ᚑくᄢቇℂᎿቇ⎇ⓥႎ๔
J. Fac. Sci. Tech., Seikei Univ.
Vol.46 No.2 (2009) pp.91-97
㧔․೎⎇ⓥ⾌ߦଥࠆ⺰ᢥ㧕♧㎮⏛᳇ࡆ࡯࠭ࠍ↪޿ߚ♧㎮⹺⼂࠲ࡦࡄࠢ⾰ߩหቯᚭ⼱ Ꮧ৻㇢*1㧘⮥↰ ⚡ਫ*2

Identification of Glycan-recognizing Proteins Using Oligosaccharide Immobilized Magnetic Beads
Kiichiro TOTANI*1㧘Shino KURATA*2
ABSTRUCT :Oligosaccharides play important roles in various biological events, such as virus infection,immunity, disease, blood type, protein quality control etc.In these processes, the glycan recognizing
proteins so called lectins are known as regulatory factors especially in a case of weak flexible
binding-dissociation of an oligosaccharide ligand. In order to select unidentified lectin candidates from
biological extracts, development of a mild enrichment procedure using oligosaccharide immobilized
magnetic beads was conducted. The mild enrichment was found to effective compared to a conventional
batch procedure in a model interaction experiment between maltose-magnetic beads and concanavalin A.
Furthermore, selections of Glc1Man9GlcNAc2-specific lectin candidates from mouse liver microsomal
extracts were carried out.
Keywords :lectin, synthetic oligosaccharide, magnetic beads(Received September 24, 2009)㧝ߪߓ߼ߦࠢ⾰ࠍ࿕ቯൻߒߚࠕࡈࠖ࠾࠹ࠖ࡯ࡆ࡯࠭ߣ↢૕᛽಴ᶧࠍ
෻ᔕߐߖ㧘࠲ࡦࡄࠢ⾰ᷙวᶧਛߩ࡟ࠢ࠴ࡦࠍๆ⌕ߐߖࠆ ♧㎮ߪ♧࠲ࡦࡄࠢ⾰߿♧⢽⾰ߩ᭴ᚑᚑಽߣߒߡ⚦⢩ౝᣇᴺߪ㧘ቯ㊂ᕈߪߥ޿߇࡟ࠢ࠴ࡦ↹ಽࠍࠛࡦ࡝࠶࠴ߢ߈߿⚦⢩⴫㕙ߦᐢߊሽ࿷ߒ㧘᭽‫↢ߥޘ‬๮⃻⽎ߩ೙ᓮࠍᜂߞ
ߡ޿ࠆ 1)‫♧ޕ‬㎮߇㑐ࠊࠆ↢๮⃻⽎ߣߒߡ࠙ࠖ࡞ࠬᗵᨴ㧘ࠆߚ߼㧘ᧂ⍮ߩ࡟ࠢ࠴ࡦ୥⵬ࠍ⛉ࠅㄟ߻↪ㅜߦ᳢↪ߐࠇߡ޿ࠆ‫ޕ‬఺∉㧘∔ᖚ㧘ⴊᶧဳ㧘࠲ࡦࡄࠢ⾰ߩຠ⾰▤ℂߥߤ߇⍮ࠄ ࠕࡈࠖ࠾࠹ࠖ࡯ࡆ࡯࠭ߩ૶↪ᣇᴺߣߒߡ৻⥸⊛ߦࠞ࡜ࠇߡ޿ࠆ߇㧘ᄙߊߩ႐ว㧘ߘߩᵴേߦߪ♧㎮⹺⼂࠲ࡦࡄࠢ⾰ߢ޽ࠆ࡟ࠢ࠴ࡦ 2)߇㑐ࠊߞߡ޿ࠆ‫ⷰߥ߁ࠃߩߎޕ‬ὐ

ࡓᴺߣࡃ࠶࠴ᴺ߇⍮ࠄࠇߡ޿ࠆ‫ޕ‬Ყセ⊛ᄢ㊂ߩ࡟ࠢ࠴ࡦ
ࠍ♖⵾ߔࠆߦߪ㧘ᠲ૞ࠍ⥄േൻߢ߈ࠆࠞ࡜ࡓᴺ߇ఝࠇߡ߆ࠄᧂ⍮ߩ࡟ࠢ࠴ࡦࠍត⚝ߒ㧘ߘߩᯏ⢻ࠍ᣿ࠄ߆ߦߔࠆ
޿ࠆ߇㧘ࡈࡠ࡯♽ߢ↪޿ࠆߚ߼ࠕࡈࠖ࠾࠹ࠖ࡯ࡆ࡯࠭ߩ
ߎߣߪ㧘♧㎮↢๮⃻⽎ࠍℂ⸃ߔࠆ਄ߢ㊀ⷐߥ⺖㗴ߣߥߞᔅⷐ㊂ߪᄙߊߥࠆ‫৻ޕ‬ᣇ㧘ࡃ࠶࠴ᴺߪᚻ૞ᬺࠍⷐߔࠆ߇㧘ߡ޿ࠆ‫ޕ‬෻ᔕኈེౝߢ♧㎮ࡆ࡯࠭ߣ࡟ࠢ࠴ࡦࠍ㐳ᤨ㑆ធ⸅ߐߖࠆ ޽ࠆ․ቯߩ♧㎮ߣ࡟ࠢ࠴ࡦߩ⋧੕૞↪⸃ᨆᴺߣߒߡ㧘ߎߣ߇ߢ߈ࠆߚ߼ๆ⌕ല₸߇㜞ߊ㧘ࠕࡈࠖ࠾࠹ࠖ࡯ࡆ࡯ࠞࡠ࡝ࡔ࠻࡝࡯ᴺ㧘ᩭ⏛᳇౒㡆ᴺ㧘⴫㕙ࡊ࡜࠭ࡕࡦ౒㡆࠭ߩᔅⷐ㊂߇ዋߥߊߡߔ߻․ᓽ߇޽ࠆ‫♧ޕ‬㎮࡝ࠟࡦ࠼߇ᴺ㧘᳓᥏⊒ାሶࡑࠗࠢࡠࡃ࡜ࡦࠬᴺ㧘ࡈࡠࡦ࠲࡞ࠕࡈࠖ
⾆㊀ߥ႐วߦߪ㧘ࡃ࠶࠴ᴺߪ᦭ലߥㆬᛯ⢇ߣߥࠆ‫߆ߒޕ‬࠾࠹ࠖ࡯ࠢࡠࡑ࠻ࠣ࡜ࡈࠖ࡯ᴺߥߤ߇Ყセ⊛ࠃߊ↪޿ࠄߒߥ߇ࠄㅢᏱߩࠕࠟࡠ࡯ࠬ߿࡜࠹࠶ࠢࠬ⵾ߩࡆ࡯࠭ߦኻࠇࠆ‫ߪࠄࠇߎࠄ߇ߥߒ߆ߒޕ‬㧘․ቯߩ࡟ࠢ࠴ࡦߣߩ⋧੕૞↪⸃ᨆߦߪ᦭ലߢ޽ࠆ߇㧘ᧂ⍮࡟ࠢ࠴ࡦࠍࠛࡦ࡝࠶࠴ߔࠆ㆙ᔃᠲ૞ߦࠃࠆ࿁෼ߢߪ㧘100%ߩࡆ࡯࠭࿁෼ߪᦸ߼ߥ޿‫ߢߎߘޕ‬㆙ᔃߦ㗬ࠄߥ޿ࡆ࡯࠭࿁෼ᣇᴺߣߒߡㄭᐕ㧘ߔࠆߩߦߪਇㆡᒰߢ޽ࠆ‫৻ޕ‬ᣇ㧘․ቯߩ♧㎮߿♧࠲ࡦࡄ*1 : ᚑくᄢቇℂᎿቇㇱ‛⾰↢๮ℂᎿቇ⑼ಎᢎ᝼

