ᚑくᄢቇℂᎿቇ⎇ⓥႎ๔
J. Fac. Sci. Tech., Seikei Univ.
Vol.45 No.2 (2008) pp.15-25
㧔ᣂછ⠪⺰ᢥ㧕♧࠲ࡦࡄࠢ⾰ຠ⾰▤ℂᯏ᭴⸃᣿ߦะߌߚวᚑൻቇ⊛ࠕࡊࡠ࡯࠴
ᚭ⼱ Ꮧ৻㇢*1

Synthetic Approach Toward Understanding Glycoprotein Quality Control System
Kiichiro TOTANI*1
ABSTRUCT :High-mannose-type oligosaccharides have been revealed to play important roles in protein
quality control. A number of intracellular proteins, such as lectins, chaperones and glycan processing
enzymes are involved in this process. These include calnexin (CNX) /calreticulin (CRT),
UDP-Glc:glycoproteinglucosyltransferase (UGGT), glucosidase II, mannosidase I, cargo receptors (VIP36,
ERGIC-53), mannosidase-like protein (EDEM, Htm1p) and ubiquitinligase (Fbs). They are considered torecognize high-mannose-type glycans with subtly different structures, although the precise specificities
are yet to be clarified. In order to gain clear understanding of these protein-carbohydrate interactions,comprehensive synthesis of high-mannose-type glycans was conducted. In addition, two approaches to
artificial glycoproteins having homogeneous oligosaccharides were investigated. Furthermore, a novel
