㄪ⠇◊✲ࡢㄢ㢟࡜ࡋ࡚㸪ࣜ࣎ࢾࢡࣞ࢔࣮ࢮ⮬యࡀ RNA ࢆศゎࡍࡿࡓࡵ㸪ᐇ㦂⣔ࡢ☜❧ࡀᅔ㞴࡜࠸࠺Ⅼࡀ࠶ࡿ㸬ࡇࢀࡲ࡛࡟㸪࠸ࡃࡘ࠿ࡢᢠ⭘⒆ᛶࣜ࣎ࢾࢡࣞ࢔࣮ࢮࢆ⏝࠸ࡓ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡀ⾜ࢃࢀ࡚࠸ࡿࡀ㸪RNA ศゎࡀ࡯࡜ࢇ࡝㉳ࡇࡽ࡞࠸ప⃰79ᗘ㸪ࡍ࡞ࢃࡕ㸪ᢠ⭘⒆స⏝ࡀ࡯࡜ࢇ࡝㐍⾜ࡋ࡚࠸࡞࠸᮲௳ୗ࡟࠾࠸࡚ࡢሗ࿌ࡢࡳ࡛࠶ࡿ㸬  ᮏ◊✲ᐊ࡛ࡣ㸪cSBL ⪏ᛶࡀࢇ⣽⬊ࢆ㸪cSBL ࡢ㛗ᮇ㛫ฎ⌮࡟ࡼࡾᶞ❧ࡋ࡚࠸ࡿ㸬ᮏᖺᗘࡣ㸪cSBL ฎ⌮࡟ࡼࡾⓎ⌧ኚ໬ࡍࡿ㑇ఏᏊࢆ㸪RNA ศゎࡢᙳ㡪ࡀ↓࠸᮲௳ୗ᳨࡛ฟࡍࡿࡇ࡜ࢆ┠ⓗ࡟◊✲ࢆ⾜ࡗࡓ㸬cSBL 㠀Ꮡᅾୗ࡛ࡶࡑࡢ⪏ᛶࢆ⥔ᣢࡍࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓ㸪」ᩘᰴ࠶ࡿ cSBL ⪏ᛶ⣽⬊ࡢ⪏ᛶᗘࢆ㸪ࢥࣟࢽ࣮ᙧ
ᡂἲ࡟ࡼࡾホ౯ࡋ㸪ࡼࡾ㧗࠸⪏ᛶࢆ♧ࡋࡓ⣽⬊ (cSR-A1 ࠾ࡼࡧ cSR-B1 ⣽⬊)ࡢ RNA ࢧࣥࣉࣝࢆㄪᩚࡋࡓ㸬㑇ఏᏊ࣐࢖ࢡࣟ࢔ࣞ࢖ࡣ㸪60K Agilent 60-mer
oligomicroarray ࢆ⏝࠸࡚⾜ࡗࡓ㸬ࡑࡢᚋᚓࡽࢀࡓ᝟ሗࢆඖ࡟㸪DAVID ࢯࣇࢺ࢙࢘࢔ࢆ⏝࠸㸪Gene Ontology (GO) ゎᯒ࠾ࡼࡧࣃࢫ࢙࢘࢖ゎᯒࢆ⾜ࡗࡓ㸬  cSBL ⪏ᛶ⣽⬊࡟࠾࠸࡚Ⓨ⌧ࡀኚ໬ࡍࡿ㑇ఏᏊࢆྠᐃࡋࡓ⤖ᯝ㸪ୖ᪼ࡲࡓࡣ
పୗࡍࡿ㑇ఏᏊ࡜ࡋ࡚㸪ࡑࢀࡒࢀ 440㸪487㸪ィ 927 㑇ఏᏊࡀྠᐃࡉࢀࡓ㸬GO
ゎᯒ࠾ࡼࡧࣃࢫ࢙࢘࢖ゎᯒࡢ⤖ᯝ࠿ࡽ㸪⣽⬊ቑṪ࡟㛵㐃ࡍࡿ㑇ఏᏊࡸ⣽⬊⭷ୖࡢ
ࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢ௚㸪≉࡟⢾ࡸ⬡㉁ࡢ௦ㅰ࡟㛵ࢃࡿ㑇ఏᏊ࡟ከࡃ
ࡢⓎ⌧ኚືࡀ࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓ㸬࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ࡛ᚓࡽࢀࡓ㑇ఏᏊࡢ୰࠿ࡽ㸪Ⓨ⌧పୗࡀ᭱ࡶ኱ࡁ࠿ࡗࡓ 5 ࡘࡢ㑇ఏᏊ (THY1㸪AKR1B15㸪
AKR1B10㸪SLC47A2㸪CBR1; Table 1) ࡟╔┠ࡋ㸪ࣜ࢔ࣝࢱ࢖࣒ PCR ἲ࡟ࡼࡾࡑࡢ⤖ᯝࢆ෌☜ㄆࡋࡓ⤖ᯝ㸪࠸ࡎࢀࡢ㑇ఏᏊࡶ cSBL ⪏ᛶ⣽⬊࡟࠾࠸࡚ࡑࡢⓎ⌧
ࡀ኱ࡁࡃ(-22 㹼-480 ಸ) ῶᑡࡍࡿࡇ࡜ࡀ♧ࡉࢀ㸪࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡢ⤖ᯝ࡜
ྠᵝ࡞ഴྥࡀᚓࡽࢀࡓ㸬ࡲࡓ㸪ྛ㑇ఏᏊࡢศᏊᶵ⬟ࢆㄪ࡭ࡓ⤖ᯝ㸪㓟໬㑏ඖ㓝⣲άᛶ࡞࡝௦ㅰ࡟㛵ࢃࡿᶵ⬟ࢆ᭷ࡋ࡚࠸ࡿࡶࡢࡀከࡃぢࡽࢀࡓ (Table 1)㸬ࡇࡢࡇ࡜࠿ࡽ㸪cSBL ࡛㛗ᮇ㛫ฎ⌮ࡉࢀࡓࡀࢇ⣽⬊࡛ࡣ㸪௦ㅰ࡟㛵୚ࡍࡿ㑇ఏᏊࡢⓎ⌧ࡀᜏᖖⓗ࡟ᙳ㡪ࢆཷࡅ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿ㸬௒ᚋࡢㄢ㢟࡜ࡋ࡚㸪௒ᅇྠᐃࡉ
ࢀࡓ㑇ఏᏊࡢ㐣๫Ⓨ⌧ࡸࣀࢵࢡ࢔࢘ࢺᐇ㦂ࢆ㏻ࡋ࡚㸪cSBL ࡢᢠ⭘⒆స⏝࡟┤᥋㛵୚ࡍࡿ㑇ఏᏊࡢྠᐃࡀ࠶ࡆࡽࢀࡿ㸬ࡇࢀࡽࡢ᝟ሗࢆඖ࡟㸪cSBL ࡢᢠ⭘⒆స⏝ᶵᗎࡢゎ᫂ࡢ௚㸪cSBL ࡢຠᯝ⮬యࢆ㧗ࡵࡿ᪉⟇ࡸ㸪᭷ຠ࡞ే⏝⸆ࡢ᥈⣴࡞࡝㸪
cSBL ࢆ฼⏝ࡋࡓ᪂つࡢࡀࢇ἞⒪ἲࡢ㛤Ⓨࡀᮇᚅ࡛ࡁࡿ࡜⪃࠼࡚࠸ࡿ㸬80

Table 1 Known molecular function of THY1, AKR1B15, AKR1B10, SLC47A2
and CBR1

㸺Ⓨ⾲ㄽᩥ㸼
A GM1b/asialo-GM1 oligosaccharide-binding R-type lectin from purplish bifurcate
mussels Mytilisepta virgata and its effect on MAP kinases.
Fujii Y, Gerdol M, Kawsar S.M.A, Hasan I, Spazzali F, Yoshida T, Ogawa Y, Rajia S,
Kamata K, Koide Y, Sugawara S, Hosono M, Tame J.R.H, Fujita H, Pallavicini A and Ozeki
Y., FEBS J., doi:10.1111/febs.15154, 2019㸺Ꮫ఍Ⓨ⾲㸼
࣭ࢼ࣐ࢬ༸ࣞࢡࢳࣥࡣ⬡㉁ࣛࣇࢺ౫Ꮡᛶ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡟ࡼࡾ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿⳢཎᰤ⣖㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪⣽㔝 㞞♸➨ 38 ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪ྡྂᒇ㸪2019 ᖺ 8 ᭶㸪せ᪨㞟 p.222
࣭Bullfrog sialic acid-binding lectin reduces the expression of EGF receptors in cancercells Tatsuta T., Sato S., Sugawara S., Hosono M.
25th International Symposium on Glycoconjugates, ࣑ࣛࣀ㸪2019 ᖺ 8 ᭶㸪せ᪨㞟
p.324
࣭Catfish (Silurus asotus) lectin enhances the cytotoxic effects of sunitinib on renal81

cell carcinoma
 Ito J., Sugawara S., Hosono M., Sato M.
25th International Symposium on Glycoconjugates, ࣑ࣛࣀ㸪2019 ᖺ 8 ᭶㸪せ᪨㞟
p.327
࣭Glycosphingolipid-binding lectin modifies tumor cell membrane transport Hosono M.
1st Japan-Europe Workshop on Glycosphingolipids and Membrane Homeostasis, ࢫࢺࣛࢫࣈ࣮ࣝ㸪2019 ᖺ 9 ᭶㸪せ᪨㞟 p.10
࣭ࢼ࣐ࢬ༸ࣞࢡࢳࣥࡣࣄࢺᏊᐑ㢕ࡀࢇ⏤᮶ HeLa ࡟࠾ࡅࡿࢫࢽࢳࢽࣈࡢྲྀࡾ㎸ࡳ࠾ࡼࡧ᤼ฟ࡟ᙳ㡪ࢆ࠾ࡼࡰࡍ
Ⳣཎᰤ⣖㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪ఀ⸨῟ a㸪బ⸨ ಙ a㸪⣽㔝 㞞♸㸦ᮾ໭་⸆኱࣭་Ꮫ㒊࣭Ἢᒀჾ⛉Ꮫㅮᗙ a㸧➨ 13 ᅇᮾ໭⢾㙐◊✲఍㸪᪂₲㸪2019 ᖺ 9 ᭶㸪せ᪨㞟 p.49
࣭cSBL ⪏ᛶ⣽⬊࡟࠾ࡅࡿ㑇ఏᏊⓎ⌧ኚ໬ ❧⏣ᓅ⏕㸪Ⳣཎ ᰤ⣖㸪⣽㔝 㞞♸➨ 92 ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍㸪ᶓ὾㸪2019 ᖺ 9 ᭶㸪₇㢟␒ྕ 1P-013
࣭ᑠ㯏⬇ⱆࣞࢡࢳࣥฎ⌮ HeLa ⣽⬊ෆ࡟ぢࡽࢀࡿᾮ⬊ᵝᵓ㐀≀࡟ࡘ࠸࡚㡲⸨ᐇဏ㸪Ⳣཎᰤ⣖㸪ᮏ⏣ᤩኴ㸪❧⏣ᓅ⏕㸪⣽㔝 㞞♸➨ 58 ᅇ᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊኱఍㸪௝ྎ㸪2019 ᖺ 10 ᭶㸪せ᪨㞟 p.95
࣭ࢩࢩ ࢔ࣝ㓟⤖ྜᛶࣞࢡࢳࣥ (cSBL) ࡣஙࡀࢇ⣽⬊࡟ ER㸪PgR ࠾ࡼࡧ HER
family ࢱࣥࣃࢡࡢῶᑡࢆక࠺⣽⬊Ṛࢆㄏᑟࡍࡿ బ⸨ ⚈Ꮚ㸪❧⏣ ᓅ⏕㸪బ⸨ ⛱அ㸪Ⳣཎ ᰤ⣖㸪㕥ᮌ ᖖ⩏㸪⣽㔝 㞞♸➨ 40 ᅇ᪥ᮏ⮫ᗋ⸆⌮Ꮫ఍Ꮫ⾡⥲఍㸪᪂ᐟ㸪2019 ᖺ 12 ᭶㸪₇㢟␒ྕ 1P-71

82

A GM1b/asialo-GM1 oligosaccharide-binding R-type lectinfrom purplish bifurcate mussels Mytilisepta virgata and its
effect on MAP kinases
Yuki Fujii1 , Marco Gerdol2 , Sarkar M. A. Kawsar3,4 , Imtiaj Hasan4,5 ,Francesca Spazzali2, Tatsusada Yoshida1 , Yukiko Ogawa1 , Sultana Rajia4,6 ,Kenichi Kamata7 , Yasuhiro Koide4 , Shigeki Sugawara8 , Masahiro Hosono8 ,and Yasuhiro Ozeki4
Jeremy R. H. Tame7 , Hideaki Fujita1 , Alberto Pallavicini2,91 Graduate School of Pharmaceutical Sciences, Nagasaki International University, Sasebo, Japan
2 Department of Life Sciences, University of Trieste, Italy
3 Department of Chemistry, Faculty of Science, University of Chittagong, Bangladesh
4 School of Sciences, Yokohama City University, Japan
5 Department of Biochemistry and Molecular Biology, Faculty of Science, University of Rajshahi, Bangladesh
6 Department of Pharmacy, Varendra University, Rajshahi, Bangladesh
7 Graduate School of Medical Life Science, Yokohama City University, Japan
8 Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan
9 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn Napoli, Italy

Keywords
bivalves; ganglioside; Mytilisepta virgata;purplish bifurcate mussels; R-type lectin
Correspondence
Y. Fujii, Graduate School of Pharmaceutical
Sciences, Nagasaki International University,
2825-7 Huis Ten Bosch, Sasebo, Nagasaki
859-3298, Japan
Tel: +81 956 20 5642
E-mail: yfujii@niu.ac.jp
Y. Ozeki, School of Sciences, Yokohama
City University, 22-2 Seto, Kanazawa-ku,
Yokohama 236-0027, Japan
Tel: +81 45 787 2221
E-mail: ozeki@yokohama-cu.ac.jp(Received 12 March 2019, revised 16
September 2019, accepted 22 November
2019)
doi:10.1111/febs.15154

A 15-kDa lectin, termed SeviL, was isolated from Mytilisepta virgata(purplish bifurcate mussel). SeviL forms a noncovalent dimer that binds
strongly to ganglio-series GM1b oligosaccharide (Neu5Acɑ2-3Galb1-3GalNAcb1-4Galb1-4Glc) and its precursor, asialo-GM1 (Galb1-3GalNAcb14Galb1-4Glc). SeviL also interacts weakly with the glycan moiety of
SSEA-4 hexaose (Neu5Aca2-3Galb1-3GalNAcb1-3Gala1-4Galb1-4Glc). A
partial protein sequence of the lectin was determined by mass spectrometry, and the complete sequence was identiﬁed from transcriptomic
analysis. SeviL, consisting of 129 amino acids, was classiﬁed as an R(icin
B)-type lectin, based on the presence of the QxW motif characteristic of
this fold. SeviL mRNA is highly expressed in gills and, in particular,
mantle rim tissues. Orthologue sequences were identiﬁed in other speciesof the family Mytilidae, including Mytilus galloprovincialis, from which
lectin MytiLec-1 was isolated and characterized in our previous studies.
Thus, mytilid species contain lectins belonging to at least two distinct
families (R-type lectins and mytilectins) that have a common b-trefoil
fold structure but differing glycan-binding speciﬁcities. SeviL displayed
notable cytotoxic (apoptotic) effects against various cultured cell lines(human breast, ovarian, and colonic cancer; dog kidney) that possess
asialo-GM1 oligosaccharide at the cell surface. This cytotoxic effect was
inhibited by the presence of anti-asialo-GM1 oligosaccharide antibodies.
With HeLa ovarian cancer cells, SeviL showed dose- and time-dependent
activation of kinase MKK3/6, p38 MAPK, and caspase-3/9. The transduction pathways activated by SeviL via the glycosphingolipid oligosaccharide were triggered apoptosis.

Abbreviations
Gb3, globotriaosylceramide; GM1, monosialotetrahexosylganglioside; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase;
MytiLec-1, Mytilus galloprovincialis ɑ-Gal-binding lectin; SeviL, Mytilisepta virgata R-type lectin; SSEA-4, stage-speciﬁc embryonal antigen 4.

2612

287 () 2612–2630 ª 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf ofFederation of European Biochemical Societies
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.

83

Y. Fujii et al.

A GM1b/asialo-GM1-binding R-type lectin of mussels

Database
Nucleotide sequence data have been deposited in the GenBank database under accession numbers MK434191, MK434192, MK434193, MK434194, MK434195, MK434196, MK434197,
MK434198, MK434199, MK434200, and MK434201.

Introduction
Many marine invertebrates possess lectins (glycanbinding proteins) with various glycan-binding properties [1–3]. In the differentiation of phylogeny, lectinmediated interactions between glycans and proteins
were adapted into various kinds of key pathways
involved in a variety of fundamental biological processes, including embryonic development, immune
responses, and cell growth regulation [4–6]. During
this functional diversiﬁcation, marine invertebrates
developed an unusually large number of lectins, many
having convergent structures that facilitate binding to
speciﬁc glycan structures exposed on the surface of target cells. This combination of functional divergenceand structural convergence has resulted in many
unique sequences and unusual glycan-binding speciﬁcities among lectins isolated from marine invertebrates[7–11].
We described in 2012 a novel lectin (termed ‘MytiLec-1’), isolated from the Mediterranean mussel Mytilus galloprovincialis (family Mytilidae), that had aunique primary structure [12]. MytiLec-1 has a b-trefoil fold [13], a 3-D structure typically found in R-type
lectins, including ricin B-chain [14]. However, MytiLec-1 bound speciﬁcally to the a-galactoside globotriose (Gala1-4Galb1-4Glc) [12,13], whereas manyother R-type lectins bind to b-galactosides such as Nacetyllactosamine. MytiLec-1 induced apoptosis in
Gb3-expressing human Burkitt’s lymphoma cells. Following the identiﬁcation of similar lectins in various
other mussel species [15–17], we referred collectively tosuch lectins as members of the ‘mytilectin family’ [18].
The taxonomic distribution of mytilectins known todate is limited to the protostome clade Lophotrochozoa
and discontinuous; members of this family have been
identiﬁed only in the subclass Pteriomorphia (phylum
Mollusca) and the order Lingulida (phylum Brachiopoda) [19].
Progress in ‘omics’ studies of mussels and otherbivalve mollusks during the past decade has greatly
enhanced our understanding of their genetics andmolecular biology, leading to major advances in basic
and applied scientiﬁc research. Mussels are a traditionalseafood consumed heavily in Europe and increasingly inother parts of the world and are widely used as ‘sentinel’organisms for biomonitoring [20]. Molecular studies
have revealed the essential role of lectins as patternrecognition receptors (PRRs) for microbe-associatedmolecular patterns (MAMPs) in the innate immune systems of mussels [21,22]. A more complete understandingof these lectins will therefore provide a useful basis for
improved mussel breeding practices and prevention of
infections. Physiological processes and the immune system in mussels are strongly correlated with exposure tobiotic and abiotic stress factors [23,24]. Numerous mussel immune system molecules including lectins wererecently shown to be functionally modulated by pathogen exposure and ocean acidiﬁcation [25,26], so thatbivalve lectins are suggested to be important molecules
which respond to the marine environment. The large
and highly diverse lectin repertoire of mytilids [27,28],
which probably includes several components yet to be
identiﬁed, will facilitate effective new approaches formonitoring health status of mussel species, associatedorganisms, and their marine environments.
Since no R-type lectin had been biochemically puriﬁed from the family Mytilidae, mytilectins were considered for some time to be the only b-trefoil lectinspresent in mytilids, and it was speculated that theirnatural function related to the innate immune
response [12,15–17]. However, the isolation of a novel
lectin from the purplish bifurcate mussel (Mytilisepta
virgata) in this study suggests the possibility ofgreater diversiﬁcation among lectins in this family.
Furthermore, the transcriptome of this species
revealed a lack of mRNAs encoding mytilectins andinstead revealed the expression of multiple distinct
mRNAs encoding proteins characterized by the presence of a ricin B-chain domain, typical of R-type lectins [29]. SeviL found from M. virgata shows
characteristics of sugar chain binding and cell toxicity
unlike any lectin reported to date. SeviL activatedintracellular signaling pathways that resulted in cell
death of mammalian carcinoma cells expressing
asialo-GM1, whereas MytiLec-1 binds to Gb3 glycan.
This is the ﬁrst report that two different b-trefoil lectin families (each with its own glycan-binding speciﬁcity) coexist in the same animal species.

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Results
Puriﬁcation of lectin (SeviL) from M. virgata
Supernatant ‘Sup 1‘(see Materials and methods ‘Lectin
puriﬁcation’) from homogenized M. virgata tissues displayed hemagglutination activity despite the absence of
MytiLec-1 from this species. Repeated homogenizationof precipitates yielded supernatants with successively
reduced activity (data not shown). The precipitates
were homogenized again with 50 mM lactose to obtainsupernatants. The hemagglutinating activity was recovered by dialyzing the supernatant ‘Sup 2’ (see Materials and methods ‘Lectin puriﬁcation’). Sup 1 and 2
were applied to a lactosyl-agarose column, and the
new lectin could be eluted with TBS containing 50 mM
lactose (Fig. 1A). The lectin was characterized as a single polypeptide with molecular mass 15 kDa by SDS/
PAGE under both reducing and nonreducing conditions (Fig. 1A) and was termed ‘SeviL’. Puriﬁcationfrom 400 g fresh tissue yielded 6.5 mg SeviL (Table 1).
Hemagglutination activity of SeviL was required tothe addition of calcium chloride (Fig. S1A), indicatingthat the activity was dependent on divalent cations
such as Ca2+. Analytical ultracentrifugation revealed
that SeviL was a tightly but noncovalently bounddimer (Fig. 1B).

Sugar-binding speciﬁcity of SeviL
Sugar-binding speciﬁcity of SeviL is summarized inTable 2. Hemagglutination activity was weakly inhibitedA

by addition of monosaccharides such as D-Gal(25 mM), D-GalNAc (25 mM), and D-Fuc (25 mM) andof disaccharides such as melibiose (25 mM) and lactose(25 mM). These ﬁndings suggest that the chirality of theC3 and C4 carbons in galactose is essential for protein–glycan interaction. Hemagglutination activity was inhibited by administration of bovine submaxillary mucin(0.125 mgmL1), but not porcine stomach mucin or
fetuin, even at concentrations > 1 mgmL1 (Table 2).
These ﬁndings suggested that SeviL does not bind to
porcine stomach mucin or fetuin, possibly because theseglycoproteins have clusters of GlcNAc or sialyllactosamine at the reducing end [30].
Deduced primary structure of SeviL
The cDNA sequence of SeviL was identiﬁed using a
combination of de novo peptide sequencing and ourearlier transcriptomics results [29]. The peptide
sequence obtained from trypsin digestion of SeviL (m/z 685.81 (MH2)2+) was LDYN(M/T/S/C) GDLVANK(Fig. S1B), which we compared with the mRNA
sequences of ﬁve M. virgata tissues determined previously. A single sequence match was found with one
proteinproduct
includingthesequence
104
LDYNGGDLVANK115 (Fig. 2A). The complete
129 amino acid residue sequence was classiﬁed as an
R-type lectin by the Pfam protein database (http://pfa
m.xfam.org/) but is unrelated to MytiLec-1. At least
two distinct lectin types (R-type lectins and mytilectins) having the b-trefoil fold structure are evidently
present in the family Mytilidae.

B

Fig. 1. Puriﬁcation of Mytilisepta virgata lectin, SeviL. (A) SDS/PAGE pattern under reducing (R) and nonreducing (NR) conditions. Numberson the left indicate molecular masses (kDa) of marker proteins (M). (B) The molecular weight of the native protein (30 kDa) obtained from
distribution of sedimentation coefﬁcient by sedimentation velocity AUC. It indicates the presence of dimers in solution, with negligible
amounts of monomer. Concentration c(M) was measured in absorption units (A280).

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A GM1b/asialo-GM1-binding R-type lectin of mussels

Table 1. Puriﬁcation of SeviL from Mytilisepta virgata.