(Associate Professor, Dept of Materials and Life Science,e-mail: ktotani@st.seikei.ac.jp)*2 : ᚑくᄢቇℂᎿቇㇱ㧠ᐕ↢⏛᳇ࡆ࡯࠭߇೑↪ߐࠇࠆ႐㕙߇Ⴧ߃ߡ߈ߚ 3)‫⏛ޕ‬᳇ࡆ࡯࠭ߪน⏛ᕈ‛⾰ࠍ฽߻ࡐ࡝ࡑ࡯ࠦࠕࠍⷫ᳓ᕈࡐ࡝ࡑ࡯ߢⷒߞߚ߽ߩߢ޽ࠅ㧘ᒝജ⏛⍹ߦࠃߞߡ෻ᔕኈེߩᐩ㕙ߦ⏛᳇ࡆ࡯࠭ࠍ◲ଢ߆ߟ㜞ല₸ߦ࿁෼ߔࠆߎߣ߇ߢ߈ࠆ‫ޕ‬㧙㧙ㅢᏱߩࡃ࠶࠴ᴺߦࠃࠆ࡟ࠢ࠴ࡦߩࠛࡦ࡝࠶࠴Ꮏ⒟ࠍ࿑㧝ߩߣߎࠈ◲ଢߦ⋡⊛࡟ࠢ࠴ࡦࠍࠛࡦ࡝࠶࠴ߔࠆᚻᴺ߇⏕A ߦ␜ߒߚ‫․ޕ‬ቯߩ♧㎮ࠍ⚿วߒߚ⏛᳇ࡆ࡯࠭ࠍ૞ᚑߒ㧘┙ߒߡ޿ߥ޿‫♧ޕ‬㎮ߣ࡟ࠢ࠴ࡦߩ⚿วߪนㅒ⊛ߢ޽ࠅ㧘ߎࠇࠍ࡟ࠢ࠴ࡦ୥⵬߇฽߹ࠇࠆ࠲ࡦࡄࠢ⾰ᷙวᶧߣߣ߽⚿ว߇ᒝ޿႐วߦߪ߶߷ⶄว૕ߩ⁁ᘒߢሽ࿷ߒߡ޿ࠆ‫ޕ‬ߦࡑࠗࠢࡠ࠴ࡘ࡯ࡉౝߢォୟᷙ๺ߒߡ♧㎮⏛᳇ࡆ࡯࠭-ߎࠇߦኻߒ⚿ว߇ᒙ޿႐วߦߪᐔⴧ߇⸃㔌⁁ᘒߦ௑߈㧘࡟ࠢ࠴ࡦⶄว૕ࠍᒻᚑߐߖࠆ‫ⶄߩߎޕ‬ว૕ࠍ⏛⍹ߦࠃࠅⶄว૕๟ㄝߦ᦭ᗧߩ㊂ߩㆆ㔌࡟ࠢ࠴ࡦ߇ሽ࿷ߔࠆߎߣߦᴉ㒠ߐߖ㧘ਇⷐߥ࠲ࡦࡄࠢ⾰⟲ࠍ฽߻ᶧ⋧ࠍ㒰෰ߒ㧘߽ߥࠆ‫⁁ߩߎޕ‬ᘒߢㅢᏱߩࡃ࠶࠴ᴺߢߩᵞᵺࠍⴕ߁ߣㆆ㔌ߣߩᶧ㊂߹ߢࡃ࠶ࡈࠔ࡯ࠍട߃ࠆ‫ߩߘޕ‬ᓟ߰ߚߚ߮ォୟ࡟ࠢ࠴ࡦߪ㒰෰ߐࠇ㧘ߎࠇࠍ➅ࠅ㄰ߔߣᒙ޿⚿วߩ࡟ࠢᷙ๺㧘⏛⍹ߦࠃࠆᴉ㒠㧘ᶧ⋧ߩ㒰෰ࠍ➅ࠅ㄰ߔߎߣߦࠃ࠴ࡦߪᦨ⚳⊛ߦ߹ߞߚߊࠛࡦ࡝࠶࠴ߐࠇߡߎߥߊߥࠆ‫ޕ‬ߞߡ⏛᳇ࡆ࡯࠭ߦ㕖․⇣⊛ߦๆ⌕ߒߚ࠲ࡦࡄࠢ⾰ࠍᵞᵺߘߎߢᚒ‫♽ߪޘ‬ౝߦ♧㎮⏛᳇ࡆ࡯࠭ߣᒙ޿࡟ࠢ࠴ࡦ߇ሽ

ߒ㧘࡟ࠢ࠴ࡦࠍࠛࡦ࡝࠶࠴ߒߡ޿ߊ‫⊛⥸৻ߪࡦ࠴ࠢ࡟ޕ‬࿷ߒߚ႐วߩ࡟ࠢ࠴ࡦỚᐲಽᏓߦᵈ⋡ߒ㧘ᒙ޿⚿วࠍᵞߦ᛫૕ߥߤߦᲧߴࠆߣ⚿วജ߇ᒙ޿߇㧘ᄙߊߩᬀ‛࡟ࠢ޿ᵹߒߡߒ߹߁࡝ࠬࠢࠍシᷫߔࠆࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺ࠴ࡦ㘃ߪᲧセ⊛ᒝ޿⚿วജ (Ka = 10 to 10 M ) ࠍ᦭ߒࠍឭ᩺ߔࠆ㧔࿑㧝B㧕‫ߩߎޕ‬ᣇᴺߪ♧㎮⏛᳇ࡆ࡯࠭-࡟ࠢ࠴ߡ޿ࠆ‫ߦࡦ࠴ࠢ࡟ߥ߁ࠃߩߎޕ‬ኻߒߡߪ࿑㧝A ߦ␜ߔࡃࡦⶄว૕ࠍ⏛⍹ߢᴉ㒠ߔࠆᎿ⒟߹ߢߪㅢᏱߩࡃ࠶࠴ᴺߣ࠶࠴ᴺ߇᦭ലߦᯏ⢻ߔࠆ߇㧘േ‛ߩ⚦⢩ౝߦሽ࿷ߔࠆ⚿ห᭽ߢ޽ࠆ߇㧘ᶧ⋧ࠍ㒰෰ߔࠆ㓙ߦᒙ޿⚿วߩ࡟ࠢ࠴ࡦ