substrate of UGGT was discovered. With these synthetic oligosaccharides, quantitative analysis of CRT,Fbs 1 and UGGT were carried out.
Keywords : glycoprotein quality control, calreticulin, Fbs1, UGGT, synthetic oligosaccharide,
high-mannose-type oligosacharide, artificial glycoprotein(Received September 25, 2008) ߒ߆ߒߥ߇ࠄߎߩࠃ߁ߥ⎇ⓥࠍታᣉߔࠆߦ޽ߚࠅ㧘♧㧝ߪߓ߼ߦ
㎮ࠨࡦࡊ࡞ߩ⺞⵾߇ߒ߫ߒ߫໧㗴ߣߥࠆ‫ߢ߹ࠇߎޕ‬㧘♧ ࠥࡁࡓߩ⸃⺒ߦࠃࠅ㧘ࡅ࠻ㆮવሶߪ੍ᗐࠃࠅ㆝߆ߦዋ㎮ߩᯏ⢻⸃᣿ߦߪ↢૕⹜ᢱ߆ࠄᓧࠄࠇࠆ♧㎮߿♧࠲ࡦࡄߥ޿ߎߣ߇᣿ࠄ߆ߦߥߞߚ‫ߩߎޕ‬੐ታߪ⠡⸶ᓟߦୃ㘼ߐࠢ⾰߇ᄙߊ↪޿ࠄࠇߡ߈ߚ߇㧘↢૕ౝߦሽ࿷ߔࠆ♧㎮ߪࠇࠆ࠲ࡦࡄࠢ⾰߇ᢙᄙߊሽ࿷ߒ㧘ⶄ㔀ߥಽሶࡀ࠶࠻ࡢ࡯
᭴ㅧߩਇဋ৻ᕈࠍ฽ࠎߢ޿ࠆ਄㧘ߘߎ߆ࠄᓧࠄࠇࠆ♧㎮ࠢࠍᒻᚑߒߡ޿ࠆ੐ࠍ␜ߒߡ޿ࠆ‫♧ޕ‬㎮ୃ㘼ߪ⠡⸶ᓟୃࠨࡦࡊ࡞ߪᓸ㊂ߢ޽ࠆ‫ߦࠇߎޕ‬ኻߒ♧㎮ߩൻቇวᚑߦߪ㧘㘼ߩઍ⴫଀ߢ޽ࠅ㧘ᄙߊߩಽᴲ࠲ࡦࡄࠢ⾰߿⤑࠲ࡦࡄࠢᓧࠄࠇࠆࠨࡦࡊ࡞ߩ㊂㧘᭴ㅧߩဋ৻ᕈ㧘㕖ᄤὼဳ♧㎮ࠍߪ♧㎮߇ઃടߐࠇߚ♧࠲ࡦࡄࠢ⾰ߢ޽ࠆ‫࠲♧ߩࠄࠇߎޕ‬วᚑߢ߈ࠆ╬㧘ᢙ‫ߩޘ‬೑ὐ߇᜼ߍࠄࠇࠆ‫ⶄߚ߹ޕ‬㔀ߥಽࡦࡄࠢ⾰♧㎮ߪ㧘࠲ࡦࡄࠢ⾰ߩ᭴ㅧ߿ᯏ⢻ߣኒធߦ㑐ㅪߒߡ߅ࠅ㧘᭽‫↢ߥޘ‬๮⃻⽎ߦ߅޿ߡ㊀ⷐߥᓎഀࠍᜂ߁ 1)‫ޕ‬ጘဳ♧㎮ߪ㧘วᚑൻቇ⎇ⓥߩᮡ⊛ߣߒߡ߽㕖Ᏹߦ⥝๧ᷓ޿‫ޕ‬ᚒ‫♧⾰ࠢࡄࡦ࠲♧ߪޘ‬㎮߇↢૕ౝߢᨐߚߔ㊀ⷐߥᓎታ㓙㧘♧ォ⒖㉂⚛߿♧ട᳓ಽ⸃㉂⚛ߩ⇣Ᏹ߇᭽‫∔ߥޘ‬ᖚഀߩ৻ߟ㧘࠲ࡦࡄࠢ⾰ߩຠ⾰▤ℂ 2)ߦᵈ⋡ߒ㧘ߎࠇߦ㑐ࠍᒁ߈⿠ߎߔߎߣ߇⍮ࠄࠇߡ߅ࠅ㧘♧㎮߇↢๮ᵴേߩ⛽ㅪߔࠆ㜞ࡑࡦࡁ࡯ࠬဳ♧㎮ 3)߿ߘߩㇱಽ᭴ㅧ 4)ߩวᚑ㧘ᜬߦᔅ㗇ߢ޽ࠆߎߣ߇┍߃ࠆ‫ߒ߆ߒޕ‬㧘ߤߩ♧㎮߇ߤߩੱᎿ♧࠲ࡦࡄࠢ⾰ߩഃ⵾ 5)ߦขࠅ⚵ࠎߢ᧪ߚ‫⃻ޕ‬࿷㧘ߎ᭽ߥ↢๮⃻⽎ߦᔅⷐߢ޽ࠆ߆ࠍቯ㊂⊛ߦ⸃ᨆߒߚ଀ߪᧂ
ࠇࠄߩಽሶࡊࡠ࡯ࡉࠍᦨᄢ㒢ߦᵴ↪ߒ㧘♧㎮ᯏ⢻ߩቯ㊂ߛዋߥߊ㧘⃻࿷߽᭽‫ޕࠆ޿ߡࠇࠊⴕ߇࠴࡯ࡠࡊࠕߥޘ‬⊛ߥ⸃ᨆߦขࠅ⚵ࠎߢ޿ࠆ‫⺰ᧄޕ‬ᢥߢߪ㧘㜞ࡑࡦࡁ࡯ࠬဳ♧㎮෸߮ߘߩ㘃✼૕ߩ᦭ᯏวᚑࠍࡌ࡯ࠬߣߒߚ♧࠲ࡦ* : ᚑくᄢቇℂᎿቇㇱ‛⾰↢๮ℂᎿቇ⑼ኾછ⻠Ꮷ
(Instructor, Dept of Material and Life Science,e-mail: ktotani@st.seikei.ac.jp)
1