Fraction
Crude extract obtained by TBS
Puriﬁed lectin
Crude extract obtained by lactose inTBS
Puriﬁed lectin

Titer
(HU)

Volume(mL)

Total
activitya

Protein conc.
(mgml1)

Proteinamount (mg)Speciﬁc
activityb

Puriﬁcation
ratio (fold)c

Recovery ofactivity (%)d

128
512
512

500
10
100

64 0005120
51 200

6.5
0.27
2.1

3525
2.7
2100

0.04
190
0.24

1
4750
1

100
8
100

10

40 960

0.38

3.8

1024

4491

80

4096

Total activity is shown by Titer 9 volume; Speciﬁc activity was shown by titer per mg of protein; Puriﬁcation ratio was shown by comparing the value of speciﬁc activity on the crude extract vs. puriﬁed lectin; d Recovery of activity was revealed by comparing the value of
total activity on the crude extract vs. puriﬁed lectin.

ab

Table 2. Saccharide and glycoprotein speciﬁcity of SeviLa.
Saccharides

Minimum inhibitory concentration (mM)

D-GalNAc
D-GlcNAc
D-Gal
D-Glc
D-Man
D-Fuc
LactoseMelibiose
Sucrose

25
N.I.b
25
> 50
> 50
25
25
25
N.I.b

c

absence of a reference genome for this species hampered an in-depth investigation. All the experiments
carried out and reported in this paper refer to SeviL-1,
as the analysis of RNA-sequencing data [29] revealed
that this variant was expressed > 4-fold higher than
SeviL-2 in all tissues.
Comparison of SeviL orthologues among mytilid
species

Glycoproteins

Minimum inhibitory
concentration (mgmL1)

Bovine submaxillary mucin
FetuinPorcine stomach mucin

0.125
N.I.c
N.I.c

Titer of SeviL was previously diluted to 16; b Inhibition was notoccurred even at 200 mM; c Fetuin and bovine submaxillary mucin
did not inhibit even at 2 mgmL1.
aTandem-repeat structure of SeviL
SeviL has a triple tandem-repeat structure with threesubdomains, each consisting of ~ 40 amino acids, with
13–21% sequence similarity, consistent with a b-trefoil
fold (Fig. 2B). A QxW motif is conserved in each subdomain of R-type lectins, and SeviL shows a similar
pattern at residues 40–42 (QxW), 79–81 (TxW), and
121–123 (ExW). The SeviL sequence included only one
Cys (C) residue, which does not form a disulﬁde bond(Fig. 1A).
A second sequence, highly homologous to SeviL andnamed SeviL-2, was identiﬁed in the M. virgata transcriptome. The two sequences only differ at 10 out of
129 amino acid residues (Fig. S1C). Due to the highheterozygosity of mussels, SeviL and SeviL-2 mayeither represent allelic variants of the same locus or
the product of distinct orthologue genes, but the

One or more SeviL orthologues are found in several
other members of the family Mytilidae, including
M. galloprovincialis, M. edulis, M. californianus, andM. trossulus, P. purpuratus and L. lithophaga, with alevel of interspecies sequence conservation ranging from
25% to 99%, depending on the species considered(Fig. S2A). Like the aforementioned case of M. virgata,some species (i.e., P. purpuratus, M. galloprovincialis,and L. lithophaga) display two sequence variants, whichwere characterized more in detail in the Mediterraneanmussel genome [32], revealing a two exons/one intron
gene organization, with the ORF entirely contained
within exon 2 (Fig. S2B). The three Trp residues of
SeviL (residues 43, 81, 123; Fig. 3) are notably conserved in all of Mytilidae SeviL-like proteins.
The distribution of SeviL-like R-type lectins andmytilectins in the different mytilid subfamilies is variable and partially overlapping (Fig. 4). Transcriptome
analysis suggests that R-type lectins (SeviL orthologues)
are present in the subfamilies Brachidontinae (Mytilisepta, Perumytilus) and Lithophaginae (Lithophaga). On
the other hand, both R-type lectins and mytilectins were
found in the transcriptomes of Mytilinae (Mytilus,
Perna), and neither of the two lectin families weredetected in the genomes of Modiolinae, Bathymodiolinae(deep-sea mussels), or Arcuatulinae. While no SeviL-like
R-type lectins could be found in the genomes of nonmytilid bivalves, mytilectin genes are present in Pectinidae (scallops). Overall, these observations reveal a287 2612–2630 ª 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf ofFederation of European Biochemical Societies

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A

B

Fig. 2. cDNA sequence and deduced amino acid sequence of SeviL. (A) The asterisk indicates the stop codon. The peptide fragment
obtained from mass spectrometric analysis is underlined. (B) Amino acid sequence alignment of the internal tandem-repeat subdomains of
SeviL. The consensus at bottom summarizes the residues shared by the three domains. The sequence alignment within the polypeptide isanalyzed by using MUSCLE program [31].

markedly discontinuous distribution of SeviL-like lectins and mytilectins in the bivalve tree of life, which suggests that their loss or retention may be dependent on
unknown environmental or ecological factors.
Expression of SeviL mRNA in M. virgata tissues
RNA-seq mapping graphs based on various tissues
collected from a pool of adult mussels (Fig. 5A) showhigh expression of SeviL (TPM > 10) in gills and2616

mantle rim. SeviL expression levels were much lower
in the digestive gland and posterior adductor muscle
and barely detectable in foot (TPM < 1). The highexpression of SeviL in mantle rim was conﬁrmed by
qRT/PCR on individual mussels (Fig. 5B). The speciﬁcity of expression of SeviL in tissues (gills and mantle) that are constantly exposed to the external
environment suggests the possibility that this lectin is
involved in recognition of glycans found on parasitic
or symbiotic microorganisms.

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A GM1b/asialo-GM1-binding R-type lectin of mussels

Fig. 3. Multiple sequence alignment of SeviL orthologues in various mytilid species, detected in publicly available genomic or transcriptomic
resources. mytvir1 (GenBank accession number MK434191) and mytvir2 (MK434192): Mytilisepta virgata; mytgal1 (MK434193) and mtgel2(MK434194): Mytilus galloprovincialis; litlit1 (MK434195) and litlit2 (MK434196): Lithophaga lithophaga; mytedu (MK434197): Mytilus edulis;
mytcal (MK434198): Mytilus californianus; myttro (MK434199): Mytilus trossulus; perpur1 (MK434200) and perpur2 (MK434201):
Perumytilus purpuratus. Names with numbers (e.g., mytvir1, mytvir2) indicate lectin variants from the same organism. Note that thesequence perpur1 and perpur2 display incomplete N-terminal and C-terminal regions, respectively. The sequence alignment within thepolypeptide is analyzed by using MUSCLE program [31].

Glycan-binding proﬁle of SeviL
The glycan-binding proﬁle of SeviL was determined byarray analysis using 52 representative glycans, as illustrated and numbered in Fig. S3A and Table S1. SeviL
bound signiﬁcantly to the GM1b oligosaccharide(Neu5Acɑ2-3Galb1-3GalNAcb1-4Galb1-4Glc; 36) andits precursor asialo-GM1 oligosaccharide (Galb1-3GalNAcb1-4Galb1-4Glc; 41) (Fig. 6B and Fig. S3B). The lectin also interacted weakly with the glycan moiety ofasialo-GM2 (GalNAcb1-4Galb1-4Glc; 43) oligosaccharide (which contains a GM1b or asialo-GM1 oligosaccharide component) and the globo-series SSEA-4 hexaose(Neu5Acɑ2-3Galb1-3GalNAcb1-3Galɑ1-4Galb1-4Glc;
51) (Fig. 6B). The total amount of SeviL bound with thesethree glycans was concentration-dependent (Fig. 6B and
Fig. S3B).
On the other hand, SeviL showed no notable binding to Thomsen–Friedenreich (TF-) antigen (Galb13GalNAc; 9) nor GM1a (Galb1-3GalNAcb1-4[Neu5Aca2-3]Galb1-4Glc; 35). SeviL displayed nosigniﬁcant interaction with N-glycans (Fig. 6A: 1–7),
O-glycans (Fig. 6A: 8 and 9), and glycosaminoglycans(Fig. 6A: 10–14), which are derived from glycoproteins.
Asialo-GM1 oligosaccharide-dependent apoptosis
Possible triggering of ganglioside-dependent signals by
SeviL was examined. Anti-asialo-GM1 pAb wasapplied to HeLa, MCF7, BT474, Caco2, and MDCK
cells (see Materials and methods ‘Mussels, cell lines,
and reagents’) and caused surface staining of each cellline except BT474 (Fig. 7A). Next, cells (105 mL1)
were incubated with various concentrations of SeviL
for 48 h, and cell viability and proportions of living
cells were determined by WST-8 assay. Increasing the
SeviL concentration from 25 to 100 lgmL1 resulted
in apoptosis (cell death) for HeLa, MCF7, Caco2, and
MDCK (Fig. 7B), but not for BT474. Cotreatment
with anti-asialo-GM1 pAb blocked the cytotoxic effectof SeviL (Fig. 7B, ‘SeviL + pAb’). These ﬁndings

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Fig. 4. Comparative distribution of SeviL/R-type (SL) lectin and mytilectin (ML) family members in bivalves. The presence of ML and SL was
conﬁrmed by genomic and/or transcriptomic data in the family Mytilidae, using Pectinidae, and Ostreidae as outgroups in the subclass
Pteriomorphia. The cladogram was drawn based on the classiﬁcation of bivalve species based on WoRMS data [33]ription levels of SeviL invarious M. virgata tissues, calculated in silico from RNA-seq data in a pool of individuals and expressed as trascripts per milion (TPM) (panelA), or determined in single individuals with qRT/PCR (panel B) (see Materials and methods). Each bar represents the mean plus standard
deviation of three technical replicates.

indicate that SeviL induced apoptosis mediated byganglioside at the cell surface.
Activation in HeLa cells of MAPK pathway andcaspasesIn HeLa cells, SeviL activated the MAPK pathway of
extracellular signal-regulated kinase (ERK)1/2 signalingcascade in dose-dependent manner, as shown by Western blotting (Fig. 8, P-ERK1/2 vs. ERK1/2). SeviL
treatment also phosphorylated p38 mitogen-activatedprotein kinase (Fig. 8, P-p38 vs. p38) and activatedcaspase-3/9 (Fig. 9, procaspase-3 and 9 vs. activatedcaspase 3 and 9). These ﬁndings suggest that SeviL
regulates cell physiological processes through similar
MAPK pathways, including MEK/ERK and p38, andactivate caspase-3 via mitochondrial cycles.
Localization of SeviL in M. virgata tissues
SeviL signals detected by the antiserum indicated its
speciﬁc presence in the outer part of the mantle rim andgills (Fig. 10A,C), but not in the foot (Fig. 10E). This
localization pattern reﬂected the transcriptional levels of
SeviL-encoding mRNA coding in mussel tissues(Fig. 5A). The signals detected by the anti-GM1 pAb
showed the same pattern of distribution as the expression of SeviL (Fig. 10B,D). These ﬁndings suggest that2618

the main sites of expression of SeviL in M. virgata
match with the location of detection of the antigens
detected by the asialo-GM1 pAb. Since SeviL was
obtained by the elution with the sugar-containing bufferfrom the mantle and gills of the mussels during the
puriﬁcation, it seems reasonable for the lectin to befound in these tissues with binding its ligands (Table 1).

Discussion
Over the past decade, glycobiological studies of nontraditional model organisms (such as bivalve mollusks)
have revealed an unexpected diversity of lectins in various taxonomic groups. In this study, we have demonstrated the presence of an R-type lectin (SeviL) in
Mytilisepta virgata, a member of the family Mytilidae.
This lectin family is characterized by a b-trefoil foldstructure and occurs across a wide range of animals,from microorganisms to humans. R-type lectins havebeen reported previously from the invertebrate phyla
Porifera [34], Annelida [35], and Echinodermata [36].
SeviL was assigned to the R-type lectin family on thebasis of sequence similarities to the prototypical ricin Bchain domain, but it displays features not found in othermembers of this family. First identiﬁed in a plant, Rtype lectins are found in a wide variety of organisms
from bacteria to mammals, and numerous structuresfrom this group have been analyzed in detail. Although

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A GM1b/asialo-GM1-binding R-type lectin of mussels

Fig. 5. Transcription levels of SeviL invarious M. virgata tissues, calculated
in silico from RNA-seq data in a pool ofindividuals and expressed as trascripts per
milion (TPM) (panel A), or determined insingle individuals with qRT/PCR (panel B)(see Materials and methods). Each bar
represents the mean plus standard
deviation of three technical replicates.

a QxW motif is conserved in each of the three subdomains, no overall consensus sequence is found like thatof C-type lectins or galectins. The primary structure of
SeviL shares less than 20% similarity with other invertebrate R-type lectins (Fig. S2C), but there is muchgreater similarity (40–90%) among mytilid proteins(Fig. S2A). Both acidic and basic amino acids are foundthroughout the sequence of SeviL, in contrast to MytiLec-1, which has acidic amino acids only on the C-terminal side of each subdomain [12]. Surprisingly, SeviL
also possesses 6 hydrophobic amino acids at the C terminus, as found with MytiLec-1 [12]. In the case of
MytiLec-1, these residues were essential for dimerization[13], but structural analysis will be needed to determine
whether the same is true of SeviL.
Several similar sequences in other Mytilidae species
besides M. virgata (Fig. 3) deﬁne a cluster of orthologues that we have named ‘SeviL-like R-type lectins’.
Curiously, the taxonomic spread of these lectins only
partially overlaps that of mytilectins [18]. While no
mytilectin was detected in the transcriptome of

M. virgata, some mussel species (such as Mytilus galloprovincialis) possess both types of lectin, and others(such as Modiolus philippinarum) have neither (Fig. 4).
The transcription of M. virgata SeviL-like lectin and
M. galloprovincialis MytiLec-1 was similarly conﬁned
to mantle and gills in both species (Fig. 5), suggestingthat these lectins have similar roles in mussel physiology. However, while only the R-type lectin family isexpressed in M. virgata, both members of the R-type
family and the mytilectin family are encoded by the
genome of M. galloprovincialis. It is presently
unknown whether these two lectin families display anoverlapping pattern of expression and are coregulated
in this species. Determining how the expression ofmussel lectins is modulated in response to external
stimuli may bring new insights into the molecular ecology of these proteins, and helping to understand therole of lectin-glycan interactions in the notable capacity of bivalve mollusks to adapt to new environments.
SeviL was puriﬁed by using a lactose-conjugatedafﬁnity column, and its ability to hemagglutinate was287 2612–2630 ª 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf ofFederation of European Biochemical Societies

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A

B

C
B

2620

Fig. 6. Glycan-binding proﬁle of SeviL.
HyLite555-labeled SeviL (0–100 lgmL1:
right upper) was subjected to glycan array
analysis combining glycan-conjugated chips
in which 52 glycan structures were
immobilized and a surface plasmon
resonance scanning detector (numbering as
in Fig. S3A and Table S1). The evanescentﬁeld ﬂuorescence occurring by the binding
between HyLite555-SeviL and the glycans(Fig. S3B) is represented as net intensities(y-axis of these graphs). (A) The chipincludes N-glycans, O-glycans,glycosaminoglycans, Lewis typeoligosaccharides, derivatives of lactose andN-acetyllactosamine and ABH-type
oligosaccharides (No. 1-28 in Table S1). (B)The chip includes ganglio-series
oligosaccharides and globo-series
oligosaccharides (No. 29-52 in Table S1).
Arrows indicate positions (glycans number
36, 41 and 51) of glycans of GM1b, asialoGM1, and SSEA-4 hexsaose, respectively.
M. Internal standard marker for
ﬂuorescence. C. The structural motif of theglycans recognized by SeviL. The coloredpart highlights the structure shared by
GM1b, asialo-GM1 and SSEA-4 hexa(ose),
whose glycans are bound by SeviL (seepanel B). GM1a, TF, and SSEA-4 tetra(ose)are glycans that are not bound by the lectin.

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A

Fig. 7. Detection of asialo-GM1 expression
on cell membranes by immunostaining. (A)Paraformaldehyde-ﬁxed cells were stainedwith anti-asialo-GM1 oligosaccharide pAb
and AF488-tagged goat anti-rabbit Ab(green). Nuclei were counterstained withDAPI (blue). Magniﬁcation: 940. Scale bar:
10 mm. (B) Cytotoxic effects of SeviL on
human ovarian cancer (HeLa), breast cancer(MCF7, BT474), colonic cancer (Caco2), anddog kidney (MDCK) cells. Cells were treatedwith SeviL at various concentrations (0–100 lgmL1) for 24 h, and cell viability(expressed as A450; see M & M/ ‘Cell
viability and cytotoxicity assays’) was
determined by WST-8 assay. SeviL + pAb:
treatment with 50 lgmL1 SeviL plus antiasialo-GM1 oligosaccharide polyclonal
antibody (pAb). pAb (Cont): anti-asialo-GM1
oligosaccharide pAb without SeviL, as
control. Data shown are mean  SE
(n = 3). P values (*P < 0.05) were obtainedwith Dunnett’s test.

B

inhibited in the presence of lactose and melibiose.
However, such inhibition required a high concentration, 20 mM or more of the sugars, roughly 100 times
the ligand concentration used in the glycan array analysis. This observation suggests that the binding by
these disaccharides does not occur under physiologicalconditions.
SeviL, in same with an R-type lectin from sea
cucumber [36], requires divalent cations such as Ca2+
for its hemagglutination activity (Fig. S1A), whereas
MytiLec-1 shows no dependence on metal ions [12]. In
contrast to members of the mytilectin family, including
MytiLec-1 and CGL, which bind to a-Gal in Gb3[13,37], SeviL strongly binds to ganglio-series GM1b
oligosaccharide (Neu5Aca2-3Galb1-3GalNAcb1-4Gal
b1-4Glc) and asialo-GM1 (Galb1-3GalNAcb1-4Gal
b1-4Glc). SSEA-4 hexaose (Neu5Aca2-3Galb1-3
GalNAcb1-3Gala1-4Galb1-4Glc) and asialo-GM2(GalNAcb1-4Galb1-4Glc), which weakly interactedwith SeviL, comprises a part of the structure of GM1b
(Fig. 6B and Fig. S3B). On the other hand, SeviL did
not bind to TF antigens (Galb1-3GalNAc), GM1a
(Galb1-3GalNAcb1-4[Neu5Aca2-3]Galb1-4Glc). These
results suggest that Galb1-3GalNAcb1-4Galb1-4Glc is
the core structure recognized by SeviL and that thepresence of Neu5Ac at the nonreducing terminus is a

desirable feature for sugar binding (Fig. 6C). Furthermore, the hydroxyl group at the C-3 position of the
3rd Gal from the nonreducing terminus is required tobe free (Fig. 6C). SeviL displayed a different carbohydrate-binding speciﬁcity with GM1b and asialo-GM1
compared with the cholera toxin, which bound to
GM1a and fucosyl-GM1 [38]. After all, this study
found that this R-type lectin of the mussels was glycosphingolipid glycan-binding speciﬁc such as ganglioside and globoside, not but glycoprotein glycans(Fig. 6A).
The binding of SeviL to two ganglio-series (GM1band its precursor asialo-GM1) and one globo-series(SSEA-4 hexaose) oligosaccharides is very interesting,since each glycan is expressed speciﬁcally by the target
antigen for Guillain–Barre syndrome (GM1b) [39],natural killer cells and basophils (asialo-GM1) [40],and glioblastoma multiforme (SSEA-4) [41] in vertebrates, respectively. SeviL has potential clinical applications, similar to those of other invertebrate R-type
lectins that recognize speciﬁc glycans such as TF antigen (Galb1-3GalNAc) and LacdiNAc (GalNAcb14GlcNAc) [42,43].
Exposure to SeviL led to increased metabolism andinduction of apoptosis in mammalian cells bearing
asialo-GM1 oligosaccharide (Fig. 7), indicating the287 2612–2630 ª 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf ofFederation of European Biochemical Societies

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Fig. 9. Induction of caspase-3/9 cleavage by SeviL. HeLa cells
were treated for 48 h with SeviL (50, 100 lgmL1), anticancer
reagent etoposide (200, 500 lM) or lysates were prepared, andcaspase-3/9 activation was detected by Western blotting. Eachexperiment was performed in triplicate.

Fig. 8. Phosphorylation of MAPKs by SeviL in HeLa cells. Cells(5 9 105) were treated with SeviL at various concentration (0–100 lgmL1) or durations (0–24 h). Phosphorylation of kinases
was evaluated by Western blotting of cell lysates. P-p38, P-ERK1/2,
and P-MKK3/6: phosphorylated forms of p38, ERK1/2 and MKK3/6
kinase, respectively. MKK3/6 is the upstream kinase of ERK1/2.

ability of the protein to regulate cell proliferation.
SeviL is a dimer like MytiLec-1 (Fig. 1B), whose selfassociation is essential for cytotoxicity. The use of ananti-asialo-GM1 polyclonal antibody (pAb), whichblocked the access to the target of SeviL, completely
abrogated the effect of the lectin (Fig. 7B). The cell
regulatory mechanisms triggered by the interaction
between SeviL and GM1b oligosaccharides will also be
2622

clariﬁed by using a speciﬁc anti-GM1b antibody infuture. SeviL activated various metabolic pathways(including MKK3/6, ERK1/2, p38, and caspase-3/9).
Both MytiLec-1 and SeviL therefore can potentiallyregulate the growth of human cancer cells by binding
their respective ligands and activating similar metabolic pathways (Figs 8 and 9). The mytilectin family is
known to play roles not only in the regulation of cell
death [44] and also cell proliferation [45], through theactivation of kinases. SeviL and the other R-type lectins isolated from Mytilidae may similarly have multiple activities, and it is possible that their expression
may be regulated by signals external to the organism.
b-Trefoil lectins of mussels have been proposed to be
involved in defense against pathogenic microorganisms
typically encountered by bivalves due to their ﬁlterfeeding habits [16,18,46]. By using immunohistochemistry techniques, the expression of SeviL was detected intissues which are in direct contact with the internal andexternal environment (Fig. 10). The comparison
between the transcriptional levels of SeviL-like lectin
and mytilectin in mussels grown in different environments may clarify the speciﬁc role of these lectins in
immune defense against invading microorganisms. The
spatial overlap between the signals detected with theanti-asialo GM1 polyclonal antibody and the antiSeviL antibody (Fig. 10) suggests the presence of similar
or identical antigens with asialo-GM1 in the mussel tissues. The autoantibody which recognizes GM1b raisedin patients affected by the Guillain–Barre syndrome
arises from infection with Campylobacter, because

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Y. Fujii et al.

A GM1b/asialo-GM1-binding R-type lectin of mussels

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

Fig. 10. Localization of SeviL and substances which reacted with anti-asialo-GM1 polyclonal antibody in M. virgata tissues. Tissue sections
derived from mantle rim, gill, and foot were applied by anti-SeviL (column anti-SeviL: A, C, E, G, I and K) and anti-asialo-GM1 (column Anti
AsGM1: B, D, F, H, J, and I) polyclonal antibodies followed by Alexa 568 (red: for the detection Anti-SeviL) or Alexa488 (green: for thedetection of As asialo-GM1) conjugated secondary anti-rabbit IgG antibody. All sections were stained also with DAPI to detect nuclei (blule:
A–F and M-R). IF: immunoﬂuoresence (A–F), phase: phase contrast (G–L) and control: applying nonimmune primary antibody (for SeviL: M,
O, and Q) and without primary antibody (for asialo-GM1 antibody reactant: N, P and R), respectively. Scale bars: 20 lm each.

speciﬁc lipopolysaccharides of the bacteria possess
structures similar to gangliosides [39]. This evidence
may support the hypothesis that the glycans recognized
by SeviL may be present both in invading microorganisms and in the tissues of species pertaining to the family Mytilidae. It has been recently reported that thelectin subunit of the cholera toxin, which is known tobind GM1a, also binds to lipooligosaccharides and iscapable of inhibiting the growth of genus Campylobacter [47]. The glycan structures of most invertebrates andmicroorganisms remain to be thoroughly investigated[48,49], and the application of structural glycobiology
approaches to these phyla will provide a signiﬁcant
improvement in the knowledge on this subject.
Besides the immune response of bivalves in response
to infection by pathogenic microorganisms, the studyof the infection and defense mechanisms enacted bythese marine bivalves against neoplasia has also met aconsiderable interest in recent years. The group of
Goff showed that horizontal transmission of cancer
cells in bivalves resulted from activation of the retrotransposon gene ‘Steamer’ [50]. Such neoplastic cells
may be propagated from one individual and transmitted to others through sea water [51]. In recent, thegroup of Metzger elucidated how the cancer cells ofmussels were transferred across the Atlantic andPaciﬁc Oceans and between the Northern and Southern hemispheres [52]. By knowing this situation, we
will have more interest in how the cancer cells transfer
into the mussels in the Asian area. In order to better
elucidate the physiological role of SeviL-like lectins,
one of our next goals will be to investigate whether
their administration to tumor cells derived frombivalves may have a signiﬁcant effect on cell growth
regulation.
SeviL and MytiLec-1 bind to b- and a-galactosides,
respectively, but it is not yet certain whether the natural ligand of these proteins is found within the organism
itself or in the surrounding environment. The physiological roles of these proteins will remain unclear, however,until their target glycans are identiﬁed. Certain mollusks
have characteristic glycosphingolipids with Gal or GalNAc at their glycan termini [53,54]. Such glycans arepotential ligands for mytilectins, and similar ligands
may exist for SeviL as well. Although the protein
appears from its sequence to be a b-trefoil, it shows limited conservation to other such proteins at the sugarbinding sites (Fig. S4). Characteristics of the molecular

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Y. Fujii et al.

structure of the protein may help understand the function of lectins by revealing the modes of endogenousand exogenous ligand binding. Although the history of
lectins in the mollusks is so long, the studies on the functional characterization of the b-trefoil fold lectin in thebivalves have just begun.

Materials and methods
Mussels, cell lines, and reagents
Mussels (M. virgata) were collected from the seashore at
Saikai City, Nagasaki prefecture, Japan, under the permission of the Saikai-Oosaki ﬁsheries union. The shells were
removed, and bodies were stored whole at 80 °C. Humancell lines HeLa (cervical cancer), MCF7 and BT474 (breast
cancer), and Caco2 (colonic cancer) were from American
Type Culture Collection (ATCC). Dog kidney cell line
MDCK was kindly provided by T. Fujiwara. Lactose, melibiose, sucrose, D-Gal, D-Glc, D-Man, D-Fuc, D-GalNAc,
D-GlcNAc, standard protein markers for SDS/PAGE, porcine stomach mucin, fetuin, and penicillin–streptomycin forcell culture were from FUJIFILM Wako Pure Chemical
Corp. (Osaka, Japan). Bovine submaxillary mucin wasfrom MP Biomedical (Illkirch-Graffenstaden, France). Lactosyl-agarose gel was from EY Laboratories (San Mateo,
CA, USA). RPMI 1640 medium and fetal bovine serum(FBS) were from Thermo Fisher Scientiﬁc (Waltham, MA,
USA). Cell Counting Kit-8 including 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) monosodium salt (WST-8), HiLyte Fluor 555
labeling kit-NH2, and Biotin Labeling kit-NH2 were from
Dojindo Laboratories (Kumamoto, Japan). HRP-conjugated b-actin monoclonal antibody (mAb) was from FUJIFILM. Anti-asialo-GM1 polyclonal antibody (pAb) (rabbit
immunoglobulin) was from Cedarlane (Ontario, Canada).
Anti-p38, antiphosphorylated p38 (pT180/pY182), antiErk1, and antiphosphorylated ERK1/2 (pT202/pY204)
monoclonal antibodies (mAbs) were from Becton Dickinson (Franklin Lakes, NJ, USA). Antiphosphorylated
MKK3/6 (MKK3(S189)6(S207) 22A8) and anticaspase-9
mAbs were from Medical and Biological Laboratories(Nagoya, Japan). Anticaspase-3 mAb was from Cell Signaling Technology (Danvers, MA, USA). HRP-conjugatedanti-mouse or anti-rabbit IgG as secondary antibody wasfrom Chemicon International (Temecula, CA, USA). Alexa
Fluor 568-labeled goat anti-rabbit IgG and Alexa Fluor
488-labeled goat anti-rabbit IgG were from Abcam (Cambridge, UK). Can Get Signal Immunoreaction Enhancer
Solution 1 and 2 were from Toyobo Co. (Osaka, Japan).
Tissue-tek compound was from Sakura Finetek Co (Tokyo,
Japan). Low ﬂuorescence and silicon-coated glass slides
were from Matsunami Glass Industry (Kishiwada, Osaka,
Japan).