วജߩᒙ޿ (Ka = 104 to 103 M-1) ࡟ࠢ࠴ࡦߪ㧘ㅢᏱߩࡃ࠶

߇ࡆ࡯࠭๟ㄝߢ⚿ว-⸃㔌ߩᐔⴧࠍ➅ࠅ㄰ߒߡ޿ࠆߎߣ࠴ᴺߦ߅ߌࠆᵞᵺᎿ⒟ߢࡆ࡯࠭߆ࠄߪ߇ࠇ⪭ߜ㧘ࠛࡦ࡝ࠍ⠨ᘦߒߡ㧘ṁᶧߩඨಽ㊂ߩ਄Ẵߺࠍ㒰෰ߒߡࡔࠬࠕ࠶࠶࠴ߔࠆߎߣ߇࿎㔍ߢ޽ࠆ‫ߥ߁ࠃߩߎޕ‬ᒙ޿࡟ࠢ࠴ࡦᵴࡊߔࠆᵞᵺᣇᴺࠍ↪޿ࠆߎߣߢ㧘ߎࠇ߹ߢᵞ޿ᵹߐࠇߡᕈߪߎࠇ߹ߢ⷗ㆊߐࠇߡ߈ߚ߇㧘ㄭᐕ㧘ᒙ޿⚿วߎߘ߇޿ߚ࡟ࠢ࠴ࡦߩࠛࡦ࡝࠶࠴ࠍ⹜ߺࠆ‫ᧄޕ‬Ⓜߢߪࡑ࡞࠻࡯↢૕ᯏ⢻ࠍᨵエߦ⺞▵ߔࠆ㊀ⷐߥ࿃ሶߢ޽ࠆߣߩ⹺⼂߇ࠬ (Mal) ⏛᳇ࡆ࡯࠭ߣࠦࡦࠞ࠽ࡃ࡝ࡦ A (ConA) ࠍࡕ࠺㜞߹ࠅ㧘ᒙ޿࡟ࠢ࠴ࡦᵴᕈࠍᬌ಴ߔࠆᚻᴺߣߒߡࡈࡠࡦ࡞ߣߒߡࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩ᦭ലᕈࠍᬌ⸽ߒ㧘ߐࠄ

75-14)

࠲࡞ࠕࡈࠖ࠾࠹ࠖ࡯ࠢࡠࡑ࠻ࠣ࡜ࡈࠖ࡯ ᴺ߿ࡈࡠ࡯ࠨߦࡑ࠙ࠬዊ⢩૕↹ಽ߆ࠄߩᧂ⍮࡟ࠢ࠴ࡦត⚝ߦᔕ↪ߒߚ5)ࠗ࠻ࡔ࡯࠲࡯ࠍ೑↪ߒߚᚻᴺ ߇㐿⊒ߐࠇߡ޿ࠆ‫৻ޕ‬ᣇ㧘ߩߢႎ๔ߔࠆ‫ޕ‬ᒙ޿ᵴᕈࠍ␜ߔᧂ⍮ߩ࡟ࠢ࠴ࡦត⚝ࠍ⋡ᜰߔ႐ว㧘⃻࿷࿑㧝 ♧㎮⏛᳇ࡆ࡯࠭ࠍ↪޿ߚ࡟ࠢ࠴ࡦࠛࡦ࡝࠶࠴ᴺ㧦 # ㅢᏱߩࡃ࠶࠴ᴺ㧘 $ ࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺ㧙㧙mg/mL WGA, 0.6mg Mal-⏛᳇ࡆ࡯࠭,1 mM CaCl2, 0.5 mM㧞㧚ታ㛎ᣇᴺ

MnCl2, 0.5 M NaCl, 20 mMTris•HCl pH 7.4]ࠍ 1 mL ⺞ ᧄ ⎇ ⓥ ߢ ߪ ⏛ ᳇ ࡆ ࡯ ࠭ ߣ ߒ ߡ Invitorogen ␠ ⵾⵾ߒ㧘4 °C ߢ 60 ಽ㑆ォୟᷙ๺ᓟ㧘⏛⍹ߢ Mal-⏛᳇ࡆ࡯Dynabeads-M280 Tosylactivated ࠍ૶↪ߒߚ‫ߪࡦ࠴ࠢ࡟ޕ‬࠭ࠍᴉ㒠࡮࿁෼ߒߚ‫ޕ‬ṁᶧ⋧ߩ㒰෰㧘ࡃ࠶ࡈࠔ࡯ (1 mM
CaCl2, 0.5 mM MnCl2, 0.5 M NaCl, 20 mMTris•HCl pHSigma ␠⵾ ConA ߅ࠃ߮↢ൻቇࡃࠗࠝࡆࠫࡀࠬ␠⵾ዊ㤈7.4) 1mL ߦࠃࠆᏗ㉼㧘4 °C ߢ 5 ಽ㑆ߩォୟᷙ๺㧘⏛⍹⢦⧘࡟ࠢ࠴ࡦ (WGA) ࠍ૶↪ߒߚ‫࠲࡞࡯ࡠ࠻ࡦࠦߚ߹ޕ‬ߦࠃࠆࡆ࡯࠭ߩᴉ㒠ࠍ 10 ࿁➅ࠅ㄰ߒ㧘㕖․⇣⊛ߦๆ⌕ߒࡦ ࡄ ࠢ ⾰ ߣ ߒ ߡ SIGMA ␠ ⵾ ࠙ ࠪ ⴊ ᷡ ࠕ ࡞ ࡉ ࡒ ࡦ(BSA) ࠍ↪޿ߚ‫ࠬ࠙ࡑޕ‬ዊ⢩૕↹ಽߪ SAM ࡑ࠙ࠬ⎇ⓥߚ࠲ࡦࡄࠢ⾰ࠍᵞᵺߒ㧘Mal-⏛᳇ࡆ࡯࠭਄ߦ࡟ࠢ࠴ࡦࠍ

ද⼏ળ߆ࠄಽਈߐࠇࠆ SAMR1 ߅ࠃ߮ SAMP10 ࠍᣣᧄࠛࡦ࡝࠶࠴ߒߚ‫ޕ‬ߦࠃߞߡ⺞⵾ߒߚ‫⏛ޕ‬᳇ࡆ࡯࠭਄ߩ࿕ቯൻߔࠆ♧㎮ߪ᧲ࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߦࠃࠆ࡟ࠢ࠴ࡦߩㆬ೎㧔৻

SLC ␠⚻↱ߢಓ⚿⢄⤳ߣߒߡ౉ᚻߒ㧘⎕⎈ᓟ㧘㆙ᔃಽ↹⥸⊛ߥᚻᴺ㧕੩ൻᚑᎿᬺ␠⵾ࡑ࡞࠻࡯ࠬ߅ࠃ߮ൻቇวᚑߒߚ ࡑࠗࠢࡠ࠴ࡘ࡯ࡉౝߢ Mal-⏛᳇ࡆ࡯࠭ (0.6 mg) ࠍࡉGlc1Man9GlcNAc2 (G1M9) ဳ㜞ࡑࡦࡁ࡯ࠬ♧㎮ࠍ↪޿ࡠ࠶ࠢࠛ࡯ࠬ෻ᔕᶧ (1 mg/mL, 1 mL) ߣ 4 °C ߢ 60 ಽߚ‫ޕ‬㑆ಣℂᓟ㧘෻ᔕᶧࠍ㒰෰ߒߚ‫⴫ߦ߁ࠃߩߎޕ‬㕙ࠍࠦ࡯࠹ࠖࡦࠣߒߚ Mal-⏛᳇ࡆ࡯࠭ߦ࡟ࠢ࠴ࡦ㘃ࠍട߃ߚ෻ᔕ♧㎮⏛᳇ࡆ࡯࠭ߩวᚑ

ᶧ[0.1 mg/mLConA, 0.1mg/mL BSA, 0.1 mg/mL WGA, 0.6 ౖဳ⊛ߥᚻᴺ㧦Mal (100 mg, 0.292 mmol) ࠍ㘻๺὇㉄mg Mal-⏛᳇ࡆ࡯࠭, 1 mM CaCl2, 0.5 mM MnCl2, 0.5 M᳓⚛ࠕࡦࡕ࠾࠙ࡓ᳓ṁᶧ (2 mL) ߦṁ⸃ߒ㧘0°C ߢ 24 ᤨ