ࡄࠢ⾰ߩຠ⾰▤ℂߦ㑐ࠊࠆ♧㎮ᯏ⢻ߩቯ㊂⸃ᨆ 6)ߦߟ޿
ߡ㧘ᦨㄭߩ⚿ᨐࠍႎ๔ߔࠆ‫ޕ‬㧙㧙ࠅ㧘᛬ࠅ⇥ߺ࠮ࡦࠨ࡯ߣߒߡᯏ⢻ߒߡ޿ࠆ‫ߩߎޕ‬᭽ߥ Glc

㧞㧚♧࠲ࡦࡄࠢ⾰ຠ⾰▤ℂᯏ᭴ߩಾ㒰ߣઃടߩ➅ࠅ㄰ߒ㧔CNX/CRT ࠨࠗࠢ࡞㧕ߦࠃߞ ⚦⢩ߦߪ࠲ࡦࡄࠢ⾰ߩ㜞ᰴ᭴ㅧߩᒻᚑ㧘ャㅍ㧘ਇ⦟࠲ߡᦨ⚳⊛ߦᱜᏱߥ᛬ࠅ⇥ߺࠍ₪ᓧߒߚ♧࠲ࡦࡄࠢ⾰ߩߺ

ࡦࡄࠢ⾰ߩಽ⸃╬ࠍ⋙ⷞߔࠆຠ⾰▤ℂᯏ᭴߇ሽ࿷ߒߡ޿

߇㧘࡟ࠢ࠴ࡦᵴᕈࠍ᦭ߔࠆࠞ࡯ࠧ࡟࠮ࡊ࠲࡯ߦࠃߞߡࠧ

ࠆ‫ߩࠄࠇߎޕ‬ㆊ⒟ߢਛᔃ⊛ߥᓎഀࠍᨐߚߔߩߪ㧘⚦⢩⾰㧘࡞ࠫ૕߳ߣㆇ߫ࠇࠆ 11)‫ޕ‬ዊ⢩૕㧘ࡒ࠻ࠦࡦ࠼࡝ࠕߥߤߦሽ࿷ߔࠆಽሶࠪࡖࡍࡠࡦ ৻ᣇ㧘CNX/CRT ࠨࠗࠢ࡞ߢᱜᏱߥ㜞ᰴ᭴ㅧࠍ₪ᓧߢߣ๭߫ࠇࠆ࠲ࡦࡄࠢ⾰⟲ߢ޽ࠆ‫( ࡦࠪࠠࡀ࡞ࠞޕ‬CNX)

߈ߥ߆ߞߚ♧࠲ࡦࡄࠢ⾰ߪ㧘⚦⢩⾰ߦㅒャㅍߐࠇಽ⸃ࠍ

ዊ⢩૕ౝߩ♧㎮ࠍ⹺⼂ߔࠆಽሶࠪࡖࡍࡠࡦߢ޽ࠅ㧘ᣂ↢

ߥ Man9GlcNAc2 ဳ♧࠲ࡦࡄࠢ⾰ߪ㧘ዊ⢩૕ౝߦሽ࿷ߔ

෸߮ߘߩนṁᕈࡎࡕࡠࠣ㧘ࠞ࡞࡟࠹ࠖࠠࡘ࡝ࡦ (CRT)ߪ㧘ฃߌࠆ㧔ዊ⢩૕㑐ㅪಽ⸃㧘ERAD㧕12)‫ޕ‬᛬ࠅ⇥ߺਇቢో♧࠲ࡦࡄࠢ⾰ߩ㜞ᰴ᭴ㅧᒻᚑߦ㊀ⷐߥᓎഀࠍᨐߚߒߡ޿