2624

Lectin puriﬁcation
Mussel mantles and gills were homogenized with 10 volumes (w/v) 150 mM NaCl containing 10 mM Tris/HCl, pH
7.5 (TBS) with 10 mM CaCl2. Supernatant (Sup 1) was collected by centrifugation at 27 500 g for 1 h at 4 °C as
described in our previous study [12], with some modiﬁcation. Precipitate was homogenized with 10 volumes (w/v)
TBS containing 50 mM lactose, and supernatant (Sup 2)
was collected as above. Sup 2 was dialyzed extensively
against TBS. Both Sup 1 and Sup 2 were applied to lactosyl-agarose column (5.0 mL), and the column was washedwith TBS until absorbance at 280 nm (A280) of efﬂuent
reached baseline level. Lectin was eluted with TBS containing 50 mM lactose.

Hemagglutination assay and sugar-binding
speciﬁcity assay
Hemagglutination assay was performed in 96-well V-shaped
plates as described previously [55]. Twenty microliters of
twofold dilution of puriﬁed lectin in TBS was mixed with
20 lL of a 1% suspension (with TBS; v/v) of trypsinized,
glutaraldehyde-ﬁxed rabbit erythrocytes, TBS, or TBS with
0.2% Triton X-100. Plates were incubated for 1 h at room
temp, and formation of a sheet (agglutination-positive) ordot (agglutination-negative) was observed and scored as
lectin titer. For sugar-binding speciﬁcity assay, 20 lL ofsugar solution (200 mM) was serially diluted with TBS andmixed with 20 lL of lectin solution (adjusted to titer 16),
trypsinized/glutaraldehyde-ﬁxed rabbit erythrocytes, orTBS containing 1% Triton X-100. Plates were incubatedfor 1 h at room temp, and minimal inhibitory sugar concentration was determined.

Protein quantiﬁcation and molecular mass
determination
Protein was quantiﬁed using a protein assay kit (Thermo
Fisher/ Pierce) based on the principle of bicinchoninic acidfor colorimetric detection [56,57], using ovalbumin as standard. SDS/PAGE [58] was performed in 15% (w/v) acrylamide gel under reducing or nonreducing conditions, andgels were stained by Coomassie Brilliant Blue R-250.

Analytical ultracentrifugation
Sample concentration was estimated as 1.0 mgmL1 from
A280 measurement. Sedimentation velocity experiments
were performed using an Optima XL-I analytical ultracentrifuge (Beckman Coulter, Brea, CA, USA) with An-50 Ti
rotor. Analytical cells (with standard Epon two-channel
centerpiece and sapphire windows) were loaded with
400 lL sample and 420 lL reference solution (50 mM
potassium phosphate, pH 7.4, 0.1 M NaCl). Prior to each287 2612–2630 ª 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf ofFederation of European Biochemical Societies95

Y. Fujii et al.

run, the rotor was kept stationary at 293 K in vacuum
chamber for 1 h for temperature equilibration. A280 scans
were performed at 10-min intervals during sedimentation at
201 600 g and analyzed using the continuous distribution (c(s)) analysis module in SEDFIT [59]. Frictional ratio (f/f0)
was allowed to ﬂoat during ﬁtting. c(s) distribution wasconverted to molar mass distribution c(M). Partial speciﬁcvolume of protein, solvent density, and solvent viscosity
were calculated from standard tables using the program
SEDNTERP [60].

Determination of primary structure of SeviL bymass spectrometry
The partial peptide sequence of SeviL was derived by Proteomics International (Nedlands, WA, Australia). 200 lg
lectin was dialyzed extensively against distilled water toremove salt, lyophilized, and digested by trypsin, and peptides were extracted by standard techniques [61]. Peptides
were analyzed by electrospray ionization mass spectrometry using Prominence nano HPLC system (Shimadzu,
Kyoto, Japan) coupled to 5600 triple time-of-ﬂight (TOF)
mass spectrometer (AB Sciex, Framingham, MA, USA).
Tryptic peptides were loaded onto Zorbax 300SB-C18 column, 3.5 mm (Agilent, Santa Clara, CA, USA) and separated on a linear gradient of water/ acetonitrile/ 0.1%
formic acid (v/v). MS/MS spectra were analyzed using
PEAKS Studio software platform v. 4.5 SP2 (Bioinformatics Solutions, Waterloo, ON, Canada) with manual interpretation.

Transcriptomic analysis of full-length cDNA
cDNA sequence of SeviL obtained as above was used toscreen de novo-assembled transcriptome data of M. virgata
obtained from four tissues (i.e., gills, mantle rim, posterior
adductor muscle, and digestive gland) from a pool of mussels collected at the seashore of Saikai city [29].
The assembled contig corresponding to the putative
mRNA lectin sequence was identiﬁed by tBLASTn (e-value
threshold was set at 0.05). The partial peptides sequences
obtained as described in the previous section were used as
queries for BLAST searches against the transcriptome. Correct assembly of the consensus transcript was conﬁrmed byback-mapping RNA-seq reads to the sequence and byassessment of uniform and homogenous mapping along theentire coding sequence. The expression level of SeviL invarious tissues was calculated in silico as TPM (transcripts
per million) using CLC Genomics Workbench v.10 RNAseq mapping tool (Qiagen, Hilden, Germany), setting
length fraction parameter to 0.75, similarity fraction
parameter to 0.98, and match/mismatch/deletion penalties
to 3/3/3. RNA-seq datasets from the tissues mentioned
above were used for this analysis [29].

A GM1b/asialo-GM1-binding R-type lectin of mussels

To further conﬁrm the tissue speciﬁcity of SeviL, qRT/PCR analyses were carried out on three individual mussels,
as described in [29]. In this case, the sequence-speciﬁc primers designed for SeviL are (50 -> 30 ): AATTTGGGGCGTAAAGACCT (forward primer) and GGACTCTCTTCCGAGGGTG (reverse), aiming at the ampliﬁcation of a
111-bp target sequence.

Sequence data availability
The cDNA sequence of SeviL, SeviL-2, and the orthologue
sequences identiﬁed in publicly available transcriptomes ofother mytilid species have been deposited in the GenBank
repository, under the accession numbers MK434191–
MK434201. The sequence alignments among each orthologue in the different species or the subdomains in thepolypeptide are analyzed by using the MUSCLE program(http://www.drive5.com/muscle) [31].

Glycan array analysis
Glycan array analysis was performed by Sumitomo Bakelite Co. (Tokyo, Japan). SeviL was ﬂuorescence-labeled(kex/em 555/570 nm) using HiLyte Fluor 555 labeling kitNH2 as per the manufacturer’s instructions. A wide rangeof 52 glycans including N-glycans, O-glycans, Lewis glycans, lactosamine, blood-type glycans, gangliosides, andglobosides were immobilized on wells. Fluorescence-labeled
SeviL at concentrations ranging from 0 to 100 lgmL1
were incubated overnight at 4 °C with shielding fromlight. SeviL-binding glycans were detected by using BioREX Scan 300, an evanescent ﬂuorescence scanner(Rexxam Co. Ltd., Osaka, Japan). Wavelength of laser
light was used for Cy3, and the exposure time was 300 ms[62].

Cell viability and cytotoxicity assays
Cells were maintained in RPMI 1640 supplemented with
heat-inactivated FBS 10% (v/v), penicillin (100 IUmL1),and streptomycin (100 lgmL1) at 37 °C. Cytotoxic effects
and cell growth following treatment with SeviL at concentrations ranging from 0 to 100 lgmL1 were determinedusing Cell Counting Kit-8 containing WST-8 [12]. Cells(2 9 104, in 90 lL solution) were seeded into 96-well ﬂatbottom plates and treated with 10 lL lectin for 24 h at
37 °C. To evaluate glycan-inhibitory effects, anti-asialoGM1 oligosaccharide pAb (50 lgmL1) was co-incubatedwith cells in addition to lectin for 24 h and then applied tothe assay system. For assay of effect on cell growth, eachwell was added with 10 lL WST-8 solution and incubated
4 h at 37 °C. Cell survival rate was determined by measuring A450 (reference: A600) with a microplate reader (modeliMark; Bio-Rad, Tokyo, Japan).

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Y. Fujii et al.

Detection of activated signal transduction
molecules and their phosphorylated forms

Statistical analysis

HeLa cells (5 9 105) were cultured with SeviL (0–100 lgmL1) for 24 h and then lysed in 200 lL RIPA buffer. Lysate was separated by SDS/PAGE and electrotransferred onto PVDF membrane as described previously [63].
Primary mAbs used were directed to p38 (1:3000; mouse),
phospho-p38 (1:3000; mouse), ERK1 (1:3000; mouse),
phospho-ERK1/2 (1:3000; mouse), phospho-MKK3/6(1:3000; mouse), caspase-3 (1:5000; rabbit), and caspase-9(1:5000; mouse). These antibodies were applied in Can Get
Signal solution 1. Membrane was incubated for 24 h at
4 °C. HRP-conjugated secondary antibody was diluted
1:5000 in Can Get Signal solution 2 [64].

Immunocytochemical analysis of asialo-GM1
oligosaccharide expression
Cells (1 9 106) were ﬁxed with 4% paraformaldehyde in
PBS for 15 min at room temperature, washed 39 with
PBS, blocked with 1% BSA in PBS for 30 min at room
temperature. They were washed 39 with PBS, incubatedwith or without 100 lL anti-asialo-GM1 oligosaccharide
pAb (dilution 1:200 with PBS) at 4 °C for 30 min, washed39 with PBS, treated with 100 lL Alexa Fluor 488-tagged
goat anti-rabbit IgG (dilution 1:200 in PBS) at 4 °C for
30 min. Cells were placed onto low ﬂuorescence glass
slides, mounted with 50% glycerol solution, and examinedby confocal microscopy. Confocal images were obtainedusing FV10i FLUOVIEW (Olympus, Tokyo, Japan).

Acknowledgements
This study was supported by the Research Promotion
Fund of Nagasaki International University and Yokohama City University. This work was also supported
by JSPS Grant-in-Aid for Scientiﬁc ResearchKAKENHI under Grant No. JP19K06239 (YO andYF) and JP18K07458 (YOg and YF). MG and AP
were supported by funding from the European
Union’s Horizon 2020 research and innovation program under grant agreement No. 678589. JRHT
thanks OpenEye Scientiﬁc Software for ﬁnancial support. YO thanked Research and Developmental Support 2018–2019 for Life Sciences from the City of
Yokohama (Life Innovation Platform, LIP. Yokohama, Project No 1842110002). YF and YO were supported by funding from Sugiyama Chemical andIndustrial Laboratory 2019. The authors are gratefulto Mr. Yasunori Imura for directing us to an excellent
M. virgata sampling area and to Dr. S. Anderson forEnglish editing of the manuscript.

Conﬂict of interest

Immunohistochemistry of SeviL expressions on
the mussel tissues

The authors declare no conﬂict of interest.

Mussel organs (gill, mantle rim, and foot) were cut intoaround 1-cm-square pieces, embedded in the Tissue-tek
compound and frozen in isopentane, cooled in by liquid
nitrogen. The frozen tissue block was sliced on 6-lm-thickwith Leitz cryostat (Leica Instruments, Nussloch, Germany), placed on silicon-coated glass slides. Sections weresequentially ﬁxed in PBS containing 4% paraformaldehyde
for 15 min at room temperature, incubated in blocking
solution containing 0.05% saponin and 1% BSA in PBS
for 30 min, incubated with or without anti-SeviL or antiasialo-GM1 oligosaccharide pAb (dilution 1:100 withblocking solution) at room temperature for 1 h. Afterwashing the tissues with PBS, they were treated with Alexa
Fluor 568-labeled goat anti-rabbit IgG or Alexa Fluor
488-labeled goat anti-rabbit IgG (dilution 1:100 in blocking
solution) at room temperature for 1 h, washed with PBS,
mounted with 50% glycerol solution, and observed by confocal microscopy FV10i FLUOVIEW. Nuclei were stained
by DAPI (364/454 nm) [64].

2626Experiments were performed in triplicate, and results presented as mean  standard error (SE). Data were subjected
to one-way analysis of variance (ANOVA) followed byDunnett’s test, using SPSS STATISTICS software package, v. 10(www.ibm.com/products/spss-statistics). Differences with
P < 0.05 were considered signiﬁcant.

Author contributions
YF, MG, JRHT, and YO designed the study; YF,
MG, FS, KK, IH, SR, SMAK HF, and YO performed the experiments; JRHT, YOg, MH, and AP
contributed new reagents/analytic tools; YF, MG,
KK, SS, YK, TY, YOg, and YO analyzed data; YF,
YO, MG, and JRHT wrote the paper.

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Supporting information
Additional supporting information may be foundonline in the Supporting Information section at the endof the article.
Fig. S1. Calcium-dependent hemagglutination and denovo sequence of SeviL.

287 2612–2630 ª 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf ofFederation of European Biochemical Societies

100

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A GM1b/asialo-GM1-binding R-type lectin of mussels

Y. Fujii et al.

Fig. S2. Pairwise sequence comparison of SeviL, andschematic organization of M. galloprovincialis locus
encoding of SeviL-like lectin.
Fig. S3. Glycan-array analysis.

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Fig. S4. A homology model of SeviL.
Table S1. List of 52 oligosaccharides used for the glycan-array analysis.

287 2612–2630 ª 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf ofFederation of European Biochemical Societies101㸺⸆ရ≀⌮໬Ꮫ㒊㛛㸼ᩍᤵ ᒣཱྀ ⰾᶞㅮᖌ ┿㘠 ἲ⩏ຓᡭ ኱㔝 リṌ⢾㙐ᵓ㐀ゎᯒࡢࡓࡵࡢᢏ⾡ᇶ┙ࡢᵓ⠏
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⪃࠼ࡽࢀ࡚࠾ࡾ㸪ࡇࢀࡲ࡛ከࡃࡢ◊✲⪅ࡀᵝࠎ࡞࢔ࣉ࣮ࣟࢳ࡟ࡼࡾ㸪ࡑࡢᶵ⬟ⓗഃ㠃ࢆ᫂ࡽ࠿࡟ࡋ࡚ࡁ࡚࠸ࡿ㸬⢾㙐ࡢᶵ⬟ࡢከࡃࡣ㸪⢾⤖ྜࢱࣥࣃࢡ㉁࡛࠶ࡿࣞ
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ࡶ㐣ゝ࡛ࡣ࡞࠸㸬㏆ᖺ㸪ࣂ࢖࢜་⸆ရ࣭ᢠయ-⸆≀」ྜయ࡜ࡋ࡚ࡢᢠయࡢ฼⏝ࡀቑຍࡋ࡚࠾ࡾ㸪⢾㙐ࡢᵓ㐀࡜ࡑࡢᶵ⬟ไᚚ࡟ࡲࡍࡲࡍὀ┠ࡀ㞟ࡲࡗ࡚࠸ࡿ㸬ᢠయ࡟⤖ྜࡋ࡚࠸ࡿ⢾㙐ࡀࡑࡢᶵ⬟࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸࡚㸪᭱㏆ࡢሗ࿌ࢆࡲ࡜ࡵ㸪≉࡟❧యᵓ㐀ࡢほⅬ࠿ࡽ㆟ㄽࡋ࡚⥲ㄝ࡜ࡋࡓ(1)㸬ࡲࡓ㸪ᢠయࡢ⢾㙐ᨵኚᢏ⾡ࡸศᯒᢏ⾡ࡀ㐍ᒎࡋࡓࡇ࡜ࡶᢠయࡢ⢾㙐ࡢᵓ㐀ᶵ⬟┦㛵ࡢ◊✲ࢆᚋᢲࡋࡋ࡚࠾ࡾ㸪ᢠయ⢾㙐ᨵኚࡢᛂ⏝ࡶྍ⬟࡟࡞ࡗ࡚࠸ࡿ㸬⌧ᅾᵝࠎ࡞࢚ࣥࢻ-ǃ-N-࢔ࢭࢳࣝࢢࣝࢥࢧ࣑ࢽ
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102ࡋ࡚ࡲ࡜ࡵሗ࿌ࡋࡓ(3,4)㸬
II㸬 C ᆺࣞࢡࢳࣥཷᐜయࡢᵓ㐀࡜ᶵ⬟࡟㛵ࡍࡿ◊✲ࣞࢡࢳࣥࡣ⢾㙐ࢆ≉␗ⓗ࡟ㄆ㆑ࡍࡿศᏊ࡜ࡋ࡚ᖜᗈࡃᶵ⬟ࡋ࡚࠸ࡿ㸬ࣞࢡࢳࣥࡣ༢⢾࡟ᑐࡋ࡚ࡢぶ࿴ᛶࡣపࡃ mM ⛬ᗘࡢゎ㞳ᐃᩘ࡛࠶ࡿࡀ㸪ከࡃࡢࣞࢡࢳࣥࡣ
࢜ࣜࢦ࣐࣮໬࡟ࡼࡾ㸪」ᩘࡢ⤖ྜ㒊఩ࢆᥦ♧ࡍࡿࡇ࡜࡟ࡼࡾぢ࠿ࡅࡢぶ࿴ᛶࢆྥୖࡉࡏ࡚࠸ࡿ㸬ࣞࢡࢳࣥࡢぶ࿴ᛶྥୖࡢࢫࢺࣛࢸࢪ࣮ࡣᕦጁ࡛࠶ࡾ㸪ಶࠎࡢࣞࢡ
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ࡵࡓ(5)㸬
ᡃࠎࡣࡇࢀࡲ࡛୺࡟ C ᆺࣞࢡࢳࣥཷᐜయࢆ୰ᚰ࡟ᵓ㐀⏕≀Ꮫ◊✲ࢆ⾜ࡗ࡚ࡁࡓ㸬≉࡟ C ᆺࣞࢡࢳࣥཷᐜయࡢඹ㏻ࡢࣇ࢛࣮ࣝࢻ࠿ࡽ⢾㙐⤖ྜࡢከᵝᛶࡣ࡝ࡢࡼ
࠺࡟⏕ࡳฟࡉࢀࡿ࠿࡜࠸࠺ၥ㢟ࢆタᐃࡋ࡚ᵓ㐀ゎᯒࢆ⾜ࡗ࡚ࡁࡓ㸬ᵓ㐀ゎᯒࢆ㏻ࡌ࡚ᚓࡓ▱ぢ࠾ࡼࡧ௚ࢢ࣮ࣝࣉࡢሗ࿌ࡶᘬ⏝ࡋ࡞ࡀࡽ㸪C ᆺࣞࢡࢳࣥཷᐜయࡢ⢾㙐ㄆ㆑࡟ࡘ࠸࡚ࡢ⌧≧ࡢ⌮ゎࢆ⥲ㄝ࡜ࡋ࡚ࡲ࡜ࡵࡓ(6)㸬
C ᆺࣞࢡࢳࣥཷᐜయࡣ⢾㙐⤖ྜ࡟ Ca2+࢖࢜ࣥࡀᚲ㡲࡛࠶ࡿࡀ㸪ඹ㏻ࡢࣇ࢛࣮ࣝ
ࢻࢆᣢࡘ࡟ࡶᣊࢃࡽࡎ㸪Ca2+࢖࢜ࣥࢆせồࡋ࡞࠸ࣞࢡࢳࣥࡶᏑᅾࡍࡿ㸬C ᆺࣞࢡࢳࣥᵝཷᐜయ࡜࿧ࡤࢀ㸪Dectin-1 ࡶࡑࡢࣇ࢓࣑࣮ࣜࡢ୍ဨ࡛࠶ࡿ㸬Dectin-1 ࡣ ǃࢢࣝ࢝ࣥࡢཷᐜయ࡜ࡋ࡚⮬↛ච␿ࢩࢫࢸ࣒࡜ࡋ࡚ࡢᙺ๭ࢆᯝࡓࡋ࡚࠸ࡿ㸬ᅜ㝿ඹྠ◊✲࡟ࡼࡾ㸪Dectin-1 ࡣ ǃ ࢢࣝ࢝ࣥ࡜ࡣ␗࡞ࡿ✀㢮ࡢࣜ࢞ࣥࢻ࡜ࡶ⤖ྜࡋᚓࡿࡇ࡜ࢆ♧ࡋࡓ(7)㸬
III㸬 NMR ἲ࡟ࡼࡿ⢾㙐ࡢᵓ㐀ゎᯒᢏ⾡ࡢᵓ⠏
NMR ἲࡣ㸪⢾㙐ࡢ໬Ꮫᵓ㐀Ỵᐃ㸪≉࡟࢔ࣀ࣐࣮ࡢỴᐃ࣭⢾⤖ྜ㒊఩ࡢྠᐃ࡟࠾
࠸࡚ጾຊࢆⓎ᥹ࡍࡿ㸬ࡇࢀࡣ௚ࡢ᪉ἲ࡛ࡣྠᐃࡀⴭࡋࡃᅔ㞴࡛࠶ࡾࠊNMR ἲ࡟ࡼࡿ㎿㏿࠿ࡘ☜ᐇ࡞ゎᯒࡀ㔜せ࠿ࡘࣘࢽ࣮ࢡ࡞఩⨨ࢆ༨ࡵࡿ㸬ᮏᖺᗘࡣ㸪⢾⬡㉁⢾㙐ࡸࢢࣜࢣ࣮ࢩࣙࣥ⏘≀ࡢᵓ㐀ゎᯒ⤖ᯝࢆ௨ୗሗ࿌ࡍࡿࡇ࡜ࡀ࡛ࡁࡓ㸬࣏ࣜከ⢾㸦LPS㸧ࡣ㸪Toll ᵝཷᐜయ㸦TLR㸧4-MD-2 」ྜయࢆ௓ࡋ࡚ᐟ୺ࡢ⮬↛ච␿⣔ࢆ่⃭ࡍࡿࢢ࣒ࣛ㝜ᛶ⣽⳦⣽⬊⾲㠃ࡢᡂศ࡛࠶ࡿ㸬ࣟࢻࣂࢡࢱ࣮ᒓࡢ LPS
ࡣ࢚ࣥࢻࢺ࢟ࢩࣥάᛶࢆᣢࡓࡎ㸪㏫࡟࢔ࣥ࢔ࢦࢽࢫࢺ࡜ࡋ࡚ᶵ⬟ࡍࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿ㸬ᮏ◊✲࡛ࡣ IL-8 ࡢ⏘⏕ࢆಁࡋ㸪㏫࡟ IL-6 ࡢ⏘⏕ࢆᢚไࡍࡿാࡁࢆࡶ
ࡘ Rhodobacter azotoformans ࡢ࣏ࣜ࢜ࣜࢦ⢾㸦LOS㸧⏬ศ࡟ὀ┠ࡋ࡚ᵓ㐀ゎᯒࢆ⾜ࡗࡓ㸬 ヲ⣽࡞ NMR ࠾ࡼࡧ㉁㔞ศᯒࡢඹྠ◊✲ࡢ⤖ᯝ㸪㏻ᖖぢฟࡉࢀࡿ࣊ࣉ
ࢺ࣮ࢫࢆྵࡲࡎ㸪ࢢࣝࢡࣟࣥ㓟ࢆྵࡴ▷࠸⢾㙐࠿ࡽ࡞ࡿ᪂つ LOS ࡢᵓ㐀ࢆỴᐃࡋࡓ(8)㸬