NaCl, 20 mMTris•HCl pH 7.4] ࠍ 1 mL ⺞⵾ߒ㧘4 °C ߢ㑆ᠣᜈߒߚ‫ޕ‬෻ᔕ⚳ੌᓟ㧘෻ᔕᶧࠍᷫ࿶Ớ❗ߒ㧘᳓ߢ➅60 ಽ㑆ォୟᷙ๺ᓟ㧘⏛⍹ߢ Mal-⏛᳇ࡆ࡯࠭ࠍᴉ㒠࡮࿁ࠅ㄰ߒଏᴣߔࠆߎߣߢ♽ౝߩ὇㉄߅ࠃ߮ࠕࡦࡕ࠾ࠕࠍ㒰෰ߒߡࠣ࡝ࠦࠪ࡞ࠕࡒࡦ૕ࠍᓧߚ‫ޕ‬ᓧࠄࠇߚࠣ࡝ࠦࠪ࡞
ࠕࡒࡦࠍࠫࠝࠠࠨࡦ㧦᳓ (1:1) ᷙวṁᇦ (5mL)ߦṁ⸃ߒ㧘᳖಄ਅ㧘὇㉄᳓⚛࠽࠻࡝࠙ࡓ (247 mg, 2.94 mmol) ߅ࠃ෼ߒߚ‫ޕ‬ṁᶧ⋧ߩ਄Ẵߺ 0.5 mL ಽࠍ㒰෰㧘ࡃ࠶ࡈࠔ࡯

(1 mM CaCl2, 0.5 mM MnCl2, 0.5 M NaCl, 20 mM Tris•HClpH 7.4) 0.5 mL ߦࠃࠆᏗ㉼㧘4 °C ߢ 5 ಽ㑆ߩォୟᷙ๺㧘߮Ⴎൻࠢࡠࡠࠕ࠮࠴࡞ (12.2µL, 0.15 mmol) ࠍ 15 ಽߏߣ⏛⍹ߦࠃࠆࡆ࡯࠭ߩᴉ㒠ࠍ 20 ࿁➅ࠅ㄰ߒ㧘㕖․⇣⊛ߦๆߦ㧟࿁ട߃ߚ‫ޕ‬᳖಄ਅ㧘ᤨ㑆ᠣᜈᓟ㧘෻ᔕᶧࠍᷫ࿶Ớ❗

⌕ߒߚ࠲ࡦࡄࠢ⾰ࠍᵞᵺߒ㧘Mal-⏛᳇ࡆ࡯࠭਄ߦ࡟ࠢ࠴

ߒᱷᷲࠍ SepPak C-18 (Waters Co.) (H2O to 20% MeOH)

ࡦࠍࠛࡦ࡝࠶࠴ߒߚ‫ޕ‬®

ߦߡ♖⵾ߒ㧘ࠢࡠࡠࠕ࠮࠴࡞૕ࠍᓧߚ‫ޕ‬ᰴ޿ߢࠢࡠࡠࠕ
࠮࠴࡞૕ࠍ 30% ࠕࡦࡕ࠾ࠕ᳓ṁᶧ (5 mL) ߣ 24 ᤨ㑆5&5ࡐ࡝ࠕࠢ࡝࡞ࠕࡒ࠼㔚᳇ᵒേ෻ᔕᓟ㧘ࠕࡦࡕ࠾ࠕࠟࠬࠍᷫ࿶⇐෰ߒ Mal-Gly ࠍ 30.8 mg ෻ᔕᶧ㧘ࠛࡦ࡝࠶࠴ᓟߩ਄Ẵߺ↹ಽ㧘ࠛࡦ࡝࠶࠴ᓟߩ
ࡆ࡯࠭ Ộ↹ಽࠍߘࠇߙࠇ 2 x ࠨࡦࡊ࡞ࡃ࠶ࡈࠔ࡯ (0.1(26%, 3 Ꮏ⒟) ᓧߚ‫ޕ‬Mal-Gly: MALDI-TOF MS calcd. For

C14H26N2O11m/z 398.15, found 398.26.

M Tris-HClpH6.8, 4% SDS, 12% 20 ࡔ࡞ࠞࡊ࠻ࠛ࠲ࠝ࡯࡞,µg) 㧘ࠣ࡝ࠪࡦ (0, 50, 100, 250 or 500 µg) 㧘ࠍ 0.1M borate

100 °C 5 ಽ㑆ࠗࡦࠠࡘࡌ࡯࠻ᓟ㧘ATTO ⵾ e•PAGEL (15%)

10% ࠣ࡞࠮ࡠ࡯࡞, ࡉࡠࡕࡈࠚࡁ࡯࡞ࡉ࡞࡯)ߦᏗ㉼ߒ㧘 Dynabeads M-280 Tosylactivated (3 mg) 㧘Mal-Gly (50pH9.5 (1.3mL) ߦṁ⸃ߒߚ‫ޕ‬ォୟᷙ๺ߒߟߟ 37 °C ߢ 24ࡊ࡟ࠠࡖࠬ࠻ࠥ࡞ߦࡠ࡯࠺ࠖࡦࠣߒ㧘20mA ߢᵒേߒߚ‫ޕ‬ᤨ㑆ࠗࡦࠠࡘࡌ࡯࠻ᓟ㧘⏛⍹ߢࡆ࡯࠭ࠍᴉ㒠ߐߖ㧘ࡃ࠶ࡈࠔ࡯ࠍ [PBS pH7.4, 0.1% BSA] ߦ੤឵ߒߚ‫ޕ‬ォୟᷙ๺㧘ᵒേ⚳ੌᓟ㧘㑐᧲ൻቇ⵾ Silver Stain KANTO III ߢᨴ⦡ߒ㧘࠲ࡦࡄࠢ⾰ߩᵒേࡃࡦ࠼ࠍᬌ಴ߒߚ‫ޕ‬⏛⍹ߦࠃࠆࡆ࡯࠭ߩᴉ㒠㧘ࡃ࠶ࡈࠔ࡯ߩ੤឵ࠍߐࠄߦ㧟࿁➅ࠅ㄰ߒ㧘♧㎮⏛᳇ࡆ࡯࠭ࠍᓧߚ‫ޕ‬ࡃ࠶࠴ᴺߦࠃࠆ࡟ࠢ࠴ࡦߩㆬ೎ ࡑࠗࠢࡠ࠴ࡘ࡯ࡉౝߢ Mal-⏛᳇ࡆ࡯࠭ (0.6 mg) ࠍࡉࡠ࠶ࠢࠛ࡯ࠬ෻ᔕᶧ (1 mg/mL, 1mL) ߣ 4 °C ߢ 60 ಽ㑆ಣℂᓟ㧘⏛⍹ߢᴉ㒠ߒ㧘෻ᔕᶧࠍ㒰෰ߒߚ‫߁ࠃߩߎޕ‬ߦ⴫㕙ࠍࠦ࡯࠹ࠖࡦࠣߒߚ Mal-⏛᳇ࡆ࡯࠭ߦ࡟ࠢ࠴ࡦ