ࠆࡑࡦࡁࠪ࠳࡯࠯ I (Man’ase I)ߦࠃࠅ Man8(B)GlcNAc2

ࠆ‫ᧄޕ‬㗄ߢߪߎࠇࠄߩ࡟ࠢ࠴ࡦ᭽ࠪࡖࡍࡠࡦࠍਛᔃߣߒ

ဳ߳ߣࡊࡠ࠮ࠪࡦࠣߐࠇ㧘ࡑࡦࡁࠪ࠳࡯࠯᭽࠲ࡦࡄࠢ⾰ߚ㧘CNX/CRT ࠨࠗࠢ࡞ߣ๭߫ࠇࠆ♧࠲ࡦࡄࠢ⾰ຠ⾰▤

㧔MLPs㧕13)ߦࠃߞߡዊ⢩૕߆ࠄ⚦⢩⾰߳ߣㅒャㅍߐࠇࠆ‫ޕ‬ℂߩ᭎⇛ࠍㅀߴࠆ㧔࿑㧝㧕2a)‫ޕ‬⛯ߊ⚦⢩⾰ߦ߅ߌࠆ♧࠲ࡦࡄࠢ⾰ߩಽ⸃ߦߪ㧘࡙ࡆࠠ࠴ࡦ−ࡊࡠ࠹ࠕ࠰࡯ࡓࠪࠬ࠹ࡓ߇ਥߥᓎഀࠍᨐߚߔߣ⠨߃ ᣂ↢࠲ࡦࡄࠢ⾰ߪዊ⢩૕ߦ౉ࠆߣ⋥ߜߦN-⚿วဳ♧㎮
Glc3Man9GlcNAc2ߩઃടࠍฃߌࠆ‫♧ߩߎޕ‬㎮ߪዊ⢩૕ౝࠄࠇߡ޿ࠆ 14)‫ޕ‬ㄭᐕ⊒⷗ߐࠇߚ♧㎮ࠍ⹺⼂ߔࠆ࡙ࡆࠠ࠴
ࡦൻ㉂⚛ߪ 15)㧘ߎߩ⚻〝ࠍⵣઃߌࠆ߽ߩߣߒߡ⣉శࠍᶎߦሽ࿷ߔࠆࠣ࡞ࠦࠪ࠳࡯࠯I㧔Glc’aseI㧕ߩ௛߈ߦࠃࠅ㧘
Glc2Man9GlcNAc2߳ߣࠣ࡞ࠦ࡯ࠬ㧔Glc㧕ࠍ৻ᱷၮಾᢿ
ߐࠇࠆ2b) 7)‫ߦࠄߐޕ‬㧘Glc2Man9GlcNAc2ߪGlc’ase IIߦࠃ

ࠅGlc1Man9GlcNAc2㧘Man9GlcNAc2߳ߣ㗅ᰴ࠻࡝ࡒࡦࠣ

߮ߚ‫∛࡯ࡑࠗࡂ࠷࡞ࠕޕ‬㧘ࡄ࡯ࠠࡦ࠰ࡦ∛㧘ࠢࡠࠗ࠷ࡈ
ࠚ࡞࠻−ࡗࠦࡉ∛╬㧘᭽‫⚻␹ߥޘ‬ᄌᕈ∔ᖚߩේ࿃ߪ㧘ਇ↪࠲ࡦࡄࠢ⾰ߩ⫾Ⓧߢ޽ࠆߣ⠨߃ࠄࠇߡ߅ࠅ㧘࡙ࡆࠠ࠴ࡦൻࠍ੺ߒߚಽ⸃ᯏ᭴ߣߩ㑐ㅪ߇ᵈ⋡ߐࠇߡ޿ࠆ‫ޕ‬ߐࠇࠆ‫ߩߎޕ‬㑆㧘♧㎮ࠍ⹺⼂ߔࠆಽሶࠪࡖࡍࡠࡦCNX෸
8)

߮CRTߪ㧘Glc1Man9GlcNAc2ဳ♧㎮ࠍ⹺⼂ߒ 㧘ࠫࠬ࡞ ᣂ↢࠲ࡦࡄࠢ⾰ߩ 30㧑એ਄ߪ㧘ᱜᏱߥ㜞ᰴ᭴ㅧߦ⥋ࠄߕ⚦⢩ౝߢಽ⸃ߐࠇߡ޿ࠆ‫ޕ‬ᓥߞߡㆮવሶߩ⸃ᨆߩߺߥ