Campylobacter jejuni ࡢ࣏ࣜ࢜ࣜࢦ⢾ࡣ㸪့ங㢮࢞ࣥࢢࣜ࢜ࢩࢻ GM1 ࡜ࡢᵓ

103㐀㢮ఝᛶ࠿ࡽࢠࣛࣥࣂ࣮ࣞ⑕ೃ⩌ࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡀᥦၐࡉࢀ࡚࠸ࡿ㸬ᮏㄽᩥ࡛ࡣ㸪Campylobacter jejuni ࡢ࣏ࣜ࢜ࣜࢦ⢾㙐ࡢࢥ࢔㒊ศࡢ໬Ꮫྜᡂ࡟ᡂຌࡋࡓ㸬ྛ✀ከḟඖ NMR ἲࢆᐇ᪋ࡋ㸪࢜ࣜࢦ⢾㙐୰ࡢࢢࣜࢥࢩࢻ⤖ྜࡢỴᐃࢆ⾜࠸㸪ඹྠ◊✲࡜ࡋ࡚ᮏㄽᩥ࡟㈉⊩ࡋࡓ㸬≉࡟ᵓ㐀Ỵᐃࡢ㝿࡟၏୍Ꮡᅾࡍࡿ OH ᇶࣉࣟࢺࣥࢆྠᐃࡋ࡚ᖐᒓࡢᣐⅬ࡜ࡍࡿࡇ࡜ࢆᕤኵࡋࡓ(9)㸬
ࣇࣝࢡࢺ࣮ࢫࡣࢱࣥࣃࢡ㉁ࡢࣜࢪࣥഃ㙐࡜㠀㓝⣲ⓗ࡟཯ᛂࡋ࡚ AGE ⏘≀࡜࡞ࡿ㸬௒ᅇ㛤Ⓨࡉࢀࡓᢠయ࡜཯ᛂᛶࢆ᭷ࡍࡿ AGE ࡟ᑐࡋ࡚ NMR ゎᯒࢆ⾜ࡗࡓ⤖ᯝ㸪ࣜࢪࣥࡢഃ㙐 ǆ ఩ NH ࡀࢢࣝࢥ࣮ࢫࡢ 2 ఩࡜཯ᛂࡋࡓࢢࣝࢥ࣮ࢫࣜࢪ࡛ࣥ࠶
ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋ㸪ඹྠ◊✲࡜ࡋ࡚ᮏㄽᩥ࡟㈉⊩ࡋࡓ(10)㸬
NMR ἲࡣ㸪⢾㙐ࡢ໬Ꮫᵓ㐀Ỵᐃࡢࡳ࡞ࡽࡎ㸪❧యᵓ㐀࣭㐠ືᛶ࣭┦஫స⏝࡟㛵ࡍࡿཎᏊࣞ࣋ࣝࡢ᝟ሗࢆࡶࡓࡽࡍ㸬 ⢾㙐ࡢ᪂ᶵ⬟ࢆ㛤ᣅࡍࡿࡓࡵ࡟ࡣ㠀ኳ↛ᆺ⢾㙐ࡢྜᡂࡶ㑅ᢥ⫥ࡢ୍ࡘ࡛࠶ࡿ㸬ᮏඹྠ◊✲࡛ࡣࢩ࢔ࣝ㓟ࡢࢭࣞࣀࢢࣜࢥࢩࢻࢆ
ᑐ㇟࡜ࡋࡓ㸬ྜᡂ࡟ࡼࡾࢭࣞࣥྵ᭷⢾㙐ࡣ㸪77Se-NMR ࡟ࡼࡾゎᯒྍ⬟࡜࡞ࡿ㸬ᐇ㝿㸪ᮏࢭࣞࣀ⢾㙐ࢆࢱࣥࣃࢡ㉁࡜ࡢ┦஫స⏝ゎᯒ࡟ᛂ⏝ࡋࡓ࡜ࡇࢁ㸪ࢩ࢔ࣝ㓟⤖ྜࢱࣥࣃࢡ㉁ࡢ✀㢮࡟ࡼࡗ࡚⤖ྜࣉࣟࣇ࢓࢖ࣝࡀ␗࡞ࡿࡇ࡜ࢆ 77Se-NMR ࡟ࡼࡾ᫂ࡽ࠿࡟ࡋࡓ(11)㸬⌧ᅾࡣ㸪⢾㙐࣭⢾࣌ࣉࢳࢻࡢ NMR ಙྕࡢᖐᒓࢆ㐍ࡵ࡚࠾ࡾ㸪௒ᚋࡣ⤖ྜศᏊ࡜ࡢ┦஫స⏝ゎᯒࢆ⾜࠺ணᐃ࡛࠶ࡿ㸬IV㸬 ィ⟬໬Ꮫ࣭ࢹ࣮ࢱ࣮࣋ࢫࡢᇶ┙ᵓ⠏⢾㙐ࡢ❧యᵓ㐀࣭ࢲ࢖ࢼ࣑ࢡࢫࡢゎᯒ࡟ࡣ NMR ἲ࡞࡝ࡢᐇ㦂ⓗᡭἲ࡟ຍ࠼࡚㸪ศᏊືຊᏛィ⟬࡞࡝ࡢィ⟬໬Ꮫⓗᡭἲࡀᚲせ࡟࡞ࡿ㸬ᡃࠎࡣ௨๓࡟㸪⢾㙐ࡢ‽Ᏻᐃᵓ㐀ࢆࣞࢡࢳࣥ࡟⤖ྜࡉࢀࡿࡇ࡜࡟ࡼࡾᐇ㦂ⓗ࡟ᤊ࠼ࡿࡇ࡜ࢆ⾜ࡗ࡚ࡁࡓ㸬ᮏඹྠ◊✲࡛ࡣ㸪ศᒱᆺ⢾㙐ࡢศᏊືຊᏛィ⟬ࢆ⾜࠸㸪⢾㙐ᵓ㐀ࡢ㐪࠸ࡀࢲ࢖ࢼ࣑
ࢡࢫ࡟୚࠼ࡿᙳ㡪ࢆㄪ࡭ࡓ㸬⢾㙐ᵓ㐀ࡢ㐪࠸࡟ࡼࡾ㸪2 ᮏ㙐⢾㙐ࡢྛศᯞࡢࢲ࢖
ࢼ࣑ࢡࢫࡀ␗࡞ࡿࡇ࡜㸪ࡲࡓࡑࡢ⤖ᯝ㸪⢾㌿⛣㓝⣲ࡢᇶ㉁࡬ࡢ࡞ࡾࡸࡍࡉ࡟ᙳ㡪ࡍࡿࡇ࡜ࢆᥦ♧ࡍࡿࡇ࡜ࡀ࡛ࡁࡓ(12)㸬⌧ᅾࡣ㠀඾ᆺ⢾࡛࠶ࡿࣜࣅࢺ࣮ࣝࡢᵓ㐀≉ᛶ࡟ࡘ࠸࡚㸪ィ⟬࡜ᐇ㦂ࡢ୧㠃࠿ࡽヲࡋࡃㄪᰝࡋ࡚࠸ࡿ㸬 ࡲࡓࢹ࣮ࢱ࣮࣋ࢫ࠿ࡽ㸪⢾㙐❧యᵓ㐀࣭┦஫స⏝ࡢἲ๎ᛶࢆぢฟࡍࡇ࡜ࢆ㐍ࡵ࡚࠸ࡿ(13)㸬⌧ᅾࡣᢠ⢾㙐ᢠయࡢ❧యᵓ㐀ࢹ࣮ࢱࢆ㞟✚ࡍࡿࡓࡵࡢ᪉ἲࢆ᳨ウࡋ࡚࠾ࡾ㸪ࢹ࣮ࢱࡢศ㢮ࢆ㐍ࡵ࡚࠸ࡿẁ㝵࡛࠶ࡿ㸬ྜࢃࡏ࡚ᢠయࡢ㉸ྍኚ㡿ᇦࢆྵࡴ Fv 㡿ᇦࡢࣔࢹࣜࣥࢢᡭἲࡢ᳨ウࢆ⾜ࡗ࡚࠸ࡿࠋ
V㸬 ࡑࡢ௚ ⢾㙐ᵓ㐀ࡢไᚚ࣓࢝ࢽࢬ࣒ࡸ⢾㙐ࡢ㛵ࢃࡿ㑇ఏᛶ⑌ᝈࡢཎᅉࢆ᫂ࡽ࠿࡟ࡍࡿࡓ
ࡵ࡟ࡣࠊ⢾㙐ᵓ㐀ࢆㄪ࡭ࡿ࡜࡜ࡶ࡟ࠊ⢾㌿⛣㓝⣲࡞࡝⢾㙐⏕ྜᡂ࡟㛵ࢃࡿ୍㐃ࡢ
⺮ⓑ㉁ࡢ❧యᵓ㐀᝟ሗࡀᚲ㡲࡛࠶ࡿࠋຍ࠼࡚࢔࣑ࣀ㓟⨨᥮ࡀࡑࡢάᛶࡸ❧యᵓ㐀࡟ཬࡰࡍᙳ㡪ࢆண࣭ㄪᰝࡍࡿࡇ࡜ࡶ㔜せ࡟࡞ࡿࠋ⌧ᅾࠊඹྠ◊✲࡟ࡼࡾ⢾㙐㛵

104㐃㑇ఏᏊࡢኚ␗ࡀࡑࡢࢱࣥࣃࢡ㉁ࡢάᛶࡸᏳᐃᛶ࡟ཬࡰࡍᙳ㡪ࢆ⣔⤫ⓗ࡟ㄪ࡭ࡿࡓࡵࡢ᪉ἲࢆ᳨ウ୰࡛࠶ࡿࠋᮏᖺᗘࡣࠊ⢾㙐ࡢᵓ㐀ゎᯒᢏ⾡࡜ࡑࡢ࿘㎶࡛㛵㐃ࡍࡿᢏ⾡࡟ࡘ࠸࡚ᑗ᮶ࡢᒎᮃ࡜࡜ࡶ࡟ࡲ࡜ࡵ࡚ሗ࿌ࢆࡋࡓ(14-17)㸬

㸺Ⓨ⾲ㄽᩥ㸼
1. Yamaguchi, Y., and Barb, A. W. (2020) A synopsis of recent developments defining
how N-glycosylation impacts immunoglobulin G structure and function. Glycobiology,
30, 214-225
2. Manabe, S., Yamaguchi, Y., Matsumoto, K., Fuchigami, H., Kawase, T., Hirose, K.,
Mitani, A., Sumiyoshi, W., Kinoshita, T., Abe, J., Yasunaga, M., Matsumura, Y., andIto, Y. (2019) Characterization of antibody products obtained through enzymatic andnonenzymatic glycosylation reactions with a glycan oxazoline and preparation of ahomogeneous antibody-drug conjugate via Fc N-glycan. Bioconjug. Chem. 30, 13431355
3. ┾㘠ྐ஀㸪ᒣཱྀⰾᶞ. (2019) ADC 㛤Ⓨ࡟࠾ࡅࡿ࣮ࣜࣥ࢝タィࡢព⩏. ࡀࢇศᏊᶆⓗ἞⒪ 17, 11-154.

┾㘠ྐ஀㸪ᒣཱྀⰾᶞ. (2019) ⢾㙐ᨵኚ࡟ࡼࡿᢠయʊ⸆≀」ྜయྜᡂ. ᭶หࣇ࢓࢖ࣥࢣ࣑࢝ࣝ 48, 51-59

5.
6.

ᒣཱྀⰾᶞ. (2019) ࣞࢡࢳࣥࡢ⢾㙐ㄆ㆑࡜ࡣ. ་Ꮫࡢ࠶ࡺࡳ 269, 750-755
Nagae, M., and Yamaguchi, Y. (2019) Structural aspects of carbohydrate recognition
mechanisms of C-type lectins. Curr. Top. Microbiol. Immunol., 1-30
7. Manabe, Y., Marchetti, R., Takakura, Y., Nagasaki, M., Nihei, W., Takebe, T., Tanaka,K., Kabayama, K., Chiodo, F., Hanashima, S., Kamada, Y., Miyoshi, E., Dulal, H. P.,
Yamaguchi, Y., Adachi, Y., Ohno, N., Tanaka, H., Silipo, A., Fukase, K., and Molinaro,
A. (2019) The core fucose on an IgG antibody is an endogenous ligand of Dectin-1.
Angew. Chem. Int. Ed. 58, 18697-18702
8. Kanie, Y., Yamaguchi, Y., Hayashi, A., Uzawa, J., Hatakeyama, M., Hidaka, Y., Toda,
N., Nakamura, S., and Kanie, O. (2019) Structural analysis of a novel
lipooligosaccharide (LOS) from Rhodobacter azotoformans. Carbohydr. Res. 473,
104-114
9. Yoshida, F., Yoshinaka, H., Tanaka, H., Hanashima, S., Yamaguchi, Y., Ishihara, M.,
Saburomaru, M., Kato, Y., Saito, R., Ando, H., Kiso, M., Imamura, A., and Ishida, H.
(2019) Synthesis of the core oligosaccharides of lipooligosaccharides from
Campylobacter jejuni: A putative cause of Guillain-Barre syndrome. Chem. Eur. J. 25,
796-805
10. Ohno, R., Ichimaru, K., Tanaka, S., Sugawa, H., Katsuta, N., Sakake, S., Tominaga, Y.,
Ban, I., Shirakawa, J., Yamaguchi, Y., Ito, E., Taniguchi, N., and Nagai, R. (2019)
Glucoselysine is derived from fructose and accumulates in the eye lens of diabetic rats.
J. Biol. Chem. 294, 17326-17338
11. Suzuki, T., Hayashi, C., Komura, N., Tamai, R., Uzawa, J., Ogawa, J., Tanaka, H. N.,

105

Imamura, A., Ishida, H., Kiso, M., Yamaguchi, Y., and Ando, H. (2019) Synthesis andglycan-protein interaction studies of Se-sialosides by 77Se NMR. Org. Lett. 21, 63936396
12. Nakano, M., Mishra, S. K., Tokoro, Y., Sato, K., Nakajima, K., Yamaguchi, Y.,
Taniguchi, N., and Kizuka, Y. (2019) Bisecting GlcNAc is a general suppressor of
terminal modification of N-glycan. Mol. Cell. Proteomics 18, 2044-2057
13. Yamaguchi, Y. (2019) A struggle of the last eleven years after getting JSCR award.
Trends Glycosci. Glycotechnol. 31, SE50-SE52, SJ50-SJ52
14. Yamaguchi, Y. (2019) Ion mobility-mass spectrometry (IM-MS). Glycoscience: Basic
Science to Application, pp.6-7
15. Yamaguchi, Y. (2019) Conformational analysis of glycans. Glycoscience: Basic
Science to Application, pp.40-41
16. Yamaguchi, Y. (2019) Computational science (supercomputer) and AI (artificial
intelligence). Glycoscience: Basic Science to Application, pp.42-43
17. Yamaguchi, Y. (2019) Core technologies for elucidating the role of glycans in disease.
Glycoscience: Basic Science to Application, pp.265-266
㸺Ꮫ఍Ⓨ⾲㸼
࣭Asp-Asp ࣔࢳ࣮ࣇ࡟࠾ࡅࡿ㠀㓝⣲཯ᛂ኱㔝 リṌ
➨ 7 ᅇ≀⌮࣭ศᯒ⣔ⱝᡭ◊✲⪅ࢭ࣑ࢼ࣮㸪௝ྎ㸪2019 ᖺ 2 ᭶࣭ࣜࣥ㓟஧Ỉ⣲࢖࢜ࣥࢆゐ፹࡜ࡍࡿ Asp-Ser 㓄ิ࠿ࡽࡢࢫࢡࢩࣥ࢖࣑ࢻ⏕ᡂ኱㔝 リṌ㸪┿㘠 ἲ⩏㸪㧗ᶫ ኸᐅ᪥ᮏ⸆Ꮫ఍➨ 139 ᖺ఍㸪༓ⴥ㸪2019 ᖺ 3 ᭶㸪₇㢟␒ྕ p.143࣭ⓑෆ㞀ࡢ⑓ែᙧᡂ࡟㛵୚ࡍࡿࢡࣜࢫࢱࣜࣥ୰ Asp ṧᇶ␗ᛶ໬ࡢங㓟࡟ࡼࡿゐ፹ᶵᵓ㹼ィ⟬໬Ꮫⓗ࣭⏕໬Ꮫⓗ᳨ウ㹼┿㘠 ἲ⩏㸪బࠎᮌ 㞞ே㸪⏣୰ ኱㸪኱㔝 ㈼୍㸪኱㔝 リṌ㸪᱒㉺ ுኴ㸪ຍ⸨๰㸪⸨ᮧ ົ㸪ᰘ⏣ ಙஅ㸪㧗ᶫ ኸᐅ ᪥ᮏ⸆Ꮫ఍➨ 139 ᖺ఍㸪༓ⴥ㸪2019 ᖺ 3 ᭶㸪せ᪨㞟 p.161
࣭ Solution NMR and docking simulation analysis of interaction between tau
aggregates and low-molecular-weight compounds
Yoshiki YamaguchiInternational Academic Forum on Neuroinflammation and Psychopharmacology,
Nantong, China, May 2019, ₇㢟␒ྕ 3࣭㘄๣ほᐹ࡟࠾ࡅࡿ㉮ᰝ㟁Ꮚ㢧ᚤ㙾⏝ࡢᗈ㡿ᇦ᩿㠃స〇ἲ᠕ᮏ ⋢ᜨ a㸪ῦ㔝 ዉὠᏊ a㸪ᮅẚዉ ಇ㍜ a㸪┿㘠 ἲ⩏㸦᪥ᮏ㟁Ꮚᰴᘧ఍♫ a㸧᪥ᮏ⸆๣Ꮫ఍➨ 34 ᖺ఍㸪ᐩᒣ㸪2019 ᖺ 5 ᭶㸪せ᪨㞟 p.42
࣭㘄๣ᡂศࡢศᕸศᯒ࡟⏝࠸ࡿ᩿㠃ヨᩱࡢస〇࡜ࡑࡢศᯒἲ

106┿㘠 ἲ⩏㸪᠕ᮏ ⋢ᜨ a㸪ῦ㔝 ዉὠᏊ a㸪㛗㇂㒊 ♸἞ a㸪ᮅẚዉ ಇ㍜ a㸦᪥ᮏ㟁Ꮚᰴᘧ఍♫ a㸧᪥ᮏ⸆๣Ꮫ఍➨ 34 ᖺ఍㸪ᐩᒣ㸪2019 ᖺ 5 ᭶㸪せ᪨㞟 p.65
࣭⢾㙐ࡢᵓ㐀ゎᯒ࡜ࡑࡢᶵ⬟ࡢゎ᫂ᒣཱྀ ⰾᶞ᪥ᮏ⮚ჾ〇⸆㸦ᰴ㸧≉ูㅮ₇఍㸪රᗜ㸪2019 ᖺ 7 ᭶㸪₇㢟␒ྕ 2࣭Ỉ⁐ᾮ୰࡟࠾ࡅࡿ⢾⬡㉁ࡢ⬺࣑ࢭࣝ໬࡜ NMR ἲ࡟ࡼࡿᛶ≧ゎᯒᒣཱྀ ⰾᶞ➨ 38 ᅇ᪥ᮏ⢾㉁Ꮫ఍ᖺ఍㸪ྡྂᒇ㸪2019 ᖺ 8 ᭶㸪₇㢟␒ྕ WS-04
࣭⢾㙐ᵓ㐀ࡢከᵝᛶ࡜ࡑࡢ⏕≀Ꮫⓗព⩏ࡢ᥈ồᒣཱྀ ⰾᶞ
➨ 13 ᅇᮾ໭⢾㙐◊✲఍㸪᪂₲㸪2019 ᖺ 9 ᭶㸪₇㢟␒ྕ S-1
࣭ࢢࣝࢥࢩࣝࢭ࣑ࣛࢻศゎ㓝⣲࡟ࡼࡿ᪂つ⬻ෆࢫࢸ࣮ࣟࣝ௦ㅰ≀࣭ࢫࢸ࣮ࣟࣝ㓄⢾యࡢ௦ㅰไᚚ⛅ᒣ ኸᏊ a, ஭ᡭ ୕ὠᏊ b, ୰す ᝋ㑻 c, ୖᮧ ⣖ோ c, ᒣཱྀ ⰾᶞ, Peter Greimeld, ୖཱྀ ⿱அ a, 㧗ᶫ Ⰻ㍜ c, ᖹᯘ ⩏㞝 b(⌮໬Ꮫ◊✲ᡤ ⬻⚄⤒⛉Ꮫ◊✲ࢭࣥࢱ࣮ ⚄⤒⣽⬊ືែ◊✲ࢳ࣮࣒ a, .⌮໬Ꮫ◊✲ᡤ బ⏥⣽⬊᝟ሗ◊✲ᐊ b, ி㒔኱Ꮫ኱Ꮫ㝔 ་Ꮫ◊✲⛉ ⮫ᗋ⚄⤒Ꮫ c, ⌮໬Ꮫ◊✲ᡤ ⬻⚄⤒⛉Ꮫ◊✲ࢭࣥࢱ࣮ ⣽⬊ᶵ⬟᥈⣴ᢏ⾡◊✲ࢳ࣮࣒ d)➨ 92 ᅇ᪥ᮏ⏕໬Ꮫ኱఍㸪᪂₲㸪2019 ᖺ 9 ᭶㸪₇㢟␒ྕ 1P-011 (1T10a-06)
࣭ Solution NMR and docking simulation analysis of interaction between tau
aggregates and small chemicals
Yoshiki Yamaguchi
Asian Pacific Prion Symposium2019 (APPS2019), Wako, October 2019, ₇㢟␒ྕ O3
࣭⢾㙐ࣉ࣮ࣟࣈࢆά⏝ࡋࡓᵓ㐀ゎᯒᒣཱྀ ⰾᶞ
Pioneering Project: Chemical Probe㸦⏕࿨⌧㇟᥈⣴ศᏊ㸧➨஧ᅇྜྠྜᐟࢭ࣑ࢼ࣮㸪ẚ௻㸪2019 ᖺ 10 ᭶㸪₇㢟␒ྕ OP-3࣭ィ⟬໬Ꮫ࡟ࡼࡿࣜࣅࢺ࣮ࣝࡢࢥࣥࣇ࢛࣓࣮ࢩࣙࣥ✵㛫ࡢゎᯒ኱㔝 リṌ㸪┿㘠 ἲ⩏㸪ᒣཱྀ ⰾᶞ
➨ 58 ᅇ᪥ᮏ⸆Ꮫ఍ᮾ໭ᨭ㒊኱఍㸪௝ྎ㸪2019 ᖺ 10 ᭶㸪₇㢟␒ྕ PE-08࣭⢾㙐ࡢ㧗ḟᵓ㐀࡜┦஫స⏝ཎ⌮ࡢゎ᫂࡟ྥࡅ࡚
ᒣཱྀ ⰾᶞ
➨ 17 ᅇ⢾㙐⛉Ꮫࢥࣥࢯ࣮ࢩ࢔࣒ࢩ࣏ࣥࢪ࣒࢘㸪ி㒔㸪2019 ᖺ 10 ᭶㸪せ᪨㞟
p.14
࣭᏶඲㔜Ỉ⣲໬ࢩࢡࣟࢹ࢟ࢫࢺࣜࣥࡢ໬Ꮫ㓝⣲ྜᡂ࡜⢾⬡㉁ࡢ NMR ゎᯒ

107ᒣཱྀ ⰾᶞ a, ┾㘠 ྐ஀ a, Peter Greimel a, ఀ⸨ ᖾᡂ a㸦⌮໬Ꮫ◊✲ᡤ a㸧
➨ 58 ᅇ NMR ウㄽ఍࣭SEST2019 ྜྠࢩ࣏ࣥࢪ࣒࢘㸪ᕝᓮ㸪2019 ᖺ 11 ᭶㸪₇㢟
␒ྕ P41࣭ṧవ཮ᴟᏊ࢝ࢵࣉࣜࣥࢢ

ᐃ࡟࠾ࡅࡿ㓄ྥຊࠊᗘᏳᐃᛶࠊ᭷ᶵ⁐፹⪏ᛶࢆවࡡഛ࠼ࡓ᪂つ㓄ྥ๣ෆ⏣ ⣖அ a, ᒣཱྀ ⰾᶞ, ᾏ⪁ྡ ಖ⏨ b, బࠎᮌ 㧗⩏ b, ▼⏣ ᗣ༤ a㸦⌮໬Ꮫ◊✲ᡤ a, ≀㉁࣭ᮦᩱ◊✲ᶵᵓ b㸧
➨ 58 ᅇ NMR ウㄽ఍࣭SEST2019 ྜྠࢩ࣏ࣥࢪ࣒࢘㸪ᕝᓮ㸪2019 ᖺ 11 ᭶㸪₇㢟␒ྕ L3-3
࣭NMR structural analysis of glycolipid in aqueous solution
Yoshiki Yamaguchi a, Shino Manabe a, Peter Greimel a, Yukishige Ito a
㸦RIKEN a㸧
11th ACGG Conference, Busan, Korea, November 2019, ₇㢟␒ྕ SS 8-3࣭⢾㙐ᵓ㐀ゎᯒࡢᇶ♏࡜ࡑࡢᐇ㝿ᒣཱྀ ⰾᶞ
➨ 410 ᅇ CBI Ꮫ఍ㅮ₇఍㸪⏣⏫㸪2019 ᖺ 11 ᭶㸪₇㢟␒ྕ 2
࣭An NMR approach to investigate glycolipid structure
Yoshiki Yamaguchi
TMPU-AS Joint Glycoscience Symposium, Taiwan

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Glycobiology, 2020, vol. 30, no. 4, 214–225
doi: 10.1093/glycob/cwz068
Advance Access Publication Date: 10 September 2019
Review

Review

A synopsis of recent developments deﬁning
how N-glycosylation impacts immunoglobulin G
structure and function
Yoshiki Yamaguchi

2 and Adam W Barb

3,1

2 Tohoku Medical and Pharmaceutical University, Sendai, Japan, 981-8558 and 3 Department of Biochemistry andMolecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, 30602
1 To whom correspondence should be addressed: Tel: 706-542-1773; Fax: 706-542-1718; e-mail: abarb@uga.edu

Received 22 April 2019; Revised 26 July 2019; Editorial Decision 21 August 2019; Accepted 21 August 2019

Abstract
Therapeutic monoclonal antibodies (mAbs) are the fastest growing group of drugs with 11 new
antibodies or antibody-drug conjugates approved by the Food and Drug Administration in 2018.
Many mAbs require effector function for efﬁcacy, including antibody-dependent cell-mediated cytotoxicity triggered following contact of an immunoglobulin G (IgG)-coated particle with activating
crystallizable fragment (Fc) γ receptors (Fcγ Rs) expressed by leukocytes. Interactions between
IgG1 and the Fcγ Rs require post-translational modiﬁcation of the Fc with an asparagine-linked
carbohydrate (N-glycan). Though the structure of IgG1 Fc and the role of Fc N-glycan composition
on disease were known for decades, the underlying mechanism of how the N-glycan affected Fcγ Rbinding was not deﬁned until recently. This review will describe the current understanding of how
N-glycosylation impacts the structure and function of the IgG1 Fc and describe new techniques thatare poised to provide the next critical breakthroughs.
Key words: antibody, Fc gamma receptor, immunoglobulin G, N-glycosylation

Introduction

Complex molecules have a long history in human health thatbegins with undefined mixtures used as vaccines and arriving at
a seminal moment in 1922 with the administration of a bovinepancreatic extract to successfully treat a young male diabetic (Bliss
2007). Each basic discovery, from defining insulin as a protein tosequencing the polypeptide chains to identifying residues that promote stability, has led to the improvement of human health throughenhancing insulin formulations, production and from protein engineering (Brange et al. 1991). The development of modern insulin
variants informs the future development of IgG analogs as therapeutics. MAbs are predominantly built on an IgG scaffold thoughatomic-level details governing critical interactions with the immune
system remain undefined. Based on the recent development of mAbs
and protein therapeutics in general, it is clear that basic descriptionsof IgG structure/function relationships will drive development of thenext generation of drugs.

Monoclonal antibodies (mAbs) are powerful drugs for cancers,
autoimmune disorders and infection in addition to many otherdiseases. Positive drug attributes including high specificity andtolerance promote the development of mAbs for a wide variety oftargets. Antibodies are complex molecules with multiple polypeptide
chains and post-translational modifications; human immunoglobulin
G1 (IgG1) is a 150 kDa heterotetramer with two conserved Asnlinked carbohydrate chains (N-glycans) and 16 disulfide bonds(Figure 1) (Liu and May 2012). Furthermore, proper 3D structureis critical for mAb function and an important quality attributeconcerning commercial mAb production (Berkowitz et al. 2012).
Thus, the development, evaluation, validation and quality control ofmAbs as drugs differs substantially from small molecule therapeutics
that previously represented the pharmaceutical industry’s primary
products.

© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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215

Figure 1. The IgG1 Fc N-glycan is essential for binding to the Fc γ receptors. (A) IgG1 Fc is a heterotetramer consisting of two Fabs and a single Fc that binds
receptors. (B) The Fc N-glycan forms an interface with Cγ 2 residues through noncovalent intramolecular interactions. The volume of interacting residues isshown with a gray background, and individual N-glycan and protein residues are highlighted. Blue squares represent N-acetylglucosamine residues; green
circles, mannose; and yellow circles, galactose.

correlation between the degree of galactose modification on IgG1
and rheumatoid arthritis provided the first indication that Fc Nglycosylation may impact antibody structure and function (Parekh
et al. 1985). More recently, strong connections between the additionof a core fucose residue to autoimmune disorders among many
IgG studies in serum and in vitro demonstrated that IgG N-glycan
composition is a crucial factor in human health and disease (Figure 2,
residue “0”) (Shields et al. 2002; Lauc et al. 2013; Chung et al. 2014;
Kapur et al. 2014; Mahan et al. 2016; Sonneveld et al. 2017; Clercet al. 2018; Doherty et al. 2018).
Surprisingly, the first atomic-resolution structure showed nodirect contact between the Fc N-glycans and the Fcγ R (Sondermann
et al. 2000). Later x-ray crystallography studies revealed small
differences in IgG Fc quaternary structure, and solution nuclear
magnetic resonance (NMR) spectroscopy showed subtle changes
in Fc structure and Fcγ R binding that correlated with N-glycan
composition (Krapp 2003; Yamaguchi et al. 2006). These results did
not reveal how N-glycan composition impacted receptor affinity, afact complicated by the observation that the Fc N-glycans appeared
immobilized through contacts with the polypeptide (Huber et al.
1976). However, this interpretation was challenged by NMR data
showing that polypeptide contacts only partially restricted motionof the N-glycan under more physiological conditions and led to the
hypothesis that the N-glycan modulated receptor binding through anindirect mechanism (Barb and Prestegard 2011).
Here we will review the most recent developments toward understanding how N-glycan composition impacts IgG1 Fc structure andreceptor binding. An article that thoroughly covers this entire history
would prove too expansive for this format. The understanding of IgG
Fc structure and function has changed dramatically in recent years,
and recent studies will form the focus of this review.

Two heavy polypeptide chains and two light chains form an IgG
heterotetramer (Figure 1). IgG binds to a target through specifically
optimized loops contained in the antigen binding fragments (Fabs).
The C-terminal halves of the heavy chains form the crystallizable
fragment (Fc) that binds various surface-borne and soluble receptors. IgG Fc contains an N-glycan at Asn297 that is required forproper interactions with Fc γ receptors (Fcγ Rs) and the therapeutic
efficacy of mAbs that elicit antibody-dependent cell-mediated cytotoxicity (Nose and Wigzell 1983; Guillerey et al. 2016). Humans
express as many as five activating Fcγ Rs including the high-affinity
Fcγ RI (CD64) and the low-affinity activating receptors Fcγ RIIa,Fcγ RIIc (expressed by ∼ 20% of individuals (Ernst et al. 2002;
Breunis et al. 2008)), Fcγ RIIIa and Fcγ RIIIb (CD32a, CD32c, CD16a
and CD16b, respectively). Humans also express a single inhibitory
receptor Fcγ RIIb (CD32b). IgG also binds the C1q component ofcomplement to elicit complement-dependent cytotoxicity. Though
C1q binding requires oligomerized IgG and binds to a different Fc
surface than the Fcγ Rs (Ugurlar et al. 2018), there are reports thatN-glycan composition affects efficacy (Peschke et al. 2017). The
neonatal Fc receptor and TRIM21 also bind IgG Fc and binding
may be influenced by N-glycan composition (James et al. 2007;
Jennewein et al. 2019). A few groups provided data that indicated DC-SIGN and CD23 bind sialylated IgG (reviewed in Pincetic
et al. 2014), though recent results cast these conclusions into doubt(Temming et al. 2019).
N-glycan composition impacts mAb recognition, and the composition of serum IgG shows strong correlations to disease. The
predominant structure found on serum IgG1 is a complex-type, corefucosylated, biantennary N-glycan (Figure 2). Modifications at the
non-reducing termini can add one or two galactose residues thateach may be modified with one N-acetylneuraminic acid residue. A

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Y Yamaguchi and A Barb

Figure 2. The different Fc N-glycan compositions provide Fc with variable afﬁnity for CD16a. (A) N-glycan compositions as discussed in the text; note theabbreviated identiﬁer to the left of the cartoon ﬁgures. (B) IgG1 Fc with each different N-glycan binds with different afﬁnity to CD16a. These K D values wereadapted directly from (Subedi and Barb 2016) but are consistent with other reports (Yamaguchi et al. 2006; Thomann et al. 2015; Dekkers et al. 2017). GlcNAc,
N-acetylglucosamine; Neu5Ac, N-acetylneuraminic acid.

The structure and function of IgG1 Fc is mediated byinteractions between the Fc polypeptide and N-glycan
residues

any other residue in the glycan (Hanson et al. 2009). The structural
relationship of this unique glycoform and the IgG1 Fc structure will
be discussed below.

IgG1 Fc N-glycan composition impacts Fcγ R binding. Systematicinvestigations into the role of Fc N-glycan composition on Fcγ Rbinding emerged in the past few years. Though it was known that
Fcγ R binding was sensitive to glycosylation, these recent efforts
probed interactions for the entire set of human Fcγ Rs with highsensitivity and a large number of Fc glycoforms. One common
feature of these studies is the unique sensitivity of CD16 affinity
to Fc N-glycan composition (Thomann et al. 2015; Subedi and Barb
2016; Dekkers et al. 2017). Though CD32 likewise requires Fc Nglycosylation for binding, CD16a and CD16b show a much greater
sensitivity to IgG1 Fc N-glycan composition with longer N-glycans
promoting tighter interactions (Figure 2B).
The most dramatic affinity differences are due to the additionof a fucose residue to the N-linked GlcNAc residue that reduces
affinity for CD16a in vitro (Shields et al. 2002; Shinkawa et al. 2003).
Two studies recapitulated the negative impact of core fucosylation
on CD16 affinity with a large number of Fc glycoforms, though
one study using isolated Fc showed a smaller 4 to 8-fold reductionof binding (Subedi and Barb 2016) compared to another using fulllength antibodies with a 40-fold reduction (Dekkers et al. 2017).
These differences may reflect additional sensitivity of the full-length
antibody to Fc N-glycan fucosylation. These studies also showeda moderate benefit of galactosylation (∼2-fold) and no consistentmeasurable impact resulting from sialylation. One study reported
only a minimal effect of bisecting N-acetylglucosamine (linked β1–4
to the (3) mannose residue) (Dekkers et al. 2017).
Human IgG1 Fc displaying only a single N-acetylglucosamine
residue attached to N297 is produced in situ by the digestion of
IgG1 containing an afucosylated N-glycan by EndoS ((1)GlcNAc-Fc;
Figure 2B) (Collin and Olsen 2001). Surprisingly, this Fc glycoform
binds to CD16a, though with reduced affinity compared to Fc witha larger N-glycan (Subedi and Barb 2015; Okbazghi et al. 2016).
Furthermore, human IgG trimmed to the single GlcNAc residue iscapable of clearing B cells in a mouse xenograft model (Kao, et al.
2015). These results may be explained by the identification of thefirst N-glycan residue as contributing a greater degree of stability thanInteractions at the Fc N-glycan/polypeptide interface stabilize N-glycan
motion. Following the definition of how Fc N-glycan composition

affects receptor binding and the identification of N-glycan motion,
multiple studies investigated N-glycan motion further to probe therelationship between N-glycan composition, motion and receptor
binding affinity. An all-atom solvated molecular dynamics study
demonstrated that the galactose residue attached to the (α1–6mannose) branch of a complex-type N-glycan on Fc exhibitedmotion (Frank et al. 2014). These simulations were initializedwith the starting coordinates observed by x-ray crystallography
and showed the detachment of this glycan branch from theprotein surface, further supporting the earlier observation by NMR
and revealing how the glycan might deform to accommodate
conformational exchange (Barb and Prestegard 2011).
Studies on the motion of the Fc N-glycan with multiple glycoforms in solution show that composition impacts glycan interactions.
Measurements of the galactose residues before and after sialylation
using NMR spectroscopy showed that the modification slightly
stabilized the (α1–6mannose) branch (Barb et al. 2012). A later study
investigated the motion of the branch N-acetylglucosamine residues
during N-glycan maturation starting with a hybrid Mannose5 + Nacetylglucosamine form and ending with a core-fucosylated, complex
type N-glycan with N-acetylglucosamine termini (Barb 2015). These
results indicated that each remodeling step increased the contact
between polypeptide and N-glycan residues during the remodelingof an oligomannose precursor to a final disialylated complex-typeN-glycan and are consistent with previous observations in vitro
and in vivo (Butler et al. 2003; Bowden et al. 2012). This result
suggests that the αmannose residues on the (α1–6mannose) branchof the oligomannose N-glycan likely form unfavorable contacts withthe polypeptide and each subsequent glycosyltransferase reaction
exploited different contact surfaces.
Hydrogen-deuterium exchange mass spectrometry (HDX)
experiments supported the conclusion that extending the Fc Nglycan increases intramolecular interactions, revealing a reduction in111

N-glycosylation in IgG Fc structure and function

217

Figure 3. IgG1 Fc Cγ 2 domain structure. Residues that are within 5 Å of CD16a are highlighted in red. These contacts are draw for residues that form contacts ineither the Fc a or b chain. Strands are labeled with letters.

by greater motion than wild-type Fc. An additional experimentdemonstrated that these double Phe mutants, enzymaticallyremodeled to display G2F N-glycans, also bound CD16a with lessaffinity that the single Phe mutants that bound with less affinity
than the wild-type Fc (Subedi, Hanson, et al. 2014). Thus, weakerintramolecular interactions between N-glycan and polypeptide
residues led to greater N-glycan motion and weaker receptor binding.
A 2D NMR fingerprint of the wild-type and mutant Fcs using[15 N]-Tyr labeling indicated that Fc structure was largely preservedin the mutants though one Tyr residue at position 300, near thesite of N-glycan attachment to Asn297, showed perturbation of
the local structure. A follow-up study indicated that the chemical
environment surrounding Tyr300 is uniquely sensitive to the Nglycan composition and that the presence of an IgG1 Fc N-glycan
largely impacts structure of the C strand and C’E loop, minimallyaffecting on other areas of the protein (shown in Figure 3) (Subedi
and Barb 2015). Furthermore, the N-glycan stabilized C’E loop
motions on a μs-ms timescale.
Extensive molecular dynamics simulations show comparable
results with oligomannose-type N-glycans (Lee and Im 2017). These
glycans exchange with the Fc surface and form fewer intramolecular
contacts than complex type N-glycans particularly at the nonreducing termini. Notably, the presence of a glycan stabilized theC’E loop, leading to stronger receptor interactions.
Thus, the Fc N-glycan dampens motion of the C strand
and C’E loop. This dampening is enhanced by extending the Nglycan to utilize a greater number of intramolecular interactions.
Though CD16a does not directly contact the bulk of the Fc Nglycan, and certainly not the non-reducing termini, CD16a does
contact the C’E loop (Sondermann, P., Huber, R., et al. 2000).
These results are consistent with the HDX-MS results discussed
previously indicating the presence of greater polypeptide structural
heterogeneity in Fcs with less stabilizing N-glycan contacts (Kiyoshi
et al. 2018; More et al. 2018). One exception to the statementthat extending the N-glycan enhances contacts and CD16a binding
affinity is modification of the core N-acetylglucosamine residue
with fucose. This topic will be specifically addressed in the“IgG sialylation and fucosylation affects IgG Fc structure andfunction” section below.

deuterium uptake in the P245-T256 peptide following galactosylation (Kiyoshi et al. 2018). The galactose residue directly interacts
with residues on this peptide, potentially reducing deuterium uptake
by occluding solvent. The authors indicate these data representchanges in the Cγ 2/Cγ 3 domain orientation. A comparable HDXMS study followed a greater number of N-glycan compositions,
including aglycosylated, (1) GlcNAc, Mannose5 and a mixture of
Mannose8 -Mannose12 and identified less polypeptide flexibility
in Fcs with longer N-glycans (More et al. 2018). Interestingly, theauthors identified decreased flexibility in two key regions, the N297containing C’E loop and the region surrounding N315 and thatthese two surfaces mediate aggregation. Consistent with the CD16abinding data presented above, this study also reported a substantial
reduction in deuterium uptake for the (1)GlcNAc-Fc when comparedto the aglycosylated Fc.
Thus far, these data indicate a relationship between N-glycan
length and Fc affinity for CD16a. Furthermore, a longer N-glycan
appears to stabilize glycan motion and Fc structure. An experimentto mutate phenylalanine residues at the intramolecular interface
directly probed this relationship. Prior studies showed that mutating residues at the interface increased N-glycan processing during
expression, potentially indicating increased motion of the N-glycans
on mutated Fcs and greater exposure to glycan modifying enzymes in
the Golgi (Lund et al. 1996; Yu, Baruah, et al. 2013). The observationof Kelly and coworkers that aromatic residues form the strongest
interactions with carbohydrate residues through dispersive interactions led to the choice to mutate the Phe241 and Phe243 residues(Chen, Enck, et al. 2013). Mutated Fcs exhibited greater N-glycan
processing, with the proteins containing two Phe to Ser mutations
showing the greatest processing (Subedi, Hanson, et al. 2014). NMR
analyses of mutant Fcs, enzymatically remodeled to have nearlyhomogeneous G2F glycans, indicated N-glycans on mutant Fcs experienced increased mobility resulting from reduced intramolecular
contacts.
Interactions at the Fc N-glycan/polypeptide interface stabilize Fc
polypeptide motion and receptor binding. The IgG1 Fc Phe mutants

showed greater N-glycan motion with double mutants revealing
greater motion than single mutants and single mutants characterized

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Y Yamaguchi and A Barb

between the Cγ 2 domains for Fc with truncated N-glycans (Leeand Im 2017). It is also possible that disorder of protein loops orquaternary structure changes contributed to changes in Fc tumbling.
Relative orientation of the Cγ 2 and Cγ 3 domains were also analyzed by stable isotope-assisted solution NMR spectroscopy (Subedi
and Barb 2015). Residual dipolar couplings (RDCs) can be used
to define the relative orientation of each observable amide N-Hbond vector for 15 N-labeled samples. RDCs from selectively 15 Nlabeled Fc samples (15 N-Tyr and 15 N-Lys labeled) were measured
for both glycosylated and aglycosylated (T299A) human IgG1 Fc.
RDCs reveal little difference between the predominant quaternary
structures of glycosylated and aglycosylated Fc in solution.
SAXS and NMR analysis were usually analyzed assuming asingle averaged conformation without consideration for a distribution of multiple sampled conformations. Recent analysis using
single molecule Förster Resonance Energy transfer (FRET) is advantageous, since this technique provides a histogram of the conformations present in solution allowing conformational populations to
be detected (Kelliher et al. 2014; Piraino et al. 2015). To introduce
a probe into Fc, Ser258 of human IgG-Fc was mutated into Cys
and the acceptor and donor fluorescent dyes were introduced togive antibody-dye conjugates with homo- and hetero-combinationsof acceptor and donor dyes. A comparison of the FRET efficiency
histograms obtained for glycosylated and deglycosylated human IgG
indicates that the Fc region becomes more flexible and can assume
a wider variety of structures upon deglycosylation with PNGase
F or EndoS. This observation further supports the idea that x-ray
crystallographic data of deglycosylated Fc captures one out of many
Fc conformations in solution.
Then why does removing the Fc N-glycan reduce the affinity witheffector molecules? Aglycosylated Fc likely samples a set of multiple
conformations while glycosylated Fc experiences a relatively limited
range of mobility. The more flexible nature of aglycosylated Fc may
be unfavorable for the interaction with effector molecules such as theFcγ Rs. From a local viewpoint, the orientation of C’E loop, whichharbors the Fc N-glycosylation site at Asn297, is thought to be critical
for the binding to Fcγ Rs. Solution NMR spectroscopy can provide
information on macromolecular motion by measuring the relaxation
parameters. By comparing the relaxation rates of glycosylated (WT)and aglycosylated (T299A) human IgG1 Fc, it was found that the C’E
loop is relatively more flexible in aglycosylated Fc upon comparison
to glycosylated Fc (Subedi and Barb 2015). This is consistent with the
x-ray crystallographic data, in which human aglycosylated Fc structure displays high crystallographic temperature factors (B-factors) for
the C’E loop, relative to the core of the protein (Borrok et al. 2012).
These observations suggest that the conformation of the C’E loop is
stabilized by the presence of the N-glycan and the loop conformation
is essential for forming the Fcγ R interface.

Controversy: Does the N-glycan contribute to Fc quaternary structure?

Evidence to support the stabilization of the IgG1 Fc C strand andC’E loop through intramolecular contacts mediated by the N-glycan
appears well established. However, recent evidence both supports andcontradicts the hypothesis that the N-glycan affects Fc quaternary
structure. Recent studies in this area have focused on comparing
glycosylated Fc with Fc that contains either a shortened (1) GlcNAc
N-glycan or completely lacks modification.
It has been assumed that deglycosylation alters Fc conformation,
thereby reducing affinity for proteins that elicit effector functions.
This assumption has been examined by x-ray crystallographic analysis of deglycosylated Fc. The crystal structures of aglycosylated
human IgG1 Fc (produced in Escherichia coli) (Borrok et al. 2012)and enzymatically deglycosylated murine IgG1-Fc (PNGase F treatment) (Feige et al. 2009) showed a closed conformation of the two
Cγ 2 domains, as judged from Pro239-Pro239 Cα distances ranging
from 18.9Å to 19.6 Å. Hence, an idea was proposed that the closed
conformation prevents interaction with Fcγ Rs and the N-glycan at
Asn297 stabilizes the open conformation of Fc. Another model of Fc
with the N-glycan truncated to a single GlcNAc residue following
digestion with EndoS revealed a similarly collapsed conformation(Baruah et al. 2012); however, this glycoform binds CD16a withmoderate affinity (Subedi and Barb 2015), indicating that the quaternary structures observed by crystallography fail to clearly separate Fc
forms capable of binding receptor from inert forms.
In contrast to these observations, a crystal structure of fully
deglycosylated human IgG1 Fc (produced in E. coli) was revealedwith an open conformation with the Pro239-Pro239 Cα distance of
27.6 Å (PDB ID: 3DNK). A later study reported a crystal structure of
enzymatically deglycosylated human IgG4-Fc (PNGase F treatment)that also adopted an open conformation with the Pro239-Pro239
Cα distance of 29.1 Å (Davies, Jefferis, et al. 2014). The results areinconsistent with the earlier crystallographic results. Taken together,
it is likely that deglycosylated Fc adopts a set of conformations(including open and closed) and the observed structures in the
crystal may be highly dependent on the crystal packing environment.
Therefore, the crystal structures of deglycosylated Fc might not reflect
the predominant conformation sampled in solution. Thus, otherbiophysical techniques are required to characterize conformation in
the solution state.
One approach is small-angle x-ray scattering (SAXS), a method
to analyze the conformation of macromolecules in solution. Several
groups reported SAXS analysis of glycosylated and aglycosylated Fc.
The radius of gyration (Rg ) was obtained from the SAXS analysis,
which is defined as the root mean square distance of all atoms
from the common center of mass. Borrok et al. (2012) reported
that the glycosylated human IgG1 Fc displayed a Rg of 27.4 Å,compared to 28.3 Å for aglycosylated Fc (E. coli expression). Theysuggest that in solution the absence of a glycan promotes a more
open conformation. Yageta et al. (2019) performed a similar SAXS
analysis of glycosylated and aglycosylated Fc (E. coli expression)that is consistent with the former result with Rg values of 26.4 Å
and 28.9 Å, respectively. Owing to the availability of many Fc
crystal structures, these authors analyzed the theoretical SAXS profiles based on individual crystal structures. The data indicated thatboth glycosylated and aglycosylated Fc adopt a “semi-closed” Cγ 2
domain orientation. The conclusion is rather different from thatof Borrok et al., but they reasonably assumed that the scatteringfrom the N-glycan itself reduces the Rg of glycosylated Fc comparedwith the aglycosylated Fc, without any changes in the Cγ 2 domain
orientation. Comparable MD simulations identified smaller distances

Fc engineering targeting the C’E loop and glycan remodeling. The
ultimate goal of a structural definition is to generate sufficient data todesign Fcs with altered properties. A number of studies support therole of the C’E loop and N-glycan in receptor binding and Fc stability.
A study by Isoda et al. (2015) tested the effect of each amino acid type
substituted at position 296. This residue is a Tyr in human IgG1 Fc
and located immediately adjacent to the glycosylated Asn297 residue.
Tyr300 may form contacts with the core fucose residue (Matsumiya
et al. 2007). With the exception of a Trp substitution, all residues
reduced affinity for CD16a and CD16b. A crystal structure of the
Fc Tyr296Trp variant indicated a presence of greater contacts thatslightly increase affinity for CD16a.

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glycosylation sites were similarly mutated to abolish N-glycosylation.
Although the CD16a glycan composition differs between the tworeports (high mannose vs complex), carbohydrate–carbohydrateinteractions were observed. Ten sugar residues were observed withthe oligomannose-type Asn162-glycan (Ferrara et al. 2011) and eightresidues were detected in the complex with the complex-type Asn162glycan (Mizushima et al. 2011).
Recently, Falconer et al. (2018) reported the crystal structure ofafucoyslated IgG1 Fc in complex with CD16a with Mannose5 Nglycans, which was different from the previous reports. Among the
CD16a glycoforms (Mannose5 , Mannose9 and complex-type), theMannose5 form showed the highest affinity toward afucosylated Fc.
In fact, CD16a isolated from NK cells contains a substantial amountof oligomannose and hybrid-type glycans (Patel et al. 2018). In the
crystal structure of the complex, in contrast with earlier reports,
electron density was detected only from single N-acetylglucosamine
residue attached to Asn162 of CD16a. It is unclear why the intermolecular N-glycan contacts were not observed. It is possible thatintermolecular N-glycan contacts in the previous cases formed as aresult of the crystal contacts. To better understand this point, theanalysis of N-glycan dynamics is required.
MD simulations were performed to examine the effect of Fc afucosylation on the dynamics of the CD16a Asn162-glycan (Sakae et al.
2017). The root mean square fluctuation of Asn162-complex-typeglycan was significantly higher (5.5 Å) in the fucosylated Fc systemthan in the afucosylated Fc system (3.7 Å). This can be interpreted asa significant disruption of proper carbohydrate-carbohydrate interactions upon Fc fucosylation. MD simulations were also performed
with CD16a displaying a Mannose5 glycoform with either fucosylated or afucosylated Fc (Falconer et al. 2018). The addition ofan Fc core fucose residue decreased the volume sampled by the
Asn162-linked glycan from 10,800 Å3 to 9,100 Å3 . Falconer et al.
interpreted these results to indicate that the Fc core fucose restricts
the conformational space of Asn162, introducing a fucose-dependent
energetic penalty upon binding to Fc. This idea is opposed to the previously proposed mechanism that direct intermolecular glycan-glycan
contacts stabilized the complex (Ferrara et al. 2011; Mizushima et al.
2011). The mechanism of affinity enhancement upon defucosylationis still controversial, but we must pay attention to the interpretationof crystal structures which rather reflect a snapshot from many
possible conformations and are often affected by unwanted crystal
contacts.

The directed replacement of both the Gln295 and Tyr296 residues
increased Fc thermal stability and decreased N-glycan processing(Chen et al. 2016). Based on an idealized structure, the Fc Gln295Phe
Tyr296Ala variant revealed reduced N-glycan processing evident
from the appearance of high levels of minimally-processed hybridtype N-glycans rarely observed on IgG1 Fc. The enhanced thermal stability resulted from stabilization of the Cγ 2; however, thismutation decreased binding to CD32a, CD16a and CD16b. It isevident that decreased Fcγ R affinity in a mAb would severely limit
efficacy if Fcγ R-mediated effector function is desired because the highconcentration of serum IgG (∼10 mg/mL = 67 μM) is far above thedissociation constant for IgG1 (50–400 nM).
A mutation to the Cγ 3 domain that disrupted Fc dimer formation also increased N-glycan processing (Rose et al. 2013). The Fc
Tyr403Glu variant of IgG1 and IgG4 showed increased N-glycan
processing similar to the Phe243 variant, indicating that Fc dimer
formation contributes to restricted processing of the Fc N-glycan.
These results are important for efforts to alter Fc properties bymodifying N-glycan processing during protein expression.