㘃ࠍട߃ߚ෻ᔕᶧ [0.1 mg/mLConA, 0.1mg/mL BSA, 0.1㧙㧙㧟㧚⚿ᨐߣ⠨ኤ ߪߓ߼ߦ Mal-Gly7)ߩวᚑࠍⴕߞߚ㧔ࠬࠠ࡯ࡓ㧝㧕‫ࡑޕ‬࡞࠻࡯ࠬࠍ㘻๺὇㉄᳓⚛ࠕࡦࡕ࠾࠙ࡓ᳓ṁᶧߦṁ⸃ߒ㧘♧㎮⏛᳇ࡆ࡯࠭ߩวᚑ

᳓ṁᶧਛߢㅪ⛯⊛ߦ⊒↢ߒߡ޿ࠆࠕࡦࡕ࠾ࠕߦࠃߞߡㆶ ᧄ⎇ⓥߢߪࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩ᦭ലᕈࠍᬌ⸽ߔࠆ

రᧃ┵ࠍࠣ࡝ࠦࠪ࡞ࠕࡒࡦ߳ߣᄌ឵ߒߚ 8)‫࡞ࠪࠦ࡝ࠣޕ‬ߚ߼ߩࡕ࠺࡞ߣߒߡ Mal-⏛᳇ࡆ࡯࠭ߦࠃࠆ ConA ߩࠛࡦࠕࡒࡦߪኈᤃߦട᳓ಽ⸃ߐࠇࠆߚ߼㧘෻ᔕᶧࠍᷫ࿶Ớ❗

࡝࠶࠴ࠍㆬᛯߒߚ‫ޕ‬ConA ߪಽጘߒߚα-Man ᭴ㅧࠍ߽ߟᓟߚߛߜߦႮൻࠢࡠࡠࠕ࠮࠴࡞ߣ෻ᔕߐߖ㧘♧㎮ߩㆶర㜞ࡑࡦࡁ࡯ࠬဳ♧㎮ߥߤߦᒝജߦ⚿วߔࠆ߇㧘ᧃ┵ߦࠢࡠࡠࠕ࠮࠴࡞ၮࠍዉ౉ߒߚ‫࠮ࠕࡠࡠࠢߢ޿ߟޕ‬Glcα1-4Glc ߩ⚿ว᭽ᑼࠍ߽ߟࡑ࡞࠻࡯ࠬߣߪᒙߊ⚿วߔ࠴࡞૕ࠍࠕࡦࡕ࠾ࠕ᳓ṁᶧߣ෻ᔕߐߖ㧘ᧃ┵ߩࠢࡠࡠၮࠆߎߣ߇⍮ࠄࠇߡ߅ࠅ (Ka = 104 M-1)6)㧘Ყセ⊛ᒙ޿࡟ࠢࠍࠕࡒࡁൻߔࠆߎߣߢࠣ࡝ࠪࡦ߳ߣᄌ឵ߒ㧘Mal-Gly ߩ࠴ࡦᵴᕈߩࡕ࠺࡞ࠤ࡯ࠬߣߒߡㆡߒߡ޿ࠆ‫৻ޕ‬ᣇ㧘⏛᳇วᚑࠍቢੌߒߚ‫ᧄޕ‬෻ᔕߪᡴ₸߇ਃᎿ⒟ 26%ߣᔅߕߒ߽ࡆ࡯࠭ߣߒߡߪ㧘☸ሶᒻ⁁߇ဋ৻߆ߟ⴫㕙߇Ṗࠄ߆ߥߚ㜞ߊߥ޿߇㧘ߎࠇߪࠣ࡝ࠦࠪ࡞ࠕࡒࡦ߇↢ᚑ⋥ᓟ߆ࠄട߼ߦ㕖․⇣⊛ߥๆ⌕߇ዋߥߊ㧘ౣ⃻ᕈߦఝࠇߚ Invitrogen᳓ಽ⸃ࠍᆎ߼ࠆߚ߼ߢ޽ࠅ㧘ട᳓ಽ⸃↢ᚑ‛ߢ޽ࠆࡑ࡞

␠ߩ Dynabeads M280 Tosylactivated ࠍㆬᛯߒߚ‫ࡆߩߎޕ‬࠻࡯ࠬ߇ 40%⒟ᐲ࿁෼ߐࠇߡ޿ࠆ‫ޕ‬ߡ޿ࠆߚ߼㧘♧㎮ࠍ⚿วߒߚ႐ว㧘⚦⢩⴫㕙♧㎮ࠍᮨ୮ࠃ߁ߥࠕࡒࡁ♧ߩ႐ว㧘ੑ૏ߩࠕ࠮࠻ࠕࡒ࠼ߩ㔚ሶๆᒁߒߚ⦟޿ࡕ࠺࡞ߣߥࠆߎߣ߇ᦼᓙߐࠇࠆ‫⴫࠭࡯ࡆߚ߹ޕ‬ᕈߦࠃߞߡࠣ࡝ࠦࠪ࡞ࠕࡒࡦ߇቟ቯൻߐࠇࠆ‫⎇ᧄޕ‬ⓥߢ

㕙ߩᵴᕈൻၮߣߒߡ࠻ࠪ࡞ၮ߇ࠦ࡯࠹ࠖࡦࠣߐࠇߡ߅ࠅ㧘ߪ㜞ࡑࡦࡁ࡯ࠬ♧㎮․⇣⊛ߥᧂ⍮࡟ࠢ࠴ࡦࠍㆬ೎ߔࠆߚ

♧㎮ߩㆶరᧃ┵ߦࠕࡒࡁၮࠍዉ౉ߒߡ߅ߌ߫㧘ਔ⠪ࠍᷙ߼ߦ Glc1Man9GlcNAc2 (G1M9)-⏛᳇ࡆ࡯࠭ࠍ૬ߖߡ⺞®

 ৻ᣇ㧘ㆶరᧃ┵ߩ♧ᱷၮ߇ N ࠕ࠮࠴࡞ࠣ࡞ࠦࠨࡒࡦߩ࡯࠭ߪ⋥ᓘ⚂ 2.8 µm ߢ߶߷⚦⢩ߣห╬ߩᄢ߈ߐࠍ᦭ߒߗࠆߛߌߢਛᕈ᧦ઙਅߢ߽ࠞ࠶ࡊ࡝ࡦࠣߔࠆߎߣ߇ߢ߈ࠆ‫♧ޕ‬㎮ߩㆶరᧃ┵ߦࠕࡒࡁၮࠍዉ౉ߔࠆ߽ߞߣ߽⋥ធ⊛ߥᣇᴺߪࠣ࡝ࠦࠪ࡞ࠕࡒࡦ߳ߩᄌ឵ߢ޽ࠆ‫ߥߒ߆ߒޕ‬߇ࠄ↢ᚑߔࠆࠣ࡝ࠦࠪ࡞ࠕࡒࡦߪᭂ߼ߡਇ቟ቯߢ޽ࠅ㧘⏛᳇ࡆ࡯࠭߳ߩዉ౉೨㚟૕ߣߒߡ㐳ᦼ଻ሽน⢻ߥ㎛ਛ㑆૕ߦߪߥࠅᓧߥ޿‫ ߢߎߘޕ‬Mal-ࠣ࡝ࠦࠪ࡞ࠕࡒࡦߦ࡝ࡦࠞ࡯ߣߒߡන⚐ߥࠕࡒࡁ㉄ߢ޽ࠆࠣ࡝ࠪࡦࠍ❗วߒ(Mal-Gly)㧘ߘߩ N ᧃ┵ߩ቟ቯߥࠕࡒࡁၮࠍ↪޿ߡ⏛᳇ࡆ⵾ߒߚ‫ߩߘޕ‬㓙ߩ㎛ਛ㑆૕ߣߥࠆ G1M9-Gly9)ߪ Mal-Glyߣห᭽ߩᚻᴺߢวᚑߒߚ߇㧘ㆶరᧃ┵߇ GlcNAc ߢ޽ࠆߚ߼㧘೨ㅀߩℂ↱ߦࠃࠅᡴ₸ߪਃᎿ⒟ 72%ߢ޽ߞߚ‫ޕ‬ ߟ߉ߦ Mal-Gly㧘G1M9-Gly ߣ߽ߦ Dynabeads®M280
Tosylactivated ߣ 0.1M borate (pH9.5) ਛߢ㧘৻᥅෻ᔕߐߖ
ࠆߎߣߢ⏛᳇ࡆ࡯࠭਄ߦ♧㎮ࠍⵍⷒߒߚ‫ߩߎޕ‬㓙㧘♧㎮-Gly ߣ Gly ࠍ⇣ߥࠆᷙวᲧߢࡆ࡯࠭ߣࠞ࠶ࡊ࡝ࡦࠣߐߖ㧘♧㎮ߩⵍⷒ₸ࠍ♽⛔⊛ߦ೙ᓮߒߚ⏛᳇ࡆ࡯࠭ࠍ⺞⵾ߒߚ‫ޕ‬࡯࠭ߣߩ⚿วࠍⴕ߁ߎߣߣߒߚ‫ޕ‬ࠬࠠ࡯ࡓ㧝 ♧㎮⏛᳇ࡆ࡯࠭ߩวᚑ㧙㧙