ࡈࠖ࠼ࠗ࠰ࡔ࡜࡯࠯ߣ౒หߒߡ♧࠲ࡦࡄࠢ⾰ࠍᱜᏱߥ᛬
9)ࠄߕ㧘ߘߩ⠡⸶ᓟߩㆊ⒟ࠍℂ⸃ߒߥߌࠇ߫㧘↢๮⃻⽎ߩࠅ⇥ߺ߳ߣዉߊ ‫ޕ‬᛬ࠅ⇥ߺᧂቢᚑߥMan9GlcNAc2♧࠲ࡦࡄࠢ⾰ߪ㧘UDP-ࠣ࡞ࠦ࡯ࠬ♧࠲ࡦࡄࠢ⾰ࠣ࡞ࠦ࡯ࠬォ⸃᣿ߦߪ⥋ࠄߥ޿‫ߩ⾰ࠢࡄࡦ࠲ޕ‬㜞ᰴ᭴ㅧᒻᚑ߆ࠄಽ⸃
߹ߢ㧘↢๮ᵴേߩᩮᐙࠍᚑߔ࠲ࡦࡄࠢ⾰ߩຠ⾰▤ℂᯏ᭴

⒖㉂⚛(UGGT)ߦࠃࠅGlcࠍઃടߐࠇ㧘ౣ߮CNX෸߮CRTߩࠪࡖࡍࡠࡦ૞↪ࠍฃߌࠆ10)‫ߩߎޕ‬㓙UGGTߪ㧘᛬ࠅ⇥ߩ⸃᣿ߪ㧘ㄭᐕߩ↢๮⑼ቇߦ߅ߌࠆ㊀ⷐߥ࠹࡯ࡑߩ৻ߟ
ߢ޽ࠆ‫ޕ‬

ߺㅜ਄ߩ♧࠲ࡦࡄࠢ⾰ߩߺࠍၮ⾰ߣߔࠆ੐߇⍮ࠄࠇߡ߅࿑㧝 ♧࠲ࡦࡄࠢ⾰ຠ⾰▤ℂᯏ᭴

㧙㧙



㧟㧚0⚿วဳ♧࠲ࡦࡄࠢ⾰㑐ㅪಽሶࡊࡠ࡯ࡉߩวᚑ

 ൻቇวᚑࠍ↪޿ࠇ߫ේℂ⊛ߦߪဋ৻᭴ㅧߩࠝ࡝ࠧ♧ࠍᄢ㊂ߦଏ⛎ߔࠆߎߣ߇ߢ߈ࠆ߇㧘㜞ࡑࡦࡁ࡯ࠬဳ♧㎮ߩࠃ߁ߥⶄ㔀ߥࠝ࡝ࠧ♧ߩోวᚑߦߪ㧘㐳ᦼ㑆ࠍⷐߒᛛⴚ

⊛ߥ࿎㔍ߣ⤘ᄢߥᎿ⒟ᢙ߇઻߁‫ౕޕ‬૕⊛ߦߪ✎ኒߦ࠺ࠩࠗࡦߐࠇߚන♧ࡈ࡜ࠣࡔࡦ࠻ߩ⺞⵾㧘ฦࡈ࡜ࠣࡔࡦ࠻ߩ

ࠞ࠶ࡊ࡝ࡦࠣ㧘⣕଻⼔ࠍ➅ࠅ㄰ߒࠝ࡝ࠧ♧วᚑࠍⴕߞߡ

޿ߊߎߣߦߥࠆ‫ߦ․ޕ‬ᚒ‫ ࠆߔߣ࠻࠶ࠥ࡯࠲߇ޘ‬N-⚿วဳ♧㎮ߩ႐ว㧘ߘߩ᭴ㅧᄙ᭽ᕈࠁ߃ߦ୘‫♧ߩޘ‬㎮ࠍ߭ߣߟ߭ߣߟวᚑߒߡ޿ߊߎߣߪ㕖Ᏹߦᚻ㑆ߩ߆߆ࠆ૞ᬺߢ޽ ߎߩᣇᴺߪ10gએ਄ߩࠬࠤ࡯࡞ߢ߽Manβ(1→4)GlcNAc