IgG sialylation and fucosylation affects IgG Fc structureand function
Structural consequences of core fucosylation. Antibody-dependent cel-lular cytotoxicity is a key effector function, relying on the bindingof antigen-antibody complexes to Fcγ Rs. Most natural antibodies
are highly fucosylated. It was discovered that antibodies lacking corefucosylation show a large increase in affinity for CD16a leading to an
enhanced ADCC activity. This discovery stimulated the developmentof therapeutic antibodies with desired activity by specifically increasing or decreasing fucosylation. Many therapeutic antibodies are,however, modified with core fucose, which may indicate that Fcγ Rmediated activity is not desired for therapeutic purpose. One possible
mechanism explaining affinity enhancement by defucosylation was
revealed from x-ray crystallographic analysis, using afucosylated Fc
and glycosylated CD16a. Ferrara et al. (2011) reported a crystal
structure of afucosylated human IgG1 Fc in complex with a glycosylated CD16a. Afucosylated Fc was prepared by introducing the
GnTIII gene into the host cell to produce bisected and afucosylated Nglycans. For the preparation of the human CD16a, three out of five Nlinked glycosylation sites (38, 74 and 169) were removed by changing
the Asn residues (38, 74 and 169) to Gln. Asn162 and Asn45 were
kept because they are essential for the affinity toward IgG1 and forexpression, respectively. Further, the CD16a was expressed in thepresence of kifunensine, producing the oligomannose-type N-glycans.
In the crystal structure of the afucosylated Fc-glycosylated CD16acomplex, unique intermolecular carbohydrate–carbohydrate interactions appeared, connecting N-glycans of the receptor the afucosylated
Fc N-glycan. In order to understand the regulatory mechanism of
IgG core fucosylation, fucosylated Fc was used for comparison.
In contrast to the complex with afucosylated Fc, carbohydrate–
carbohydrate contact area was significantly decreased in the complex
structure of fucosylated Fc with glycosylated CD16a. Core fucose
linked to the Fc is oriented toward the second N-acetylglucosamine
residue of the Asn162-glycan and as a result the Asn162-glycan mustmove and hence carbohydrate–carbohydrate contact is not properly
formed.
Independently, Mizushima et al. (2011) reported the crystal
structure of afucosylated IgG1-Fc in complex with glycosylated
CD16a. In this case, CD16a possesses a complex-type biantennarycomplex glycan at Asn45 and Asn162 and the other remaining N-

Structural consequences of sialylation. Another topic regarding thestructure and function of the Fc N-glycan is sialylation. In general,sialic acid can be incorporated in α2–3 or α2–6 linkages to a Gal
residue. Looking at the Fc part of natural IgG, sialylated glycans
are only a small population and are connected only through α2–6linkages. α2–3 Neu5Ac onto the α1–6 branch of the Fc glycan ispredicted to have a big impact on the native glycan-polypeptide interface. In vivo studies have shown that intravenous immunoglobulin G(IVIG) enriched in (α2–6) sialylation of the Fc glycan increased antiinflammatory activity by up to 10-fold when compared to nonenriched IgG (Anthony, Nimmerjahn, et al. 2008; Anthony et al. 2011).
When IVIG was treated with a sialidase to remove the modification,
the anti-inflammatory activity was abrogated (Kaneko et al. 2006).
Furthermore, ADCC activity is enhanced by (α2–6) sialylation in
the absence of core fucosylation in in vitro and in vivo assays(Li et al. 2017).
How sialylation regulates the Fc activity is still unclear, but several
mechanisms were discussed and proposed. One proposed mechanism

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Y Yamaguchi and A Barb

immunotherapy, exemplified by the anti-PD1 therapeutic antibodies(pembrolizumab/Keytruda® and nivolumab/Opdivo® ).
Davies, Rispens, et al. (2014) reported the high-resolution crystal
structure of human IgG4 Fc, prepared from papain digestion of serum
IgG4 and using recombinant Fc. The overall topology of human
IgG4 Fc is very similar to human IgG1 Fc fragments. The Asn297
glycans were facing each other in the cavity formed between two
Cγ 2 domains. Shortly thereafter, the crystal structure of full-length
human IgG4 antibody S228P (pembrolizumab) was reported at 2.3 Å
resolution (Scapin et al. 2015). Looking at the Fc region, the orientation of one Cγ 2 domain is different from the previously reported
IgG4 Fc structure. In the structure of the full-length antibody, theorientation of one Cγ 2 domain (chain B) displayed a rotation of
120o relative to the position observed in the isolated Fc, maintaining
the immunoglobulin fold. Consequently, the Asn297 glycan on chain
B is more solvent-exposed than chain A. This nature is verified bymeasuring the deglycosylation rate of IgG4 and a reference IgG1.
The rate of IgG4 deglycosylation by PNGase F is faster than for
IgG1, suggesting that IgG4 N-glycan is more solvent-exposed thanin IgG1. However, one cannot rule out the possibility that thisunusual Cγ 2 conformation represents only one of many possible
conformations.
IgG3 is the third most abundant human IgG subclass. It contains
a long hinge region which is thought to provide additional flexibility
toward antigen binding (Vidarsson, G., Dekkers, G., et al. 2014).
A high-resolution (1.8 Å) crystal structure utilized human IgG3 Fc
expressed with a modified yeast strain that added homogeneous
Mannose5 N-glycans (Shah et al. 2017). Out of the five sugar
residues, the first three sugar residues corresponding to Mannose(β1–4)N-acetylglucosamine(β1–4)N-acetylglucosamine revealed electron
density. The terminal mannose residues were not observed. The
protein-carbohydrate interactions for the three visible sugar residues
were identical to those previously reported for human IgG1 Fc
structures (Deisenhofer 1981, Nagae and Yamaguchi 2012).
Human IgG2 is known to form structural isomers that originate
from alternative disulfide bond formation of between the cysteines
in the hinge region (Wypych et al. 2008). It is suggested that theactivity of each human IgG2 isomer will be different (Dillon et al.
2008), and hence this point needs to be analyzed in detail. So far two
IgG2 Fc crystal structures were reported, one is isolated after papain
digestion and the other is from a recombinant construct (Teplyakov
et al. 2013). The human IgG2 Fc structures are very similar to human
IgG1 Fc structures. It is likely that sequence differences in the lower
high region between IgG1 and IgG2 account for differences in Fcγ Rbinding affinity.
Structural studies on non-human IgG are limited. Recently, acomprehensive study was performed for all IgG subclasses, IgG1,
IgG2, IgG3 and IgG4-Fc of an Old-World monkey, Rhesus macaque(Macaca mulatta, Mm) (Tolbert et al. 2019). Crystal structures of the
Fc from MmIgG1–4 were solved with resolutions from 2.8 Å to 3.5 Å.
The overall topologies of the MmIgG1–4 Fc resemble that of human
IgG1 Fc. Solution NMR spectroscopy was employed to analyze thedynamics of the Fc-glycan at Asn297, with the aid of metabolic 13 Clabeling (Tolbert et al. 2019). N-glycan dynamics were evaluatedfrom the linewidth of anomeric 1 H signals from Asn297-linked Nacetylglucosamine residues. The linewidth is in the following order:
human IgG1-Fc (125 Hz) > Mm IgG1-Fc (102 Hz) > Mm IgG2Fc (83 Hz) > Mm IgG4-Fc (75 Hz) > Mm IgG3 (66 Hz). This
indicates that among Mm IgGs, the Fc N-glycan dynamics is themost restricted in Mm IgG1. This data is supported by the observation that the N-glycan of Mm IgG1-Fc is more stabilized throughis that sialylated IgG-Fc is recognized by a specific receptor. In vivo
experiments showed that the anti-inflammatory effect of Fc required
expression of the murine C-type lectin receptor SIGN-R1 (Anthony,
Wermeling, et al. 2008). A human orthologue for SIGN-R1 is the Ctype lectin DC-SIGN, and it is also proposed to bind to sialylated Fc(Anthony, Wermeling, et al. 2008). Later, Sondermann et al. (2013)
showed that α2,6-sialylated IgG binds to the IgE receptor CD23
in a cell-based ELISA assay. The model was based on a hypothesisthat sialylation of IgG Fc leads to a conformational change, which
triggers receptor binding. It should be noted that conflicting results
have been reported (Yu, Vasiljevic, et al. 2013; Temming et al. 2019),
thus further analysis will be required for the identification of bonafide receptor(s). Other candidate receptors are implicated, including
DCIR (Massoud et al. 2014) and Siglecs (von Gunten and Simon
2008; Seite et al. 2010).
Attention has been paid to examine the effect of sialylation on theconformational property of Fc. Several crystal structures of sialylated
Fc have been reported from several groups (Crispin et al. 2013;
Ahmed et al. 2014; Chen et al. 2017). Overall, the conformations of
sialylated Fc are within the range of Fc structures without sialylation.
In a representative crystal structure of sialylated Fc, electron densityof Neu5Ac residue is observed only on the (α1–6Mannose) branch
and Neu5Ac interacted through water-mediated hydrogen bonds
with residues found at the interface formed between the Cγ 2 andCγ 3 domains (Chen et al. 2017). The observed interaction between
Neu5Ac and residues at the Cγ 2-Cγ 3 interface may modulate theorientation of the Cγ 2-Cγ 3 domain and affect the binding affinity
toward Fcγ Rs and the neonatal Fc receptor (Chen et al. 2017).
Based on the observation that certain N-glycan compositions caused
increased variation in Fc crystal structures, Bjorkman and coworkers
suggested that sialylation increases conformational flexibility of theCγ 2 domain which is associated with anti-inflammatory activityof the Fc (Ahmed et al. 2014). However, this hypothesis does not
explain how the conformational flexibility of Fc affects receptor
binding. Thus, the mechanism behind the anti-inflammatory activityof sialylated IgG is still an open question.

Studies on other IgGs
There are four human IgG antibody subclasses: IgG1, IgG2, IgG3 andIgG4, which are homologous to over 90% at the amino acid level(Vidarsson et al. 2014). The global structures of the four human IgG
subclasses are thus similar. However, the subclasses have sequencevariations especially in the hinge region and N-terminal Cγ 2 domain(Vidarsson et al. 2014). This variation is likely linked to differingaffinities for the specific Fcγ Rs and different abilities to activatecomplement (Bruhns et al. 2009; Vidarsson et al. 2014). So far,most studies have been conducted for human IgG1 subclasses anda large set of human IgG1 crystal structures have been reported.
In contrast, the structural information on other subclasses is rather
limited.
Human IgG4 is the least abundant of the four classes of IgG inserum, but displays unique biological properties. One is heavy chain
exchange, also known as Fab-arm exchange, to form a bispecific
but monovalent antibody (Aalberse and Schuurman 2002; Davies
et al. 2013). IgG4 binds Fcγ receptors with lower affinity than
IgG3 with the exception of Fcγ RI (Bruhns et al. 2009) and does
not activate complement (van der Zee et al. 1986); these properties
make IgG4 suitable for therapeutics when effector functions are
undesired (Davies and Sutton 2015). For this reason, the IgG4
subclass is collecting a lot of attention as a preferred subclass for115

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221

Paramagnetic lanthanide ions provide a means to study distance andorientation up to 50 Å from the ion using solution NMR spectroscopy, but some ions also provide luminescence for microscopy
and fluorescence-type solution measurements, unpaired electrons for
EPR, and electron dense nuclei for electron and x-ray diffraction (Suand Otting 2010; Koehler and Meiler 2011). This specific constructrevealed minimal motion of the paramagnetic ion relative to IgG1
Fc, expanding the utility of the tag for probing glycan and protein
motion and conformation (Barb and Subedi 2016).
An additional advance included the development of a suite of
NMR experiments to probe commercial antibodies and other therapeutic proteins. One challenge facing the pharmaceutical industry isappropriately validating the composition and structure of each product batch. Unlike small molecule drugs, three-dimensional conformation is an essential property of therapeutic proteins and is challenging
to probe with traditional techniques used by the manufacturers.
A simple mass analysis is insufficient. Developing techniques withsuitable sensitivity, resolution and throughput represents a major
challenge. Marino and coworkers developed multiple methods thatprovide sufficient resolution and are capable of characterizing pharmaceutical products using natural abundance of 13 C and 15 N. One
method utilizes the unique property of methyl groups that provide
superior detection sensitivity and resolution (Arbogast et al. 2015).
Their developments reduced experimental time to 30 min and weresensitive enough to show resolvable differences in antibodies withdifferent N-glycan composition. The next experiments correlated
backbone amide protons to the nitrogen nuclei, an approach thatis inherently less sensitive than detecting methyl correlations but
provides a fingerprint of the protein that is likely more influenced
by subtle conformational changes including changes in hydrogenbonding (Arbogast et al. 2016). These experiments required more
instrument time of ∼17 h for a single analysis, but provided agreater magnitude of changes, detecting large differences in peak
positions following deglycosylation and applying a rigorous statistical analysis to the spectra (Arbogast et al. 2017). Thus, NMR
is capable of detecting large conformational differences and small
structural differences resulting from processing (Brinson et al. 2019).
A related study by a group at Pfizer identified spectral features from1
H-13 C methyl groups that correlated with aggregation (Majumder
et al. 2018). Aggregation is another significant problem in pharmaceutical production, and is specific to individual mAb clones.
This report indicates that surprisingly melting temperature did notcorrelate with aggregation behavior in a pilot study using threemAbs, but spectral features measured by NMR did. This result may
represent a significant advance toward characterizing therapeuticproteins, though future studies with a larger number of proteins
will determine if spectral features are reliable probes of aggregation
behavior.
Though the common application of mass spectrometry provides
mass information and generally does not probe molecular shape,advances in ion mobility mass spectrometry are providing new
insight into protein structure and function. Tian and Ruotolo (2018)
reported the unfolding of different antibody glycoforms in the gas
phase. Though they observed greater sensitivity using the isolated
Fc fragment, this team differentiated antibodies that differed only inglycoform.

glycan-polypeptide interaction than Mm IgG2–4. Interestingly, Mm
IgG1 experienced the most glycan processing as compared to otherMm IgGs, suggesting that the Mm IgG1 N-glycan is more accessible
to glycosyltransferases (galactosyltransferase, sialyltransferase etc.)
and this may be correlated with the slowest dynamics of Mm IgG1
Fc glycan.
Mice express different IgG subclasses that vary by strain andinclude IgG1, IgG2a, IgG2b, IgG2c and IgG3. Subclass nomenclature
has arisen independently for each species and there is no general relationship between the subclasses from different species. A recent study
indicates that mouse Fcγ RIV is the homolog of human Fcγ RIIIa(Dekkers et al. 2018). However, much less is known about mouse
IgG2 or mouse Fcγ RIV. Crystal structures of mouse IgG2c Fc havebeen reported recently (Falconer and Barb 2018) and compared withthat of mouse IgG2b Fc. These two Fcs showed a high degree ofsimilarity with differences in loop residues including Tyr296. Solution
NMR analysis of the Asn297-linked N-acetylglucosamine residue
shows differences between mouse IgG2b, mouse IgG2c and human
IgG1, in terms of anomeric 1 H chemical shift, and that correlates
to receptor binding affinity. Even in highly conserved molecules,
structural and functional differences are present which should befurther investigated to fully establish structure/function relationships
across important model species.

Noteworthy technical achievements
Recent technical advances have provided a wealth of new tools toprobe Fc structure and motion. Progress is focused in two primaryareas. Developments in IgG1 Fc expression, purification and glycoengineering are substantial; this topic is directly related to pharmaceutical development and too broad to appropriately cover here (seeLoos and Steinkellner 2012; Subedi et al. 2015; Mimura et al. 2018).
The second major group of advances results from improvements toanalytical techniques or new analyses of IgG. These reports have notthemselves elucidated new aspects of the IgG Fc structure/function
relationship; however, these techniques warrant a separate section
highlighting significant progress in the past few years that may be
a foundation for future advances.
Solution NMR studies designed to probe protein motion requireassignment of the backbone nuclei. This allows the extraction of
atomic-level detail regarding protein structure and motion. Katoand coworkers reported a major advance with the backbone resonance assignment of the glycosylated IgG1 Fc fragment (Yagi et al.
2015). Ordinarily, Fc would be a challenging NMR target due tothe size of the molecule alone (∼52 kDa). However, the challenges
are magnified because appropriately glycosylated IgG1 Fc cannot
be expressed in prokaryotic hosts and mammalian hosts are often
used instead. This requirement introduces two additional limitations.
First, proteins expressed in mammalian hosts, like the CHO cells
used in this study, cannot be deuterated because high deuterium
content of the medium is toxic to the host. Second, labeling expressed
protein with stable 13 C and 15 N isotopes is much more expensive
in a mammalian host compared to a prokaryotic host. The authors
reported the nearly complete assignment of IgG1 Fc (99% of HN,
N and Cα). This assignment provided a crucial starting point for
at least two studies described above (Subedi et al. 2014; Subedi and
Barb 2015).
IgG1 Fc binds multiple protein ligands through many different
interfaces (Hanson and Barb 2015). The Fc-binding portion of one
ligand, protein A, was developed to simultaneously bind IgG1 Fc
and lanthanide ions with high affinity (Barb and Subedi 2016).

Future considerations
Fundamental properties of IgG emerged in the past few years thatdefine how Fc composition impacts structure and receptor binding.

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Y Yamaguchi and A Barb

Promotion of Science Kakenhi (grant numbers JP16H04758 and
JP19H03362) to Y.Y.

This may appear surprising due to the importance and intense
scrutiny focused on this molecule since the description of IgG in 1939
and high-resolution models of the Fab and Fc in 1973 and 1976,
respectively (Tiselius and Kabat 1939; Poljak et al. 1973; Huber et al.
1976). Recent advances were largely driven by developments in physical techniques and Fc preparation methods, allowing researchers to
break free from the bounds imposed by the crystal lattice. It is thenworthwhile to consider which remaining undefined aspects may lead
to better therapies once defined. Here we identify three primary areas
for future research.
1. Future studies of unglycosylated antibodies may provide insight
into which conformations are disfavored by intramolecular N-glycan
interactions. A quantitative analysis of conformation states sampled
by the Fcγ R-binding loops of aglycosylated Fc that defines the populations of each state will guide Fc engineering to limit the exposureof residues that promote aggregation and potentially enhance Fcγ Rbinding affinity. Similarly, it is likely that the Fc N-glycan completely
dissociates from the protein surface and becomes accessible to glycan
modifying enzymes. The structure of this state, and the definition of
the conformational rearrangements that promote sampling this state,
will impact Fc engineering to modify N-glycan composition during
expression.
2. IgG1 is now a well-defined molecule, though some questions
still remain. Much less is known about IgG2,3,4, IgD, IgM and IgE.
Each of these share an N-glycan at a position homologous to N297
of IgG1 Fc and display an aromatic residue similar to F243 (Subedi,
G.P., Hanson, Q.M., et al. 2014). IgE likewise shares a comparable
receptor binding mode though the role of N-glycosylation in receptor interactions is less well defined thatn IgG (Shade et al. 2019).
It is currently not known how N-glycosylation impacts structureand function for these less-studied antibodies. Furthermore, little is
known about antibodies from other species which may provide complementary evolutionarily-selected solutions to stability and receptor
binding. Unique among the five human antibody classes, IgA ismuch different, lacking homologous glycosylation, the key aromatic
residue, and IgA displays a different receptor binding mode (Herret al. 2003).
3. Differences between the behavior of IgG1 and isolated Fc andhow Fc modulates interactions between two antibodies may provide
insight into improving Fc designs. The 10-fold greater impact ofIgG1 Fc fucosylation when compared to isolated Fc in measurementsof CD16a binding indicates that the presence of Fab domains may
impact Fc activity. Detailed studies to define these interactions willlead to a more complete definition of antibody function. Hints tothe presence of intra- and inter-antibody interactions exist, including
the observation that IgG1 forms hexamers on surfaces (Saphire et al.
2001; Ugurlar et al. 2018). N-glycans are poised to modulate theseinteractions as well, though sensitive techniques must be developed
to investigate these phenomena further.
It is likely studies of antibody structure and function will continue
for decades into the future. The results of these studies stand to
improve existing therapies and create engineered mAb backbones
for a wide range of future clinical applications, if investigators are
sufficiently bold and inventive to identify key features and residues
that may be altered for improved drug properties.

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Funding
This work was supported by the National Institutes of Health (grant
number R01 GM115489) to A.W.B. and by Japan Society for the117N-glycosylation in IgG Fc structure and function

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Structural Aspects of Carbohydrate
Recognition Mechanisms of C-Type
Lectins
Masamichi Nagae and Yoshiki Yamaguchi

Contents
1
2
3

Introduction..........................................................................................................................
C-Type Lectin Fold .............................................................................................................
Sugar Binding Motifs: EPN and QPD................................................................................
3.1 Other Monosaccharide Binding Modes .....................................................................
4 Oligosaccharide Recognition...............................................................................................
4.1 Oligosaccharide Recognition via EPN Motif ............................................................
4.2 Oligosaccharide Recognition of QPD Motif-Containing C-Type Lectins ................
4.3 Oligosaccharide Recognition of C-Type Lectin-like Domains .................................
4.4 Genetic Variants of C-Type Lectins ..........................................................................
5 Functional Oligomerization of C-Type Lectin Domains....................................................
6 Conclusion and Future Perspective.....................................................................................
References ..................................................................................................................................

Abstract Carbohydrate recognition is an essential function occurring in all livingorganisms. Lectins are carbohydrate-binding proteins and are classiﬁed into several
families. In mammals, Ca2+-dependent C-type lectins, such as b-galactosidebinding galectin and sialic acid-binding siglec, play crucial roles in the immune
response and homeostasis. C-type lectins are abundant and diverse in animals. Their
immunological activities include lymphocyte homing, pathogen recognition, andclearance of apoptotic bodies. C-type lectin domains are composed of 110–130
amino acid residues with highly conserved structural folds. Remarkably, individual
lectins can accept a wide variety of sugar ligands and can distinguish subtle
M. Nagae (&)
Department of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku,
Tokyo 113-0033, Japane-mail: mnagae@mol.f.u-tokyo.ac.jp
Y. Yamaguchi (&)
Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Miyagi
981-8558, Japane-mail: yyoshiki@tohoku-mpu.ac.jp
Current Topics in Microbiology and Immunology
https://doi.org/10.1007/82_2019_181© Springer Nature Switzerland AG 2019

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structural differences in closely related ligands. In addition, several C-type
lectin-like proteins speciﬁcally bind to carbohydrate ligands in Ca2+-independentways. The accumulated 3D structural evidence clariﬁes the unexpected structural
versatility of C-type lectins underlying the variety of ligand binding modes. In thisissue, we focus on the structural aspects of carbohydrate recognition mechanisms of
C-type lectins and C-type lectin-like proteins.

1 Introduction
Carbohydrates, most often linked to proteins or lipids, cover the surface of all living
cells and are fundamental determinants of health and disease. Carbohydrate-binding
proteins or lectins interact with glycans in speciﬁc ways, eliciting many important
cellular responses including those involved in immune cell homeostasis. The Ca2+dependent or C-type lectins are the largest and most diverse lectin family found inanimals (Zelensky and Gready 2005; Drickamer and Taylor 2015). Well-studied
members include galectin and siglec in mammals. C-type lectins share highly
homologous structural modules in their carbohydrate recognition domains. These
domains are ubiquitously found both in soluble and membrane proteins. More than
100 human proteins contain at least one C-type lectin domain. The C-type lectinfamily is subdivided into 16 groups (Groups 1–16) based on phylogenetic relationships and domain architecture (Cummings and McEver 2015) (Fig. 1a). Most ofthese groups have a single C-type lectin domain. Exceptions with 8–10 C-type
lectin domains in their polypeptides (e.g. macrophage mannose receptor andDEC-205) are found in Group 6. Also, many proteins have C-type lectin domainsthat lack the conserved Ca2+ binding site, designated as “C-type lectin-likedomains,” and thus do not always bind carbohydrate ligands (e.g., Group 5 inFig. 1a). This has allowed for distinction between sugar-binding C-type lectins andthe broader family of C-type lectin-like domains (Zelensky and Gready 2005).
C-type lectins accept a wide variety of carbohydrate and non-carbohydrate
ligands such as lipids and proteins via their lectin or lectin-like domains. Some
C-type lectins play speciﬁc roles in glycoconjugate recognition, with the aglycon
moiety of the ligand often contributing to the interaction. From an immunologicalpoint of view, various C-type lectins work as pattern recognition receptors (PRRs)
which recognize highly conserved speciﬁc molecular signatures called
pathogen-associated molecular patterns (PAMPs) and damage-associated molecularpatterns (DAMPs) that are crucial for discrimination of self from non-self (Varki
2017; Sancho and Reis e Sousa 2013). In order to understand the physiologicalroles of C-type lectins in detail, 3D structural information is essential. We describe
our current knowledge of the carbohydrate recognition mechanism of C-type lectins
at monosaccharide, oligosaccharide, and polysaccharide levels.

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Structural Aspects of Carbohydrate Recognition Mechanisms …

(a)3

Group 6
Multi-CTLD
Endocytic receptor

Group 3
Collectin

R-type lectin
Fn type 2Group 4

Group 12EMBP

Stalk region SelectinGroup 2ASGPR &DC receptor

Collagen likedomainGroup 5
NK receptor
EGF domain

N

SCR motifnStalk region

N
Stalk regionnMembrane

N

C

EPN

DC-SIGN, hDCIR
SIGN-R1, mDCIR2 MBP-A, C
Dectin-2, BDCA-2 SP-A, D
Langerin, MINCLEQPDASGPR, SCARA

NC

L-selectin (n=2)
E-selectin (n=6)
P-selectin (n=9)

DEC-205 (n=3)
MR (n=1)

Dectin-1
CLEC-2

Calcium
independent

EMBP

(c)

(b)
Long loop

MannoseCa2+β 4 β3
β2
α2

β5
β1

180°

α1

EPN motif
WND motif

N
C

MBP-A
(PDB code: 2MSB)

N

Glu193C

Fig. 1 a Domain architecture of representative C-type lectin-containing proteins described in thisissue. This ﬁgure is prepared from the review (Zelensky and Gready 2005) with modiﬁcations.
C-type lectins are classiﬁed into three sugar-binding motifs (EPN, QPD, and calcium
independent). Fn type 2: Fibronectin type 2, SCR: Short consensus repeat, EGF: Epidermal
Growth Factor, ASGPR: Asialoglycoprotein receptor, SRACLA: scavenger receptor C-type lectin.
b Overall structure of C-type lectin domain (Rat mannose binding protein A (MBP-A), PDB code
2MSB). Polypeptide, carbohydrate and calcium ions are shown in ribbon, stick, and spheremodels, respectively. The long loop region is colored in cyan. Disulﬁde bonds are shown in yellow
stick model. c Close-up view of the primary binding site of MBP-A. The coordination andhydrogen bonds are depicted with red dotted lines

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2 C-Type Lectin Fold
Since the pioneering work on the mannose-binding protein was reported (Weiset al. 1992), hundreds of atomic structures have been elucidated for C-type lectins.
As of July 2019, over 250 atomic structures of C-type lectins were deposited in theProtein Data Bank. Overall fold and disulﬁde bond pattern are highly conserved
among C-type lectins and C-type lectin-like domains. The C-type lectin domain istypically composed of 110–130 amino acid residues and the overall fold is formedby two a-helices and six or seven b-strands forming two antiparallel b-sheets(Fig. 1b). A long loop is found around the “top face” and inserted between two
b-strands (b2 and b3 in typical cases). This loop is characteristic of C-type lectins,
playing crucial roles in calcium coordination and sugar recognition (colored in cyanin Fig. 1b). C-type lectin domains accept one to four calcium ions (Zelensky andGready 2005). The sugar-binding calcium ion is located at the top face of the
C-type lectin domain. In the sugar-binding site, the calcium ion makes coordination
bonds with both the lectin domain and bound monosaccharide (Fig. 1c). The othercalcium ions mainly stabilize the 3D structure and occasionally form a secondary
sugar-binding site.