10 ࿁➅ࠅ㄰ߒߚᓟߩ਄Ẵߺ↹ಽ㧔࡟࡯ࡦ 3㧦10mL ᵞᵺ㧕/CN ⏛᳇ࡆ࡯࠭ߣ %QP# ߦࠃࠆࡕ࠺࡞ታ㛎ߦߪ㧘᦭ᗧߩࡃࡦ࠼ߪᬌ಴ߐࠇߕలಽߦᵞᵺ߇ⴕࠊࠇߡ
޿ࠆߎߣ߇ಽ߆ߞߚ‫ ࡦ࡯࡟ޕ‬4 ߦߪ Mal-⏛᳇ࡆ࡯࠭߆ࠄ⏛᳇ࡆ࡯࠭਄ߩ♧㎮ⵍⷒ₸ߩᬌ⸛ ⏛᳇ࡆ࡯࠭਄ߩᦨㆡ♧㎮ⵍⷒ₸ࠍ᳿ቯߔࠆߚ߼ߦ㧘♧㎮฽᦭㊂ߩ⇣ߥࠆ a-e ߩ 5 ⒳㘃ߩ Mal-⏛᳇ࡆ࡯࠭ࠍ↪޿ߡࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߦࠃࠅ ConA ߩㆬ೎ታ㛎ࠍⴕߞߚ㧔࿑㧞㧕‫ౕޕ‬૕⊛ߦߪ߹ߕ ConA ߣ Mal-⏛᳇ࡆ࡯࠭ߩ Ộᶧ 1mL ߦኻߒ㧘⏛⍹ߦࠃࠆࡆ࡯࠭ߩᴉ㒠ᓟ㧘ᶧ㊂ߩߩṁ಴↹ಽࠍ␜ߒߚ߇㧘ConA ߦ↱᧪ߔࠆࡃࡦ࠼ߪ∥〔⒟ᐲߦߒ߆ᬌ಴ߐࠇߥ߆ߞߚ‫ޕ‬ ৻ᣇ㧘ࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺࠍ↪޿ߚ႐วߩ⚿ᨐࠍ࿑
3B ߦ␜ߒߚ‫ߩߎޕ‬ᣇᴺߩᵞᵺᎿ⒟ߦ߅ߌࠆ 1 ࿁ߩࡃ࠶ࡈ
ࠔ࡯੤឵㊂ߪࡃ࠶࠴ᴺߩඨ㊂ߢ޽ࠆߚ߼㧘ਔᚻᴺߩᵞᵺ

ඨಽࠍᣂߒ޿ࡃ࠶ࡈࠔ࡯ߦ੤឵ߒߡ޿ߊࡑࠗ࡞࠼ࠛࡦ࡝
࠶࠴ࠍ 20 ࿁➅ࠅ㄰ߒలಽߦᵞᵺߒߚ‫ޕ‬ᵞᵺᓟߩࡆ࡯࠭

㊂ࠍ৻⥌ߐߖࠆߴߊࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩᵞᵺ࿁ᢙࠍࡃ࠶࠴ᴺߩ 2 ୚ߦ⸳ቯߒߚ‫⚿ߩߘޕ‬ᨐ㧘ᵞᵺᓟߩ਄Ẵߺ Ộᶧߩ਄Ẵߺ↹ಽ߅ࠃ߮ࡆ࡯࠭߆ࠄߩṁ಴↹ಽࠍ㧘ࡆ࡯࠭ a-e ߩߘࠇߙࠇߦኻߒߡ SDS-PAGE ߢಽᨆߒߚߣߎ

ࠈ㧘ࡆ࡯࠭ a, b, c, d ߪ਄Ẵߺ↹ಽ㧔࡟࡯ࡦ 2, 4, 6, 8㧕ߦࡃࡦ࠼߇ᬌ಴ߐࠇߕ㧘㕖․⇣⊛ߥ⚿วࠍలಽߦ㒰෰ߢ߈ߡ޿ࠆߎߣ߇ಽ߆ߞߚ‫৻ޕ‬ᣇ㧘ࡆ࡯࠭ e ߪ 20 ࿁ࠛࡦ࡝࠶࠴ᓟߩ਄Ẵߺ↹ಽߦ߽ ConA ߩࡃࡦ࠼߇ᱷߞߡ߅ࠅ㧔࡟