ࠆ‫ߥ߁ࠃߩߎޕ‬࿎㔍ࠍస᦯ߔࠆߚ߼ߦࡄ࡜࡟࡞วᚑ߿⥄⚿วࠍቢ⅜ߥ┙૕ㆬᛯᕈ߆ߟ80%એ਄ߩ෼₸ߢ᭴▽ߢ߈16)㧘ࠆ㕖Ᏹߦ᦭↪ߥᣇᴺߢ޽ࠆ‫ޕ‬ታ㓙㧘ᧄᴺࠍ㎛෻ᔕߣߒߡ

޿߹ߛ㤡᣿ᦼߢ޽ࠅ㧘⃻ᤨὐߢⶄ㔀ߥ♧㎮ߩวᚑᴺߣߒ

ࠫࠪࠕ࡝࡞ൻⶄวဳ♧㎮㧔1㧕㧔ᑼ㧞㧕ߩวᚑࠍ㆐ᚑߒ20)㧘

ߡᦨㆡߥㆬᛯ⢇ߣߪߥࠅᓧߡ޿ߥ޿‫ߢߎߘޕ‬ታ㓙ߩวᚑߘߩታ↪ᕈࠍ⸽᣿ߒߡ޿ࠆ‫ޕ‬ᚒ‫ߩ⥄⁛ߩߎߪޘ‬IADᴺࠍߦ߅޿ߡᦨᄢ㒢ߩല₸ൻࠍ⋡ᜰߔߣ㧘ലᨐ⊛ߥ౒ㅢਛ㑆ᱞེߦ♧࠲ࡦࡄࠢ⾰ຠ⾰▤ℂߦଥࠆ㜞ࡑࡦࡁ࡯ࠬဳ♧㎮૕ߩ࠺ࠩࠗࡦߣ෼ᢢ⊛ߥวᚑ࡞࡯࠻ߩ᭴▽߇㕖Ᏹߦ㊀ⷐ

ߩ✂⟜⊛ߥวᚑࠍⴕ޿㧘ߐࠄߦߘࠇࠄࠍੱᎿ♧࠲ࡦࡄࠢߦߥߞߡߊࠆ‫ޕ‬⾰߿ᯏ⢻ᕈ♧㎮ࡊࡠ࡯ࡉ߳ߣዷ㐿ߒߡ㧘♧㎮↢๮⃻⽎ߩ ߹ߚࠝ࡝ࠧ♧วᚑߦ߅޿ߡᦨ߽⇐ᗧߔࠆߴ߈ᩮᧄ⊛ߥ

ቯ㊂⸃ᨆ߳ߩ⿷߇߆ࠅߦߔࠆߎߣࠍ⋡ᜰߒߚ‫ޕ‬േวᚑߩታ⃻ߦะߌߚ⎇ⓥ߇⋓ࠎߦⴕࠊࠇߡ޿ࠆ߇

໧㗴ὐߪ㧘O-ࠣ࡝ࠦࠪ࠼⚿วߩ┙૕ㆬᛯ⊛ߥ᭴▽ߢ޽ࠆ‫ޕ‬ߥ߆ߢ߽᭽‫ ߥޘ‬N-⚿วဳ♧㎮ߦ౒ㅢߒߡሽ࿷ߔࠆβ-ࡑ
ࡦࡁࠪ࠼⚿ว㧔࿑㧞ਅ✢ㇱ㧕ߩ᭴▽ߪ߽ߞߣ߽࿎㔍ߥ߽ߩߩ߭ߣߟߢ޽ࠅ㧘┙૕ㆬᛯ⊛߆ߟ㜞෼₸ߥวᚑᴺߩ㐿⊒߇ᒝߊᦸ߹ࠇߡ޿ࠆ 17)‫ޕ‬ᚒ‫ߩߎߪޘ‬໧㗴ࠍ⸃᳿ߔࠆߚ
߼ߦಽሶౝࠕࠣ࡝ࠦࡦォ⒖ᴺߦᵈ⋡ߒߚ‫ߩߎޕ‬ᣇᴺߪ
ర‫ޘ‬Barresi ߣ Hindsgaul ࠄߦࠃߞߡឭ᩺ߐࠇߚ᭎ᔨߢ޽