3 Sugar Binding Motifs: EPN and QPD
A calcium ion forms multiple coordination bonds with amino acid residues that are
well conserved among C-type lectins. The residues that deﬁne the sugar binding arecalled motifs. The asparagine and aspartate from the WND (Trp-Asn-Asp) motif
and one carbonyl side chain (Glu side chain in Fig. 1c) form coordination bonds
with Ca2+ in all C-type lectins. In addition, the EPN (Glu-Pro-Asn) or QPD(Gln-Pro-Asp) motif contributes to calcium coordination and forms thesugar-binding site. These motifs have two carbonyl groups separated by a proline
residue. Two adjacent hydroxyl groups from a monosaccharide make coordination
bonds with the calcium ion and hydrogen bonds with the EPN or QPD motif(Zelensky and Gready 2005). C-type lectin-like domains lack these conserved
motifs and do not bind a calcium ion.
EPN and QPD motifs deﬁne the monosaccharide speciﬁcity of C-type lectins.
Hence, C-type lectins can be classiﬁed into three groups: (i) EPN motif-containing
C-type lectins, (ii) QPD motif-containing C-type lectins, and (iii) C-type lectin-likedomains without these two motifs. EPN motif-containing C-type lectins usuallyaccept D-mannose, D-glucose, L-fucose, and N-acetyl-D-glucosamine (GlcNAc)
through equatorial 3-OH and 4-OH groups (left panel in Fig. 2a). In contrast, QPD
motif-containing C-type lectins bind N-acetyl-D-galactosamine (GalNAc) and Dgalactose through equatorial 3-OH and axial 4-OH groups (right panel in Fig. 2a).
In both cases, bound monosaccharide is stabilized through hydrogen bonds with thecarboxyl (–COO−) and amide (–CONH) groups from these motifs. Importantly,

124Structural Aspects of Carbohydrate Recognition Mechanisms …

5

(a)

Gal

E185
Man

OH3

Q239

N187

OH4

OH4

OH3

QPD motif

EPN motif

(b)

D241

W243

W100
Gal

Gal
OH3

OH3 OH4

OH4

S88 E86

Q239
D241

EPS motif(c)

QPD motif

K313

(d)

Sulfated
Galactose

Neu5Ac
N288
K299

N307

N297

EPN motif(e)(f)
GalNAc
Y66

KPD

N118

HeparinD106

H120

hydrogen donors and acceptors are switched in the two cases. In this way, thehydrogen bond network deﬁnes the position and orientation of the boundmonosaccharide. In fact, replacing the EPN motif with QPD in mannose binding
protein A (MBP-A) changes the binding ability to favor galactose. This result
proves the role of these motifs in monosaccharide speciﬁcity (Drickamer 1992). In a

125

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M. Nagae and Y. Yamaguchi

JFig. 2 a Monosaccharide recognition by EPN (MBP-A, PDB code 2MSB, left panel) and QPD

motifs (ASGPR, PDB code 5JPV, right panel). The amino acid residues which contribute tocalcium ion coordination and sugar binding are shown in stick models. The positions of EPN and
QPD motifs are indicated. b Galactose recognition by TC14 (PDB code 1TLG, left panel) and
ASGPR (PDB code 5JPV, right panel). The tryptophan residues (W100 in TC14 and W243 in
ASGPR) are shown in stick model and make stacking interactions with galactose rings. c Sulfatedgalactose recognition by langerin (PDB code 3P5I). The amino acid residues interacting with thesulfate group are highlighted. d Sialic acid recognition by murine SIGN-R1 (PDB code 4CAJ).
Coordination and hydrogen bonds are depicted with red dotted lines. An additional hydrogen bondwith asparagine (N288) is also shown. e Heparin disaccharide recognition by EMBP (PDB code
2BRS). One disaccharide unit (colored in white) is surrounded by three EMBP molecules (shownin semitransparent surface models, and colored in green, cyan, and magenta). Direct intermolecularhydrogen bonds are shown in red dotted lines. f Calcium-independent GalNAc recognition by
SPL-2 (PDB code 6A7S). Direct intermolecular hydrogen bonds are shown in red dotted lines.
Y66 contacts the GalNAc residue

ligand-free form, two water molecules occupy the positions which will be takenover by 3-OH and 4-OH groups of the binding sugar residue, forming aneight-coordinated calcium ion (Ng et al. 1996; Feinberg et al. 2000). The orientation of the sugar ring at the primary binding site is affected by the surrounding
amino acid residues. Occasionally, the bound sugar occurs as a mixture of the twoorientations in a single crystal structure (Ng et al. 2002).

3.1

Other Monosaccharide Binding Modes

Monosaccharide speciﬁcities of C-type lectins are generally deﬁned by these two
motifs, but there are several known exceptions. A tunicate lectin TC14 from
Polyandrocarpa misakiensis has an EPS motif and a sea cucumber lectin CEL-IV
from Cuvumaria echinate has the EPN motif. Contrary to the motif rule, they bindgalactose at the primary binding site (Poget et al. 1999; Hatakeyama et al. 2011). In
each structure, a tryptophan side chain stacks with the apolar face of the galactose
ring (left panel in Fig. 2b). In the case of galactose recognition by the QPD motif,
the sugar is typically stabilized with the tryptophan side chain, which is located on
the opposite side (right panel in Fig. 2b). Consequently, the orientation of thegalactose ring observed in TC14 and CEL-IV is inverted compared with the typicalgalactose-QPD motif interaction.
A second example is the mammalian C-type lectin receptor langerin which isexpressed on Langerhans cells and mediates carbohydrate-dependent uptake ofpathogens. Langerin has an EPN motif but, atypically, it accepts glycans withterminal 6-sulfated galactose. A crystal structure of langerin complexed with 6SO4Galb1-4GlcNAc shows that the galactose residue coordinates a calcium ion andthe sulfate group forms salt bridges with two lysine residues located close to theprimary binding site. This electrostatic interaction appears to compensate for the126Structural Aspects of Carbohydrate Recognition Mechanisms …

7

nonoptimal binding of galactose with the EPN motif (Fig. 2c) (Feinberg et al.
2011).
Another exception is the interaction between SIGN-R1 and sialic acid (Neu5Ac)(Silva-Martin et al. 2014). SIGN-R1, also known as CD209a, is a murine C-type
lectin receptor with an EPN motif and is expressed in myeloid cells. A crystal
structure of SIGN-R1 in complex with Neu5Ac shows that the carboxylic group,
not the hydroxyl groups, of the sialic acid makes coordination bonds with thecalcium ion of SIGN-R1 and hydrogen bonds with adjacent amino acid residues(Fig. 2d). One asparagine residue (N288) contributes to sialic acid binding, independent of calcium coordination.
C-type lectin-like domains without an EPN or QPD motif are predicted to lacktypical sugar-binding ability. Nevertheless, several do bind carbohydrate ligands.
For instance, the eosinophil major basic protein (EMBP) is a constituent of theeosinophil secondary granule and has a C-type lectin-like domain. Surface plasmon
resonance assay demonstrated that EMBP directly binds to heparin and heparansulfate, but not to hyaluronic acid (Swaminathan et al. 2005). A crystal structure ofhuman EMBP in complex with heparin disaccharide has been reported(Swaminathan et al. 2005). The authors introduced the disaccharide ligand bysoaking into ligand-free crystals. Three EMBP molecules in the crystal latticecontact one heparin disaccharide unit. The major contact site is located close to theprimary binding site of typical C-type lectins and the bound disaccharide unit ismainly stabilized by electrostatic interactions and hydrogen bonds (Fig. 2e).
One more example is the bivalve lectins SPL-1 and SPL-2, which show highafﬁnities for GlcNAc or GalNAc containing glycans. Intriguingly, RPD and KPD
motifs are found in SPL-1 and SPL-2, instead of EPN or QPD. Crystal structures of
SPL-2 in complex with GalNAc demonstrated that the sugar binds near the putative
primary binding site. However, the interaction mode is different from typical sugar
binding via an EPN or QPD motif (Unno et al. 2019). 3-OH and 4-OH groups of
GalNAc make hydrogen bonds with the putative primary binding site of the C-type
lectin, and the acetamido group is sandwiched by tyrosine and histidine side chains
via a stacking interaction (Fig. 2f).
A shrimp C-type lectin MjGCTL has a QAP (Gln-Ala-Pro) motif which waspredicted to not to have calcium binding ability. However, a recent study shows thatthe lectin has sugar-dependent hemocyte encapsulation activity (Alenton et al.
2017). The carbohydrate recognition mechanism of this C-type lectin-like domain
could be different. Structural analysis of carbohydrate recognition mechanisms withnovel motifs will expand our understanding of this lectin family.

4 Oligosaccharide Recognition
C-type lectins have individual speciﬁcities despite the high sequence and structuralsimilarities. For instance, langerin binds a diverse range of carbohydrates including
high-mannose-type glycan, fucosylated blood group antigens, and glycans with

127

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M. Nagae and Y. Yamaguchi

terminal 6-sulfated galactose. Meanwhile, a C-type lectin receptor DCAR speciﬁcally recognizes phosphatidylinositol mannoside (PIM), a mycobacteria glycolipid,
promoting a Th1 response during infection (Toyonaga et al. 2016). A fundamental
question is how highly conserved C-type lectins recognize various types of glycan
ligands.
Monosaccharide binding preference is deﬁned by an EPN or QPD motif, while
the speciﬁcity toward oligo- and polysaccharides is determined by the secondary
binding site which is located near the primary binding site. The amino acid residues
coordinating calcium ions are highly conserved, while the amino acid residues
located within *15 Å from calcium ion are less so. In particular, the secondary
binding site is often formed by amino acid residues on the top face, where threeb-strands (b2–b4), the long loop region and a part of two a-helices are located. The
speciﬁc residues in the secondary binding site usually contribute additional interactions, or conversely, work to discourage binding of certain ligands.
From a structural aspect, it is important to understand how each C-type lectin
attains speciﬁcity for the target glycan. To get a broader picture, 3D structures ofC-type lectin domains in complexes with oligosaccharide ligands were extracted
from the PDB and these are summarized in Table 1. In this chapter, we describe thestructural basis for the speciﬁc oligosaccharide recognition mechanism of C-type
lectins.

Table 1 List of 3D structures of C-type lectins and C-type lectin-like domains complexed withglycan ligands. The glycan complex structures were extracted to include more than just thedisaccharide units. Crystal structures of C-type lectins in ligand free forms and complexed withmonosaccharide or non-carbohydrate ligands were omitted
Receptor

Source(i) EPN motif
High-mannose-type N-glycan
MBP-A
Rat
MBP-A
Rat
MBP-A
Rat
(H189V)
MBP-A
Rat
SP-D
Human
SP-D (R343V) Human
DC-SIGN
Human
DC-SIGN
Human

LigandPDB
ID

References

Man5
Mana1-3Man
Mana1-3Man

2MSB
1KWY
1KWZ

Weis et al. (1992)
Ng et al. (2002)
Ng et al. (2002)

Man6GlcNAc2
Mana1-2Man
Mana1-2Man
Man4
Man6

1KX1
3G83
3G84
1SL4
2IT5

Ng et al. (2002)
Crouch et al. (2009)
Crouch et al. (2009)
Guo et al. (2004)
Feinberg et al.
(2007)
Feinberg et al.
(2007)
Sutkeviciute et al.
(2014)

DC-SIGN

Human

Man2

2IT6

DC-SIGN

Human

Pseudo-dimannose mimic

2XR5(continued)

128

Structural Aspects of Carbohydrate Recognition Mechanisms …

9

Table 1 (continued)
Receptor

SourceLigandPDB
ID

References

DC-SIGN

Human

Pseudo-trimannose mimic

2XR6

Dectin-2

Human

Man9

5VYB

L-selectin

Human

Man5GlcNAc2

5VC1

L-selectin

Human

Man5GlcNAc2

3CFW

Langerin

Human

Mana1-3Man (Man5)

3P5D

Langerin

Human

Mana1-2Man

3P5F

Sutkeviciute et al.
(2014)
Feinberg et al.
(2017)
Wedepohl et al.
(2017)
Mehta-D’souza
et al. (2017)
Feinberg et al.
(2011)
Feinberg et al.
(2011)

Complex-type N-glycan
DC-SIGN
Human

GlcNAc2Man3

1K9I

DC-SIGNR

Human

GlcNAc2Man3

1K9 J

hDCIR
mDCIR2
BDCA-2

Human
Mouse
Human

GlcNAcb1-2Man
Bisected glycan
Galb1-4GlcNAcb1-2Man

5B1X
3VYK
4ZET

Codakine

Bivalve

Biantennary glycan

2VUZ

SP-D

PorcineBiantennary glycan

6BBE

Fucose-containing glycan
P-selectin
Human
P-selectin
Human
E-selectin
Human
DC-SIGN
Human
DC-SIGNR
Human
MBP-A (K3)Rat
MBP-A (K3)Rat
MBP-A (K3)Rat
E-selectin
Human
Langerin
Human

PSGL-1
Sialyl-LewisX (soaked)
Sialyl-LewisX (soaked)
Lacto-N-fucopentaose III
LewisX
3’-NeuAc-LeX
3’-sulfo-LeX
4’-sulfo-LeX
Sialyl-LewisX
Gala1-3[Fuca1-2]Gal

1G1S
1G1R
1G1T
1SL5
1SL6
2KMB
3KMB
4KMB
4CSY
3P5G

Somers et al. (2000)
Somers et al. (2000)
Somers et al. (2000)
Guo et al. (2004)
Guo et al. (2004)
Ng and Weis (1997)
Ng and Weis (1997)
Ng and Weis (1997)
Preston et al. (2016)
Feinberg et al.
(2011)

Glucose-containing glycan
SP-D
Human
MincleCow

MaltoseTrehalose (Glca1-a1Glc)

1PWB
4KZV

MincleTrehalose monobutylate

4ZRV

Shrive et al. (2003)
Feinberg et al.
(2013)
Feinberg et al.
(2016)

Cow

Feinberg et al.
(2001)
Feinberg et al.
(2001)
Nagae et al. (2016)
Nagae et al. (2013)
Jegouzo et al.
(2015)
Gourdine et al.
(2008)
van Eijk et al.
(2018)(continued)

129

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M. Nagae and Y. Yamaguchi

Table 1 (continued)
Receptor

SourceLigandPDB
ID

References

MincleCow

Trehalose

4ZRW

Mincle

Cow

KMJ1

5KTI

MincleCow

Brartemicin

5KTH

Langerin

Human

Laminaritriose

3P5H

Langerin

Human

Maltose (Glca1-4Glc)

3P7H

SIGN-R1

Mouse

Dextran sulfate

4C9F

Feinberg et al.
(2016)
Feinberg et al.
(2016)
Feinberg et al.
(2016)
Feinberg et al.
(2011)
Chatwell et al.
(2008)
Silva-Martin et al.
(2014)

OthersCEL-IVSea cucumber

3ALU

CEL-IVSea cucumber

Rafﬁnose(Gala1-6Glcb1-2Fru)
Melibiose (Gala1-6Glc)

Langerin

Human

6SO4-Galb1-4GlcNAc

3P5I

Langerin

Human

GlcNAcb1-3Galb1-4Glc

4N33

Langerin

Human (K313I)

GlcNAcb1-3Galb1-4Glc

4N35

Langerin

Human (N288D/
K313I)

GlcNAcb1-3Galb1-4GlcNAc

4N38(ii) QPD motif
RSL
SCARA4

Rattlesnake
Mouse

Lactose (Galb1-4Glc)
LewisX

1JZN
2OX9

ASGPR

Human

Lactose (Galb1-4Glc)

5JPV(iii) C-type lectin-like domains
EMBP
Human

Heparin

2BRS

Dectin-1
CLEC-2

Glcb1-3Glcb1-3Glc
O-glycosylated podoplanin

2CL8
3WSR

4.1

Mouse
Human

3ALT

(Hatakeyama et al.
2011)
Hatakeyama et al.
(2011)
Feinberg et al.
(2011)
Feinberg et al.
(2013)
Feinberg et al.
(2013)
Feinberg et al.
(2013)Walker et al. (2004)
Feinberg et al.
(2007)
Sanhueza et al.
(2017)
Swaminathan et al.
(2005)
Brown et al. (2007)
Nagae et al. (2014)

Oligosaccharide Recognition via EPN Motif

The structural basis for the speciﬁc oligosaccharide recognition of EPN
motif-containing C-type lectins has been well studied. Here, we introduce therecognition mechanism of EPN motif-containing C-type lectins toward representative oligosaccharides such as high-mannose type, complex-type N-glycans,

130Structural Aspects of Carbohydrate Recognition Mechanisms …

11

sialyl-LewisX, and glucose-containing glycans. The primary binding sites of corresponding C-type lectins bind mannose, fucose, and glucose residues of theseoligosaccharides, and the secondary binding sites deﬁne their speciﬁcities.

4.1.1

Oligosaccharide Recognition of EPN Motif-Containing C-Type
Lectins Through Mannose(I) Recognition of high-mannose-type N-glycan through mannose
High-mannose-type N-glycans are often found in viral and fungal glycoproteins aswell as in nascent mammalian glycoproteins. A representative structure of mammalian high-mannose-type N-glycan is shown as Man9GlcNAc2 which contains
mannose residues of a1-2, a1-3, and a1-6 linkages (Fig. 3a). The Mana1-2 Man
unit is a common terminal structure on mannans of yeast and other fungi. Therefore,
this disaccharide unit can be a target for several C-type lectins working as patternrecognition receptors in the immune system. Crystal structures of 16 EPN
motif-containing C-type lectins have been reported in complexes withhigh-mannose-type glycans (Table 1). Of these, mannose-binding protein A(MBP-A) and pulmonary surfactant protein D (SP-D) are soluble proteins categorized as the collectin family (Group 3). DC-speciﬁc intercellular adhesion
molecule-3-grabbing nonintegrin (DC-SIGN), langerin, and DC-associated C-type
lectin-2 (Dectin-2) are type II membrane proteins which belong to the NK receptors
family (group 5). L-Selectin is an adhesion receptor of the selectin family (Group 4)(Fig. 1a). The interaction modes of these lectins with high-mannose-type glycans
are visualized in Fig. 3b. C-type lectins accept a1-2, 1-3 and 1-6 linkages of the
mannose disaccharide unit at their primary binding sites with several variations. In
this section, the atomic recognition modes of high-mannose-type glycan are discussed for each glycosidic linkage.
(I-1) Mana1-2Man unit recognition
There are two known binding modes for the a1-2 linkage: (a) mannose at the
non-reducing end (non-reducing mannose) bound to the primary site (SP-D wild
type and MBP-A in Fig. 3b), and (b) mannose at the reducing end (reducing
mannose) bound to the primary site (Dectin-2, SP-D R343 V mutant, DC-SIGN
and langerin in Fig. 3b). The surrounding amino acid residues determine the choiceof the binding modes. In SP-D, Arg313 is located close to the primary site and thereplacement of this arginine with valine dramatically changes the disaccharide
binding mode (Crouch et al. 2009). The R343 V mutant accepts reducing mannose
at the primary binding site and forms additional hydrogen bonds with the
non-reducing mannose via the secondary binding site (Fig. 3b). Compared withmode (a), the orientation of mannose in mode (b) is ﬂipped in the primary bindingsites of Dectin-2, SP-D R343 V and DC-SIGN. Dectin-2 mainly recognizes innermannose residues of Man7 glycan (Man-A and C) and the disaccharide recognitionmode seems similar to that of DC-SIGN. However, in DC-SIGN-Man2 complex,

131

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M. Nagae and Y. Yamaguchi

(a)

(b)

132

Structural Aspects of Carbohydrate Recognition Mechanisms …

13

JFig. 3 a A typical high-mannose-type glycan (Man9GlcNAc2, left panel) and biantennarycomplex-type glycan (right panel) linked to asparagine. Monosaccharide symbols follow the
SNFG (symbol nomenclature for glycans) system (Varki et al. 2015). The glycosidic linkages andresidue numbers are labeled at each position. b Summary of the interaction between
high-mannose-type glycan and C-type lectins. Structures are classiﬁed based on the glycosidic
linkages (Mana1-2Man, Mana1-3Man and Mana1-6Man) of the mannose residues. The positionsof reducing and non-reducing mannose residues are labeled as “R” and “NR,” respectively. The
residue names are also labeled. c Three cross-linked structures of C-type lectin-high-mannose
glycan complexes. Two MBP-A glycan complexes (PDB code 2MSB, left and PDB code 1KX1,
middle panels) and one Dectin-2 complex (PDB code 5VYB, right panel) are shown. The mannose
residues at the primary binding sites are labeled. d Complex-type N-glycan recognition by
BDCA-2 (PDB code 4ZET, left panel), hDCIR (PDB code 5B1X, middle panel), and mDCIR2(PDB code 3VYK, right panel). e The branch-speciﬁc interaction with complex-type N-glycan. Allfour C-type lectins (mDCIR2 (PDB code 3VYK), DC-SIGN (PDB code 1K9I), Codakine (PDB
code 2VUZ), and SP-D (PDB code 6BBE)) accept mannose residues (Man-4) of the a1-3 branchat the primary binding sites. It should be noted that the relative position of the a1-6 branch of themDCIR2 complex is different from the other C-type lectins. C-type lectin domains are shown insurface models

2-OH and 3-OH groups of the non-reducing mannose residue are adjacent tophenylalanine (F313 in Fig. 3b), which prevents further extension toward thenon-reducing side. A structure of langerin with a Mana1-2Man oligosaccharide has
also been described (Fig. 3b right panel), showing that the bound reducing mannose
residue is ﬂipped 180° compared with the other complexes in mode (b). Since thereducing mannose residue is clamped between asparagine (N287) and lysine(K299), the 1-OH group cannot be used for extension to a larger glycan. Anotherconformation has also been reported in which the non-reducing mannose resides in
the primary binding site, though the electron density of reducing mannose is
missing. Langerin preferentially binds the reducing mannose of the disaccharide,
but rather binds the non-reducing end of whole high-mannose glycan.
(I-2) Mana1-3Man unit recognition
C-type lectins can bind the terminal and inner Mana1-3Man unit of N-glycan(Fig. 3b). Langerin and MBP-A H189 V mutant recognize the non-reducing
mannose residue of Mana1-3Man unit. Langerin Mana1-3Man disaccharide unit
complex was obtained by using core Man5 oligosaccharide (Mana1-3[Mana1-3[Mana1-6]Mana1-6]Man). In this complex, non-reducing mannose tightly interacts
with the primary binding site, while the reducing ends are exposed to solvent(Fig. 3b). Wild-type MBP-A recognizes the reducing end of the Mana1-3Man
disaccharide unit, and however, introduction of an H189 V mutation causes achange in the bound residue (non-reducing end) as well as a 180° ﬂip of the boundligand (Ng et al. 2002) (Fig. 3b). The position of this histidine in MBP-A isdifferent from that of R343 in SP-D, indicating that the binding mode is determinedby a marginal balance of the surrounding residues.
DC-SIGN recognizes inner a1-3 linked mannose residues. A DC-SIGN-Man4(Mana1-3[Mana1-6]Mana1-6Man) complex shows that all four mannose residues(corresponding to Man-A, Man-B, Man-4’ and Man-3 in Fig. 3a) are uniquely

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(c)

(d)

(e)

Fig. 3 (continued)

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deﬁned. In DC-SIGN-Man6 (Mana1-2Mana1-3Man[Mana1-2Mana1-6]Man)
complex, Mana1-2Mana1-3Man, which is a part of the D2 arm, interacts with
DC-SIGN in a major conformation. In addition, only the terminal Mana1-2Man
unit (Man-A and Man-D2 residues) binds in a minor conformation. The interaction
mode of the major conformation is similar to that of the Man4 complex and these
two glycans are well superimposable (Fig. 3b). This observation indicates that
DC-SIGN tightly associates with the inner tetrasaccharide unit (Mana1-2Mana13Mana1-6Man) of high-mannose-type glycan. The mannose residue (Man-A in
Fig. 3b) coordinates with the calcium ion and both ends of the mannose residues arelocated at the secondary binding sites. The minor conformation of Man6 complex issimilar to the major conformation of Man2 complex. Hence, DC-SIGN can recognize the inner a1-3 linked mannose residue as well as terminal a1-2 linked
mannose of high mannose glycan. These multiple binding modes of DC-SIGN may
enhance its apparent afﬁnity towards glycoproteins carrying high-mannose glycan
under physiological conditions.
L-selectin is a member of the selectin family and mediates cell adhesion andsignaling in inﬂammation. A major physiological ligand of L-selectin is thought tobe sialyl-LewisX (sLeX) as described in a later section. In addition to this ligand,L-selectin binds inner a1-3 linked mannose residues of high-mannose-type glycan.
Interestingly, the binding mode is different from that of DC-SIGN. Crystal structures of N-glycosylated L-selectin were reported by two groups (Wedepohl et al.
2017; Mehta-D’souza et al. 2017). In these structures, L-selectin tightly binds to theN-glycan (Man5GlcNAc2) from the symmetry related L-selectin molecule via thesugar-binding site. The mannose at a1-3 branch (Man-4) resides in the primarybinding site and the adjacent sugar residues, GlcNAc, b-mannose (Man-3) and a1-6
branched mannose (Man-4’), interact with L-selectin via the secondary binding site
(Fig. 3b).
(I-3) Mana1-6Man recognition
To date there is only one example showing the binding mode of a1-6 linked
mannose. The 3D structure is obtained from an MBP-A-Man5 (Mana1-2Mana1-3[Mana1-3Mana1-6]Man) complex (PDB code: 2MSB). Remarkably, one
high-mannose -type glycan bridges two MBP-A molecules (discussed in the next
section). The Man-B residue at the non-reducing end of the Mana1-6Man unit
binds to MBP-A. However, there is no apparent interaction between MBP-A andthe mannose at the reducing side (Fig. 3b).
(I-4) Cross-linking by high-mannose-type glycan
High-mannose-type glycan can function as a multivalent ligand for C-type lectins.
Three cross-linked structures have been reported so far (Fig. 3c). Two out of three
are MBP-A-high-mannose-type glycan complexes, while the other structure is
Dectin-2-high-mannose-type glycan complex. Crystal structure of an MBP-A-Man5(Mana1-2Mana1-3[Mana1-3Mana1-6]Man) complex (PDB code: 2MSB) showsthat one high-mannose-type glycan is simultaneously recognized by two MBP-A
molecules (Weis et al. 1992). One MBP-A molecule binds to the Mana1-2Man unit