࡯ࡦ 10㧕㧘࡝ࠟࡦ࠼♧㎮ߩ฽᦭㊂߇ਇలಽߥߎߣ߇᣿ࠄ߆ߣߥߞߚ‫৻ޕ‬ᣇ㧘ṁ಴↹ಽߦߪ޿ߕࠇ߽ ConA ߩࡃࡦ↹ಽߦ࠲ࡦࡄࠢ⾰ߩࡃࡦ࠼߇ᬌ಴ߐࠇߥ޿ߩߦኻߒ㧔࡟
࡯ࡦ 2㧦5mL ᵞᵺ㧘࡟࡯ࡦ 3㧦10 mL ᵞᵺ㧕㧘࡟࡯ࡦ 4 ߩࡆ࡯࠭ṁ಴↹ಽߦߪ ConA ߩࡃࡦ࠼߇ߪߞ߈ࠅߣ⃻ࠇߚ‫ޕ‬ߎࠇࠍ࿑ 3A ߣᲧセߔࠆߣṁ಴↹ಽߦ߅ߌࠆࠛࡦ࡝࠶࠴ല₸ߩ㆑޿ߪ᣿⏕ߢ޽ࠅ㧘Mal ߣ ConA ߩࠃ߁ߥᲧセ⊛ᒙ޿⚿วߦኻߒߡ㧘ࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩ᦭ലᕈ߇⏕⹺ߐࠇߚ‫ޕ‬࠼߇ᬌ಴ߐࠇߚ߇㧔࡟࡯ࡦ 3, 5, 7, 9, 11㧕㧘50 kDa ઃㄭߩਇ⚐‛ߩࡃࡦ࠼߇ዋߥߊ S/N Ყߩ㜞޿ࡆ࡯࠭ b㧔࡟࡯ࡦ
5㧕߇ ConA ߩㆬ೎ߦߪᦨㆡߢ޽ࠆߣ⚿⺰ߒߚ‫ޕ‬࿑㧟 ࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩ᦭ലᕈߩᬌ⸽㧦 # ࡃ࠶࠴ᴺ㧘 $ ࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺ
↢૕᛽಴ᶧ߆ࠄߩ⋡⊛࡟ࠢ࠴ࡦߩㆬ೎
࿑㧞 /CN⏛᳇ࡆ࡯࠭ߩᦨㆡ♧㎮ⵍⷒ₸ߩᬌ⸛ ᧄ⎇ⓥߩᦨ⚳⊛ߥ⋡⊛ߪ㧘↢૕᛽಴ᶧߥߤߩⶄ㔀ߥ࠲ࡃ࠶࠴ᴺߣࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩᲧセࡦࡄࠢ⾰ᷙวᶧߩਛ߆ࠄ․ቯߩ♧㎮․⇣⊛ߥ࡟ࠢ࠴ࡦࠍ ೨㗄ߢ Mal-ⵍⷒ₸ࠍᦨㆡൻߒߚࡆ࡯࠭ b ࠍ↪޿ߡ㧘࠙ㆬ೎ߔࠆߎߣߦ޽ࠆ‫ߢߎߘޕ‬ᚒ‫ߩޘ‬ឭ᩺ߔࠆࡑࠗ࡞࠼ࠛ

ࠪⴊᷡࠕ࡞ࡉࡒࡦ (BSA) 㧘ዊ㤈⢦⧘࡟ࠢ࠴ࡦ (WGA) 㧘ConA ߩ࠲ࡦࡄࠢ⾰ᷙวᶧ߆ࠄ ConA ߩㆬ೎ታ㛎ࠍⴕߞߚ‫ߩߎޕ‬㓙㧘ㅢᏱߩࡃ࠶࠴ᴺߣࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩ
ࠛࡦ࡝࠶࠴ല₸ࠍᲧセߒ㧘ᧄᴺߩ᦭ലᕈࠍᬌ⸽ߒߚ㧔࿑

ࡦ࡝࠶࠴ᴺ߇ታ㓙ߩ↢૕᛽಴ᶧߦኻߒߡ᦭ലߢ޽ࠆ߆ࠍ್ᢿߔࠆߚ߼ߦ㧘േ‛⚵❱↱᧪ߩࡒࠢࡠ࠰࡯ࡓ↹ಽߦ
ConA ࠍᷝടߒߚࡕ࠺࡞࠲ࡦࡄࠢ⾰ᷙวᶧߦኻߒ㧘Mal-⏛᳇ࡆ࡯࠭ࠍ↪޿ߡ ConA ߩࠛࡦ࡝࠶࠴ࠍ⹜ߺߚ㧔࿑ 4㧕‫ޕ‬3㧕‫ޕ‬ࡒࠢࡠ࠰࡯ࡓ↹ಽߣߒߡߪࡑ࠙ࠬ (SAMP10㧦⠧ൻଦㅴ࠭ߩ Ộᶧ 1mL ߦኻߒߡᶧ⋧ߩ੤឵ߦࠃࠆᵞᵺࠍ 5 ࿁➅mMTris•HCl pH7.4, 0.25M Sucrose, 2 mM EDTA, 5 mM ࡃ࠶࠴ᴺߩ႐ว㧔࿑ 3A㧕㧘࠲ࡦࡄࠢ⾰ߣ Mal-⏛᳇ࡆ࡯ࠅ㄰ߒߚᓟߩ਄Ẵߺ↹ಽ㧔࡟࡯ࡦ 2㧦5mL ᵞᵺ㧕߅ࠃ߮ࡕ࠺࡞) ⢄⤳ࠍ⎕⎈ᓟ㧘㆙ᔃಽ↹ 10)ߒߚࡍ࡟࠶࠻ࠍ [202-mercaptoethanol, ptrotease inhibitor cocktail (Roche), 0.5%㧙㧙

Triton X-100] ߦߡนṁൻߒ↪޿ߚ‫↹ࡓ࡯࠰ࡠࠢࡒޕ‬ಽ (1(G1M9) ࠍ࿕ቯൻߒߚ⏛᳇ࡆ࡯࠭ߦࠃߞߡ࡟ࠢ࠴ࡦ୥⵬mg/mL) ߦ ConA (0.1mg) ࠍᷝടߒߚ࠲ࡦࡄࠢ⾰ᷙวᶧߩត⚝ࠍⴕ߁ߎߣߣߒߚ‫ޕ‬(1 mL) ߦ Mal-⏛᳇ࡆ࡯࠭ࠍട߃㧘ࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺ ߹ߕࡆ࡯࠭਄ߩᦨㆡߥ G1M9 ⵍⷒ₸ࠍ᳿ቯߔࠆߚ߼ߦ㧘ࡦ 2㧘ࡆ࡯࠭ṁ಴↹ಽࠍ࡟࡯ࡦ 3 ߦ␜ߒߚ‫⚿ߩߘޕ‬ᨐ㧘ߚࡒࠢࡠ࠰࡯ࡓ↹ಽ㧔ዊ⢩૕ࠍਥᚑಽߣߔࠆ㧕ߦኻߒ㧘ߦࠃߞߡ 20 ࿁ᵞᵺࠍ➅ࠅ㄰ߒߚᓟߩ਄Ẵߺ↹ಽࠍ࡟࡯ࡑ࠙ࠬ (SAMP10㧦⠧ൻଦㅴࡕ࠺࡞) ⢄⤳ࠃࠅ㆙ᔃಽ↹ߒṁ಴↹ಽߦ ConA ࠍ฽߻޿ߊߟ߆ߩࡃࡦ࠼߇ᬌ಴ߐࠇ㧘Mal ․⇣⊛ߥ࡟ࠢ࠴ࡦ୥⵬ࠍㆬ೎ߔࠆߎߣ߇ߢ߈ߚ‫ߎޕ‬ࠇߦࠃࠅᧄᴺߪⶄ㔀ߥ↢૕᛽಴ᶧਛߢ߽᦭ലߦߪߚࠄߊ