ࠆ߇ 18)㧘ᚒ‫ߦࠇߎߪޘ‬ᡷ⦟ࠍട߃㧘♧ଏਈ૕ߩ O-2 ૏ߦp-ࡔ࠻ࠠࠪࡌࡦࠫ࡞ၮࠍ᨞ᯅ㎮ߣߒߡዉ౉ߔࠆߎߣߢ㧘ߎࠇ߹ߢߢᦨ߽ല₸⊛ߥಽሶౝࠕࠣ࡝ࠦࡦォ⒖ᴺࠍ㐿⊒ 㜞ࡑࡦࡁ࡯ࠬဳ♧㎮ࡊࡠ࡯ࡉ  ߒߚ㧔ᑼ㧝㧕19)‫ޕ‬ ᚒ‫⾰ࠢࡄࡦ࠲♧ߪޘ‬ຠ⾰▤ℂߦ߅ߌࠆ♧㎮-࠲ࡦࡄࠢ⾰⋧੕૞↪⸃ᨆߦ↪޿ࠆၮᧄ࠷࡯࡞ߣߒߡ㧘߹ߕዊ⢩૕ဳ㜞ࡑࡦࡁ࡯ࠬ♧㎮ߩ♽⛔⊛ߥวᚑࠍⴕߞߚ㧔ࠬࠠ࡯ࡓ㧝㧕‫ޕ‬ዊ⢩૕ౝߦߪGlc’ase I߅ࠃ߮II㧘Man’ase I߅ࠃ߮II㧘

UGGTߥߤ᭽‫♧ߥޘ‬㎮ࡊࡠ࠮࠶ࠪࡦࠣ㉂⚛ߩ௛߈ߦࠃࠅ㧘ᄙ᭽ߥ㜞ࡑࡦࡁ࡯ࠬ♧㎮᭴ㅧ߇ሽ࿷ߒߡ޿ࠆ‫ᦨޕ‬ೋߩ࠲࡯ࠥ࠶࠻ߣߒߡCNX/CRTߩ࡝ࠟࡦ࠼♧㎮ߢ޽ࠆࡕࡁࠣ
࡞ࠦࠪ࡞ൻ12♧㧘ߔߥࠊߜGlc1Man9GlcNAc2 (18)ࠍㆬᛯ

ߒߚ3a)‫ߩߎޕ‬ಽሶߪࡈ࡜ࠣࡔࡦ࠻A (2)㧘B (3)㧘C (4)㧘D (8)ࠍ㗅ᰴࠞ࠶ࡊ࡝ࡦࠣߔࠆ෼ᢢ⊛ߥ࡞࡯࠻ߢวᚑߒߚ‫ࡈޕ‬࡜ࠣࡔࡦ࠻A (2) ߩวᚑߪ೨ㅀߩIADᴺࠍ㎛ߣߒߡⴕ޿㧘࿑㧞 ౖဳ⊛ߥ 0⚿วဳ♧㎮ߩ᭴ㅧࡈ࡜ࠣࡔࡦ࠻B㧘Cߣߩࠞ࠶ࡊ࡝ࡦࠣࠍ⚻ߡ11♧ߣߒ㧘⿥㜞࿶᧦ઙߦࠃࠆ⣕ࠪ࡝࡞ൻ21)ࠍⴕ߁ߎߣߢൻว‛12ࠍᓧߚ‫ߊߠߟޕ‬㜞┙૕ㆬᛯ⊛ߥα-Glcᱷၮߩዉ౉ߪ♧ଏਈ૕ߣߒߡ㧤ࠍ↪޿ࠆߎߣߢ㆐ᚑߒ㧘଻⼔ၮߩ⣕଻⼔ࠍ⚻ߡ⋡⊛‛(18)ࠍᓧߚ‫ޕ‬㧙㧙ࠬࠠ࡯ࡓ㧝 㜞ࡑࡦࡁ࡯ࠬဳ♧㎮ߩ෼ᢢ⊛วᚑ Reagents and conditions; (a) MeOTf, ClCH2CH2Cl, 82 %. (b) p-TsOH,
CH3CN, 74%. (c) Cp2HfCl2, AgOTf, toluene, ~87%. (d) Ac2O, pyr., DMAP, ~98%. (e) 10% HF•pyr, DMF, 1GPa, ~95%. (f) MeOTf,
DTBMP, ClCH2CH2Cl-cyclohexane, ~93%. (g) ethylenediamine, n-BuOH, 80°C. (h) Ac2O, pyr., 0°C. (i) NaOMe, MeOH, 50°C. (j)Pd(OH)2/C, H2, 50% AcOH, ~54%, 4 steps. (k) ethylenediamine, n-BuOH, 80 °C. (l) Ac2O, pyr., DMAP, ~94% (2 steps). (m)[Ir(COD)(PMePh2)2]PF6, H2, THF. (n) I2, NaHCO3, H2O-THF. (o) Ac2O, pyr., ~97% (3 steps). (p) Pd(OH)2/C, H2, 60% AcOH-EtOH.
(q) NaOMe, MeOH, ~89% (2 steps).