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M. Nagae and Y. Yamaguchiof the a1-3 branch (especially the Man-C residue), and the other MBP-A moleculebinds to the Mana1-3Man unit of the a1-6 branch (Man-A, left panel in Fig. 3c). In
both molecules, non-reducing mannose residues occupy the primary binding sites.
The other complex, MBP-A-Man6GlcNAc2 complex, shows that one high-mannose
glycan bridges two MBP-A trimers (Ng et al. 2002). In this MBP-A complex, one
MBP-A molecule interacts with the Mana1-2Man unit, and the other MBP-A
molecule interacts with the Mana1-6Man units of the a1-6 branches (especially the
Man-B residue, middle panel in Fig. 3c). Comparing the two MBP-A cross-linked
structures, all four MBP-A molecules recognize Man-C residues at the
non-reducing end.
The Dectin-2-high-mannose-type glycan complex differs from the MBP-A
complexes in terms of glycan binding mode. A crystal structure of Dectin-2 incomplex with Man9 glycan shows that two Dectin-2 molecules sandwich one
high-mannose-type glycan (Feinberg et al. 2017). One Dectin-2 recognizes the
Mana1-2Man unit (Man-C and D1 residues) of the D1 arm, and the other Dectin-2
molecule interacts with the same disaccharide unit (Man-A and D2 residues) of theD2 arm (right panel in Fig. 3c). The interaction modes of the two Dectin-2
molecules are the same. The reducing end of the disaccharide resides in the primarybinding site, while the non-reducing end is located at the secondary binding site.
The recognition mode of inner mannose residues seems suitable for binding fungal
mannans which have variable structures at their non-reducing ends.
(II) Complex-type N-glycan recognition via mannose
Complex-type N-glycan is synthesized from high-mannose-type glycan by a seriesof enzymatic processes (Fig. 3a). Several C-type lectin receptors encoded in the
Dectin-2 cluster on the natural killer gene complex preferentially interact withcomplex-type N-glycans, such as blood DC antigen 2 (BDCA-2), human DC
immunoreceptor (hDCIR), and murine DCIR2 (mDCIR2) (Fig. 1a). These C-type
lectins share high amino acid sequence identities; however, their ligand preferences
are slightly different.
BDCA-2 binds to galactose-terminated biantennary glycans, deﬁning an epitope
found on a limited number of bi- and triantennary glycans (Riboldi et al. 2011).
Unusually, BDCA-2 with an EPN motif binds galactosylated glycan. A crystal
structure shows why. BDCA-2 primarily recognizes a mannose residue of thetrisaccharide unit (Galb1-4GlcNAcb1-2Man) with a serine residue (S139) andadditional interactions deﬁne the speciﬁcity between BDCA-2 and Galb1-4GlcNAc(Fig. 3d (Jegouzo et al. 2015)).
In contrast, human DCIR (hDCIR) binds to GlcNAc-terminated biantennary Nglycan. The binding mode of hDCIR toward the disaccharide unit
(GlcNAcb1-2Man) is similar to that of BDCA-2 [Fig. 3d, (Nagae et al. 2016)].
However, the serine residue (S139) interacting with galactose in BDCA-2 is notconserved in hDCIR (A162). This indicates that the speciﬁcity is deﬁned by theadditional interaction with the terminal glycan residue.
BDCA-2 and hDCIR can interact with both a1-3 and a1-6 arms of biantennarycomplex-type N-glycan. mDCIR2 has unique speciﬁcity toward GlcNAc-

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17

terminated biantennary glycan with bisecting GlcNAc (Nagae et al. 2013).
mDCIR2 shows arm preference, and the galactosylation of the a1-3 branch strongly
inhibits binding. A crystal structure of a mDCIR2-bisected glycan complex
demonstrated that mDCIR2 strictly recognizes the disaccharide unit (GlcNAcb12Man) of the a1-3 arm as well as bisecting GlcNAc (Fig. 3d). The binding mode of
GlcNAcb1-2Man is similar to those of BDCA-2 and hDCIR. However, aspartate(D223) tightly interacts with the bisecting GlcNAc (Fig. 3d), which is not conserved in BDCA-2 or hDCIR. Due to the simultaneous interaction with both
GlcNAcb1-2Man and bisecting GlcNAc, mDCIR2 selects the a1-3 branch. Suchsimultaneous interaction is impossible using the a1-6 branch because it is locatedslightly too far from the bisecting GlcNAc.
As described in the previous paragraph, DC-SIGN (and DC-SIGNR) preferentially interact with high-mannose-type glycans and 3D structures of DC-SIGN havebeen reported bound to high-mannose glycan. Additionally, DC-SIGN and
DC-SIGNR bound to complex-type glycans have been reported (Feinberg et al.
2001). The interaction modes of these receptors are different from those of
BDCA-2, hDCIR, and mDCIR2. The mannose residue of the a1-3 branch (Man-4)
coordinates a calcium ion, but the orientation is ﬂipped compared with themDCIR2-bisected glycan complex (Fig. 3e). The GlcNAc residue of the a1-3
branch is located away from the secondary binding site due to this ﬂipping and thea1-6 branch is located on the surface of DC-SIGN. The mannose (Man-4’) and
GlcNAc residues of the a1-6 branch form hydrogen bonds with DC-SIGN.
The bivalve lectin, codakine, from Codakia orbicularis binds a biantennarycomplex-type glycan (Gourdine et al. 2008). The binding mode of codakine issimilar to that of DC-SIGN rather than mDCIR2 (Fig. 3e). The a1-3 branchedmannose (Man-4) makes coordination bonds with a calcium ion and the a1-6
branch interacts with codakine via its secondary binding site.
A crystal structure of glycosylated porcine SP-D demonstrates that thesugar-binding site of SP-D accepts complex-type N-glycan attached onsymmetry-related SP-D (van Eijk et al. 2018). The interaction mode is similar to
those of DC-SIGN and codakine. The a1-6 branch is strongly kinked, possibly due
to crystal packing (Fig. 3e).

4.1.2

Oligosaccharide Recognition of EPN Motif-Containing C-Type
Lectins Through Fucose

C-type lectins primarily recognize mannose residues of high-mannose andcomplex-type N-glycans. In contrast, several C-type lectins such as selectins recognize the OH3 and OH4 groups of the fucose residue in sialyl-LewisX (sLeX,
Neu5Aca2-3Galb1-4[Fuca1-3]GlcNAcb1-R). sLeX is a terminal component of Nand O-glycans on hematopoietic and endothelial cells. L-selectin prefers sLeX
modiﬁed with sulfate on the GlcNAc residue. Selectins are expressed on vascularendothelium, platelets, or leukocytes and bind to cell surface glycoproteins harboring sLeX glycans such as PSGL-1. Upon ligand binding, selectins show the

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(a)

(b)

(c)

(d)

catch-bond behavior which is essential for initial tethering and rolling along thevascular endothelium and subsequent ﬁrm adhesion (Kansas 1996).
Crystal structures of E- and P-selectins complexed with sLeX have been reported(Somers et al. 2000; Preston et al. 2016). These lectins recognize a fucose residue at138Structural Aspects of Carbohydrate Recognition Mechanisms …

19

JFig. 4 a Sialyl-LewisX recognition by P- and E-selectins. Bent conformations (low afﬁnity state)of P-selectin (PDB code 1G1R) and E-selectin (PDB code 1G1T) are shown in left panel. Extend
conformations (high afﬁnity state) of P-selectin (PDB code 1G1S) and E-selectin (PDB code
4CSY) are shown in right panel. Two amino acid residues (N83 and E88) which take different
positions in two conformations are shown in stick models. b Structural superposition of
E-selectin-sialyl-LewisX complex (PDB code 4CSY, left panel) and DC-SIGN-LNFP3 complex(PDB code 1SL5, right panel). c Structural comparisons between E-selectin (PDB code 4CSY),
MBP-A K3 mutant (PDB code 2KMB) and Langerin (PDB code 3P5G) in complexes withfucose-containing glycans. d Aglycon recognition observed in P-selectin-PSGL-1 complex (PDB
code 1G1S). The interactions between P-selectin and sulfated tyrosine residues of PSGL-1 arehighlighted and labeled. The P-selectin molecule is shown in surface model

the primary binding site and form additional interactions with adjacent residues(GlcNAc, Gal, and Neu5Ac). A structural comparison of four structures shows thatthe positions of fucose, GlcNAc, and Gal are well superimposable, while theposition of the terminal Neu5Ac is variable (Fig. 4a). Interestingly, a 3D structuraldifference is observed between sLeX co-crystallized and soaked complexes. In thesoaked complexes, asparagine (N83) makes coordination bonds with the calciumion (left panel in Fig. 4a). In contrast, glutamate (E88) makes coordination bonds
with the calcium ion in co-crystallized complexes (right panel in Fig. 4a). Thisdifference causes a positional shift of the ﬂexible loop, leading to a global conformational change from bend (low afﬁnity) to extend (high afﬁnity) forms. The
glutamate is therefore a key residue in stabilizing the high afﬁnity conformation(Mehta-D’souza et al. 2017).
The structure of L-selectin in complex with high-mannose-type glycan is similar
to the co-crystallized complex, rather than the sLeX soaked complex, even though it
assumes a bend conformation (Fig. 3b). The glutamate coordinates with the calcium ion and the ﬂexible loop takes a similar position as in the co-crystallized
complex.
Other C-type lectins, such as DC-SIGN, MBP-A mutant, and langerin, also bindto fucose-containing glycans. A crystal structure of DC-SIGN in complex withlacto-N-fucopentaose III (Galb1-4[Fuca1-3]GlcNAcb1-3Galb1-4Glc) shows thatthe position of the fucose coincides well with those of selectins. However, thepositions of GlcNAc and galactose are slightly different. This subtle difference may
be derived from differences in the secondary binding site of DC-SIGN (Fig. 4b).
The introduction of a triple mutation (K211-K212-K213) in MBP-A (K3mutant) enables it to accept a series of LewisX glycans as P- and E-selectins do (Ngand Weis 1997). However, the binding mode is quite different from those of
selectins and DC-SIGN (middle panel in Fig. 4c). In this case, 2-OH (equatorial)and 3-OH (equatorial) groups of the fucose make coordination bonds with thecalcium ion. One of three lysine residues (K211) makes a hydrogen bond with thegalactose residue.
The fucose recognition mode of langerin is also different from those of selectinsand DC-SIGN (Feinberg et al. 2011). In the structure of langerin-blood group B
trisaccharide (Gala1-3[Fuca1-2]Gal) complex, 2-OH and 3-OH groups of thefucose residue also coordinate with the calcium ion (right panel in Fig. 4c).

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The aglycon moiety of PSGL-1 contributes to the speciﬁc interaction withselectins. Of particular note, the physiological interaction between human P-selectin
and PSGL-1 requires both sLeX capped core 2 O-glycan and one or more sulfatedtyrosine residues in the N-terminal region of PSGL-1. In the crystal structure(Somers et al. 2000), human P-selectin recognizes both the sLeX attached to threonine and the sulfated tyrosine residues of human PSGL-1 (Fig. 4d). P-selectin also
binds to the N-terminus of murine PSGL-1, although the sequence is different fromhuman PSGL-1. Cell-based biochemical assays suggest that sulfation of tyrosine(Y13) and the O-glycan on T17 are necessary for murine PSGL-1 to bind optimallyto P-selectin (Xia et al. 2003). The spacing of these residues in the sequence isconsiderably closer than the corresponding residues in the human PSGL-1 sequence(Y7, Y10 and T16). It is likely that murine PSGL-1 binds to P-selectin using a
different conformation of the polypeptide.

4.1.3

Oligosaccharide Recognition of EPN Motif-Containing C-Type
Lectins Through Glucose

A representative glucose speciﬁc C-type lectin is Macrophage inducible
calcium-dependent lectin (Mincle), also known as CLEC4E. Mincle is expressed on
macrophages and interacts with trehalose-6-6′-dimycolate (TDM), a glycolipid
found on the surface of Mycobacterium tuberculosis (Matsunaga and Moody 2009;
Ishikawa et al. 2009). TDM comprises a trehalose (Glca1-a1Glc) headgroup andtwo complex branched and hydroxylated acyl chains. The acyl chains are attachedto the 6-OH groups of each of the sugar residues. Crystal structures of bovine
Mincle C-type lectin domains in complexes with a series of ligands allow visualization of the interaction modes with glycolipids. A Mincle–trehalose complex
structure shows that both glucose residues interact tightly with Mincle. The 3-OH
and 4-OH of one glucose coordinate with the calcium ion and the second glucose
contacts a second binding site via hydrogen bonds (Feinberg et al. 2013) (Fig. 5a).
The additional interaction of the aglycon moiety, such as the lipid part, should
greatly improve the speciﬁcity. The Mincle–trehalose monobutyrate complex
shows that the alkyl chain is located near the hydrophobic groove of Mincle(Feinberg et al. 2016). These hydrophobic residues likely form the extended
binding site for the lipid moiety.
In addition to Mincle, langerin, SP-D, and SIGN-R1 can bind glucose residues attheir primary binding sites. The structure of a langerin-laminaritoriose(Glcb1-3Glcb1-3Glc) complex contrasts markedly with the Mincle–trehalosecomplex structure. In the langerin complex, the 1-OH and 2-OH of the glucose at
the reducing end resides in the primary binding site and the other glucose at the
non-reducing end points away towards solvent (Feinberg et al. 2011) (Fig. 5b). The
langerin–maltose (Glca1-4Glc) complex reveals that the 3-OH and 4-OH of glucose
at the reducing end also coordinate with the calcium ion, but the orientation is
totally ﬂipped compared with that in the Mincle–treharose complex (Chatwell et al.
2008). The glucose at the non-reducing end is also exposed to solvent (Fig. 5c).

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(a)

(b)

(c)

(d)

(e)

(f)

Fig. 5 Glucose recognition mechanism of EPN motif-containing C-type lectins: a Mincletrehalose complex (PDB code 4ZRW). b Langerin-laminaritriose complex (PDB code 3P5H).
c Langerin-maltose complex (PDB code 3P7H). d SP-D-maltose complex (PDB code 3P7H).
e Dextran sulfate recognition of SIGN-R1 (PDB code 4C9F). Four complexes in the asymmetricunit are superimposed. f Two sugar-binding sites (top face and side face) of SIGN-R1 (PDB code
4C9F)

In contrast, the orientation of glucose in a SP-D-maltose complex is the same asin the Mincle complex (Fig. 5d). A crystal structure of SIGN-R1 in complex witholigo-dextran sulfate (a1-3 and a1-6 linked glucose polymer with sulfation)
demonstrates a somewhat unusual binding mode (Silva-Martin et al. 2014). The
glucose at the primary binding site is positioned differently compared with typicalC-type lectins. Although the top face of the lectin accepts at least four glucose
residues, only 4-OH of Glca1-6Glc is located within the coordination bond in the
primary binding site (Fig. 5e). In the case of sulfated glucose, ring oxygen (O5)seems to make a coordination bond with the calcium ion. It is noteworthy that“side” face of SIGN-R1 can accept the repetitive molecular patterns of thepolysaccharide chain (Fig. 5f). This binding mode seems favorable for an interaction of a small globular domain with long polysaccharide chains.

4.2

Oligosaccharide Recognition of QPD Motif-Containing
C-Type Lectins

QPD motif-containing C-type lectins have been less studied than those with EPN.
Only three structures, Rattlesnake venom lectin (RSL) in complex with lactose(Walker et al. 2004), asialoglycoprotein receptor (ASGPR) lactose complex

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M. Nagae and Y. Yamaguchi

(b)

(c)

Fig. 6 Carbohydrate recognition of QPD motif-containing C-type lectins: a RSL-lactose complex(PDB code 1JZN). b ASGPR-lactose complex (PDB code 5JQ1) and c SCARA4-LewisX complex(PDB code 2OX9)(Sanhueza et al. 2017) and scavenger receptor C-type lectin (SRACLA4) in complex with LewisX trisaccharide (Fuca1-3[Galb1-4]GlcNAc) (Feinberg et al. 2007),
are deposited in the PDB. In these structures, the 3-OH and 4-OH groups ofgalactose form coordination bonds with the calcium ion and the apolar face of thegalactose is stabilized by a stacking interaction with hydrophobic amino acidresidues (Fig. 6a–c). RSL recognizes only the galactose residue of lactose. In
contrast, the bOH1 group of the glucose residue makes a hydrogen bond witharginine (R236) in an ASGPR-lactose complex (Fig. 6a and b). In the
SRACLA4-LewisX complex, the fucose residue makes additional hydrophobic
contact with isoleucine (I712) and hydrogen bonds with lysine (K691) (Fig. 6c).
A structural comparison among the three complexes clariﬁes that the aromatic
residues (Y100 in RSL, W243 in ASGPR, and W698 in SRACLA4) are located inequivalent positions and positively charged residues (R236 in ASGPR and K691 in
SRACLA4) occupy similar positions in two of the complexes, evidently engaged inligand recognition.
It is tempting to compare the recognition modes of LewisX by the EPN and QPD
motifs. In EPN motif-containing C-type lectins, fucose coordinates a calcium ion in
the primary binding site and galactose resides in the secondary binding site (Fig. 4a,b). In the case of the QPD motif, by contrast, galactose resides in the primarybinding site and fucose is located in the secondary binding site. Interestingly, theconformation of the LewisX trisaccharide is similar in both complexes. Thisobservation suggests that both lectins recognize a stable conformation of the glycan.
Glycan array analysis revealed that SRCL preferentially binds to Lewisa andLewisX, while DC-SIGN widely accepts various types of glycan such as Lewisa,
Lewisb, LewisX, and LewisY (Feinberg et al. 2007; Guo et al. 2004). This may
originate from differences in the binding modes of galactose/fucose.

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4.3

23

Oligosaccharide Recognition of C-Type Lectin-like
DomainsC-type lectin-like domains do not bind calcium due to the lack of conserved calcium binding motifs. However, some C-type lectin-like domains directly bind tocarbohydrates in calcium independent ways. The sugar-binding modes of C-type
lectin-like domains are thus expected to be completely different from those of
typical C-type lectin domains and can be expected to be diverse.
Dectin-1 is a C-type lectin-like receptor having a single extracellular C-type
lectin-like domain, a short stalk region, a single transmembrane helix and a cytoplasmic ITAM. Dectin-1 is a b-glucan receptor and shows preference for the b1-3
linked glucose polymer (Palma et al. 2006). Since the C-type lectin-like domain of
Dectin-1 lacks a QPD or EPN motif, it loses its calcium binding ability at the
primary site. A crystal structure of Dectin-1 complexed with laminaritriose showsthat two C-type lectin domains sandwich one laminaritriose via their lateral faces(Brown et al. 2007) (Fig. 7a). Laminaritiose assumes a planar conformation and isstabilized by several hydrogen bonds. It should be noted that mutational experiments suggested that the top face of the Dectin-1 C-type lectin-like domain contributes to b-glucan binding, which is located far from the binding site observed in
the crystal structure (Dulal et al. 2018). Direct evidence, as for example solution
NMR analysis, is strongly needed to solve this discrepancy.
Another example is C-type lectin like receptor 2 (CLEC-2), also known as
CLEC1B. CLEC-2 is a type II transmembrane receptor with a short N-terminal
cytoplasmic tail containing a single tyrosine-based activation motif (hemITAM), atransmembrane segment, an extracellular stalk region and a C-type lectin-likedomain which has no calcium binding activity. Podoplanin is a transmembrane Oglycoprotein that binds to CLEC-2 in a glycosylation-dependent manner.

(a)

(b)

(c)

Fig. 7 Ca2+-independent carbohydrate recognition of C-type lectin-like domain: a Dectin-1laminaritoriose complex (PDB code 2CL8). b CLEC-2-O-glycosylated (Neu5Aca2-6[Galb1-3]
GalNAc-O-Thr52) podoplanin complex (PDB code 3WSR) and c CLEC-2-rhodocytin complex(PDB code 3WWK). Carbohydrate, Glu-Asp motif, and C-terminal Y136 are shown in stickmodels143

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M. Nagae and Y. Yamaguchi

Crystallographic analysis revealed that two consecutive acidic residues (Glu-Asp
motif) as well as an a2-6 linked sialic acid residue attached on the O-glycan interact
with the lateral face of CLEC-2 (Nagae et al. 2014). Four arginine residues of
CLEC-2 interact with the sialic acid and the two acidic residues of podoplanin(Fig. 7b). Interestingly, snake venom rhodocytin also binds to CLEC-2, however
the interaction mode is somewhat different. Although the Glu-Asp motif is conserved in rhodocytin, rhodocytin is not O-glycosylated. Instead, the carboxylate of
the rhodocytin C-terminus contributes to an electrostatic interaction with the arginine residue of CLEC-2 (Fig. 7c).

4.4

Genetic Variants of C-Type Lectins

Speciﬁc sugar recognition is affected by changes in amino acid residues.
Single-nucleotide polymorphisms (SNPs) and genetic variations are potentiallyinvolved in the susceptibility for developing disease and disease outcomes. Various
SNPs and disease causative mutations of C-type lectins have been reported (Goyal
et al. 2016). The effects of SNPs and species-dependent sugar binding are not wellstudied and are limited to a few examples. In human langerin, the W264R mutation
was found in an individual who lacks Birbeck granules in the Langerhans cells(Verdijk et al. 2005). This mutation results in the loss of mannose-binding ability(Ward et al. 2006). W264 is located inside the protein, and thus this mutation
destabilizes the local folding of langerin. At present several SNPs, A278 V,
N288D, A300P, and K313I, in the human langerin C-type lectin domain aredeposited in the SNP database (Ward et al. 2006; Feinberg et al. 2013). Of note,
K313 is one of the two lysine residues which are critical for recognition of sulfatedgalactose (Fig. 2c). The afﬁnity for sulfated galactose is dramatically reduced,
instead, the afﬁnity against terminal GlcNAc is increased (Feinberg et al. 2013).
Surfactant protein A (SP-A) consists of two isoforms, SP-A1 and SP-A2,
encoded by separate genes. SP-A defends against invading pathogens in the lung.
The missense mutations of SP-A2 C-type lectin domain result in idiopathic pulmonary ﬁbrosis (IPF), a serious lung disease affecting older adults (Wang et al.
2009). These mutations, like F198S and G231 V, dramatically reduce the expression level of the protein probably due to the disruption of normal protein folding. In
addition, the K223Q mutation in the C-type lectin domain is one of the SNPs which
are signiﬁcantly associated with tuberculosis (Malik et al. 2006). This replacement
could affect the glycan recognition of SP-A2, though as yet the mechanism has notbeen revealed.
For SP-D, three SNPs, M11T, A160T, and S270T, have been reported(Leth-Larsen et al. 2005). These residues are located in the signal peptides ofcollagen-like and C-type lectin domains. The S270T mutant is located on the
opposite side of the sugar binding site and the effect of the mutation on sugar
binding is obscure. Another example is Dectin-1. The Y238X mutation of Dectin-1
is found in Africans and western Eurasians (Ferwerda et al. 2009). The Y238X

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25

mutant is poorly expressed and lacks b-glucan activity. Y236 in mouse Dectin-1,
corresponding to human Y238, is buried inside the C-type lectin domain. Thus, themutation likely inhibits proper folding. Although the binding of b-glucan is signiﬁcantly lower in Y238X patients, fungal phagocytosis and fungal killing arenormal, suggesting the presence of alternative receptors for phagocytosis.
The species-dependent sugar recognition mechanism has also been investigated.
Human and murine langerin share 66% amino acid similarity, but show different
ligand preferences for bacterial polysaccharides (Hanske et al. 2017). A crystal
structure of murine langerin shows that the different residues map to the secondary
binding site, which possibly accounts for the different ligand speciﬁcities.

5 Functional Oligomerization of C-Type Lectin Domains
C-type lectin and lectin-like domains often form stable homo- or heterodimers thatare critical for their physiological functions. For example, C-type lectin-likedomains of NK receptors (Group 5) such as Ly49s and NKGs accept various
ligands via homo or heterodimers (Li and Mariuzza 2014). The C-type lectin
domain itself forms a monomer in solution. However, many sugar-binding C-type
lectins such as collectin and DC receptor families form multimers via their stalk orcoiled-coil regions. Biochemical studies of murine Dectin-1 suggests that monomeric C-type lectin-like domain cooperatively forms an oligomer upon b-glucan
binding (Dulal et al. 2018).
Domain swapping is a mechanism for two or more protein molecules to form adimer or higher oligomer by exchanging an identical structural element (Liu andEisenberg 2002). Oligomerization by domain swapping is often found in snake
venom C-type lectin-like proteins (Eble 2019). In these proteins, the long loop
between b2 and b3 strands extends away from the core of the protein to form adomain-swapped dimer. Various snake venom C-type lectin-like proteins form
ordered heterooligomers via domain-swapping. Interestingly, domain-swapped
heterodimers, bitiscetin, botrocetin, and rhodocetin, functionally grab their target
platelet receptors such as von Willebrand factor (vWF) A1 domain, GPIba, GPVI,
and a2b1 integrin via an extended surface formed by an extended loop region(Maita et al. 2003; Fukuda et al. 2005; Eble et al. 2017).
In mammalian C-type lectin receptors, domain-swapped dimers can form undercrystallization conditions. Crystal structures of several C-type lectin receptors arereported as domain-swapped dimers in a ligand-free state, such as CRD-4 ofmacrophage mannose receptor (Feinberg et al. 2000), BDCA-2 (Nagae et al. 2014),
and NKRp1a (Kolenko et al. 2011). These domain-swapped dimers are similar to
those of snake venom C-type lectin-like proteins. An interesting point is that thefunctional states of BDCA-2 and NKRp1a are monomeric and the domain-swapped
dimers are thought to be an inactive state.
Of note, the BDCA-2 C-type lectin domain forms a domain-swapped dimer
under three different crystallization conditions (Nagae et al. 2014). Although 1 mM

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calcium chloride was present during protein puriﬁcation, no calcium ion was foundin the calcium binding site. The side chain of E178, which is critical for calcium
coordination, points away from the putative calcium binding site. This indicates thatthe domain-swapped BDCA-2 cannot accept a carbohydrate ligand. Subsequently, acrystal structure of BDCA-2 in complex with trisaccharide was reported. BDCA-2
is a monomer in the crystal and binds the sugar at the calcium binding site (Jegouzo
et al. 2015).
In the case of NKRp1a, the extended loop points away from the central core andmediates formation of a domain-swapped dimer in the crystal (Kolenko et al. 2011).
Although the refolding buffer contains a high concentration of calcium chloride,
there is no calcium ion in the structure. In contrast, a solution structure determined
by NMR is monomeric with the loop tightly anchored to the central region(Rozbesky et al. 2016). Calcium titration analysis suggests monomeric NKRp1abinds a calcium ion weakly with a dissociation constant in the mM range.
Although the physiological relevance of domain-swapped dimer formation isstill obscure, the domain-swapped dimer is likely a metastable conformation.
Formation of the domain-swapped dimer may be used as a temporary inactive state
under certain conditions.

6 Conclusion and Future Perspective
C-type lectins accept various glycans via highly conserved binding sites. Although
the primary binding site is the most conserved, there are differences in the secondary binding site that enable various types of glycans to bind. Furthermore,accumulating of 3D structural information demonstrates that C-type lectin-likedomains also recognize sugar ligands in a calcium-independent manner. The relationship between SNP and sugar-binding activity has a direct bearing on the susceptibility for pathogens, and however, the mechanism is largely unknown.
Structural analysis of C-type lectins from individuals is a promising avenue to ourunderstanding of individual variations in the immune system.

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