♧㎮ⵍⷒ₸ࠍ♽⛔⊛ߦᄌൻߐߖߚ G1M9-⏛᳇ࡆ࡯࠭A-E ߦࠃࠆࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ࠍ೨㗄ߣห᭽ߩᚻᴺߢⴕ޿㧘࡟ࠢ࠴ࡦߩㆬ೎ࠍⴕߞߚ㧔࿑ 5㧕‫♧ޕ‬㎮ࠍᜬߚߥ޿
ࡆ࡯࠭ E ߩṁ಴↹ಽߦࡃࡦ࠼߇ోߊᬌ಴ߐࠇߥ޿ߩߦߎߣ߇ಽ߆ߞߚ‫ޕ‬ኻߒ㧔࡟࡯ࡦ 11㧕㧘G1M9 ࠍ߽ߟࡆ࡯࠭ A-D ߦߪ᣿⏕ߥࡃࡦ࠼߇ᬌ಴ߐࠇߚ㧔࡟࡯ࡦ 3, 5, 7, 9㧕ߎߣߢ㧘ࠛࡦ࡝࠶࠴ߐࠇߚࡃࡦ࠼ߪ G1M9 ․⇣⊛ߥ࡟ࠢ࠴ࡦߩน⢻ᕈ߇㜞޿߽ߩߣ⠨߃ࠄࠇࠆ‫ߩ࠼ࡦࡃߚࠇߐ࠴࠶࡝ࡦࠛߚ߹ޕ‬᣿⍎ߐ߿ࠦࡦ࠻࡜ࠬ࠻߆ࠄ㧘੹࿁ߩታ㛎᧦ઙߦߪࡆ࡯࠭
D ߇Ყセ⊛ㆡߒߡ޿ࠆߎߣ߇ಽ߆ߞߚ‫ޕ‬     ࿑㧠 ↢૕᛽಴ᶧ㧔ࡑ࠙ࠬࡒࠢࡠ࠰࡯ࡓ↹ಽ㧕ߦኻߔࠆࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩ᦭ലᕈ ࡑ࠙ࠬࡒࠢࡠ࠰࡯ࡓ↹ಽ߆ࠄߩ )/ ․⇣⊛ᧂ⍮࡟ࠢ࠴ࡦߩㆬ೎࿑㧡 )/⏛᳇ࡆ࡯࠭ߩᦨㆡ♧㎮ⵍⷒ₸ߩᬌ⸛)/ ⏛᳇ࡆ࡯࠭਄ߩ♧㎮ⵍⷒ₸ߩᬌ⸛ ೨㗄ߩᬌ⸛ߦࠃࠅ Mal-⏛᳇ࡆ࡯࠭ߣ ConA ߩ⚿วࠍࡕᱜᏱ⠧ൻ߅ࠃ߮⠧ൻଦㅴࡕ࠺࡞ࡑ࠙ࠬ↱᧪ߩዊ࠺࡞ߣߒߡࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺߩ᦭ലᕈࠍ⏕⹺ߔࠆߎ⢩૕↹ಽߦ߅ߌࠆㆬ೎࡟ࠢ࠴ࡦ୥⵬ߩᲧセߣ߇ߢ߈ߚ‫ᧄޕ‬㗄ߢߪࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ᴺࠍ↢૕᛽಴ᶧ߆ࠄߩᧂ⍮࡟ࠢ࠴ࡦߩㆬ೎ߦᔕ↪ߔࠆ‫ߪߦ߼ߚߩߘޕ‬ G1M9-⏛᳇ࡆ࡯࠭ࠍ↪޿ߚࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ߦࠃࠅ࡟ࠢ࠴ࡦត⚝ߩኻ⽎ߣߥࠆࠝ࡞ࠟࡀ࡜ࠍㆬᛯߒ㧘ߘߩⅣࡑ࠙ࠬࡒࠢࡠ࠰࡯ࡓ↹ಽ߆ࠄ࡟ࠢ࠴ࡦ୥⵬ࠍㆬ೎ߢ߈ߚႺߦሽ࿷ߔࠆᧄ‛ߩ♧㎮࡝ࠟࡦ࠼ࠍ߽ߟ⏛᳇ࡆ࡯࠭ࠍ⺞ߚ߼㧘ߟ߉ߦ⇣ߥࠆ࠻࡜ࡦࠬࠫࠚ࠾࠶ࠢࡑ࠙ࠬ㑆ߢߩㆬ⵾ߒߥߊߡߪߥࠄߥ޿‫ޕ‬ᚒ‫ߪޘ‬㐳ᐕ㧘ዊ⢩૕♧࠲ࡦࡄࠢ೎࡟ࠢ࠴ࡦ୥⵬ߩ㆑޿ࠍᬌ⸛ߒߚ㧔࿑ 6㧕‫ࠬ࠙ࡑߚ޿↪ޕ‬⾰ຠ⾰▤ℂᯏ᭴ߦ߅ߌࠆ♧㎮ᯏ⢻⸃ᨆߦ៤ࠊߞߡ߈ߚ11)‫ޕ‬ᧄᯏ᭴ߦߪᄙߊߩ࡟ࠢ࠴ࡦ߇㑐ࠊࠆ߇㧘ߎߎᢙᐕߢ߽
OS9 ߿ Malectin ߣ޿ߞߚᣂߒ޿࡟ࠢ࠴ࡦߩሽ࿷߇ᰴ‫ޘ‬12)13)

ߣ᣿ࠄ߆ߦߥࠅ㧘ߘߩࡔࠞ࠾࠭ࡓోኈߩ⸃㉼ߦߪߐࠄߦ✂⟜⊛ߥ࡟ࠢ࠴ࡦߩត⚝߇ᔅⷐߣߐࠇߡ޿ࠆ‫ᧄߢߎߘޕ‬⎇ⓥߢߪࡑ࠙ࠬ⢄⤳↱᧪ߩዊ⢩૕ࠍኻ⽎ߣߒ㧘ዊ⢩૕ဳ㜞ࡑࡦ ࡁ࡯ࠬ♧㎮ߩ߭ߣߟߢ޽ࠆ Glc1Man9GlcNAc2

ߪᱜᏱ⠧ൻࡕ࠺࡞ࡑ࠙ࠬ (SAMR1) ߣ⠧ൻଦㅴࡕ࠺࡞ࡑ

࠙ࠬ (SAMP10㧦ࠕ࡞࠷ࡂࠗࡑ࡯ࡕ࠺࡞ߣߒߡ↪޿ࠄࠇࠆ) ߢ޽ࠆ‫ޕ‬ਔࡑ࠙ࠬߩ⢄⤳ࠃࠅ᛽಴ߒߚࡒࠢࡠ࠰࡯ࡓ
↹ಽ㧔࡟࡯ࡦ 1 ߅ࠃ߮ 6㧕ߦኻߒ㧘G1M9-⏛᳇ࡆ࡯࠭ (ࡆ࡯࠭ D㧦࿑ 5 ෳᾖ)ߦࠃࠆࡑࠗ࡞࠼ࠛࡦ࡝࠶࠴ࠍⴕߞߚߣ
ߎࠈ㧘࡟࡯ࡦ 3 ߅ࠃ߮ 4 ߩࠃ߁ߥ࡟ࠢ࠴ࡦ୥⵬⟲߇ㆬ೎ߐࠇߚ‫ޕ‬⍫ශߢ␜ߒߚࡃࡦ࠼߇Ყセ⊛᣿⍎ߥ࡟ࠢ࠴ࡦ୥㧙㧙⵬ߢ޽ࠆ߇㧘଀߃߫ a ߪ G1M9 ․⇣⊛ߥ࡟ࠢ࠴ࡦࠪࡖࡍࡠࡦߣߒߡ⍮ࠄࠇߡ޿ࠆࠞ࡞ࡀࠠࠪࡦ14)(65 kDa) 㧘b ߪෳ⠨ᢥ₂ߘߩนṁᕈࡎࡕࡠࠣߢ޽ࠆࠞ࡞࡟࠹ࠖࠠࡘ࡝ࡦ 15) (461)(a)R. A. Dwek, Chem Rev., 96, 683 (1996); (b)W. J.

ࠢࡠ࠰࡯ࡓ↹ಽ߆ࠄᓧࠄࠇߚ࡟ࠢ࠴ࡦ୥⵬⟲ߪ᭎ߨ৻⥌2)

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