㧙㧙 㧢♧(11)ߪ᳢↪ᕈߩ޽ࠆ㎛ਛ㑆૕ߢ޽ࠅ㧘ߎߩൻว‛ߪࠪࠬ࠹ࠗࡦࠍ৻ࠞᚲ᦭ߔࠆᲧセ⊛ዊߐߥ(28kDa)࠲ࡦ

ߦኻߒߡ೎ㅜวᚑߒߚࡈ࡜ࠣࡔࡦ࠻ C (4-7)㧘D(8-10)ࠍࠞ

ࡄࠢ⾰ߢ޽ࠆ‫ߦ⾰ࠢࡄࡦ࠲ߩߎޕ‬ኻߒߡᄌᕈ᧦ઙਅ㧘ㆶ

3c)

3c)

3b)

3b)

࠶ࡊ࡝ࡦࠣߒߡ޿ߊߎߣߢ㧘16 㧘17 㧘19 㧘20 㧘21㧘
3a)

3b)

ర♧(18b, 22b, 23b)߆ࠄࠣ࡝ࠦࠪ࡞ࠕࡒࡦ(26)ࠍ⚻ߡ⺞⵾3b)

22 㧘23 㧘24 㧘25 ߽߶߷ห᭽ߩ࡞࡯࠻ߢวᚑࠍ㆐ᚑ

ߒߚ৻ㅪߩ࡛࡯࠼ࠕ࠮࠻ࠕࡒ࠼(27)ࠍ෻ᔕߐߖࠆߣ㧘ㅦߒߚ‫ࠅࠃߦࠇߎޕ‬ਥⷐߥዊ⢩૕ဳ♧㎮ࠍోߡ᭴ㅧ߇⏕ታ߿߆ߦ⋡⊛ߩੱᎿ♧࠲ࡦࡄࠢ⾰(28)ߩ↢ᚑ߇⏕⹺ߐࠇߚߥวᚑຠߢឥ߃ࠆߎߣ߇ߢ߈ߚ‫ޕ‬

5b)

‫ޕ‬

 ╙ੑߩࠕࡊࡠ࡯࠴ߣߒߡᚒ‫ࠢࡄࡦ࠲ߪޘ‬-࡝ࠟࡦ࠼㑆 ੱᎿ♧࠲ࡦࡄࠢ⾰ࡊࡠ࡯ࡉ5)ߩᒝജߥ⋧੕૞↪ࠍ೑↪ߔࠆࠕࡊࡠ࡯࠴ࠍᬌ⸛ߒߚ㧔ࠬࠠ࡯ࡓ㧞㧘ࠕࡊࡠ࡯࠴ B㧕‫㉄⪲ࡠ࠼ࡅࠫߪߦ⊛⋡ߩߎޕ‬ㆶ ᄤὼߩ♧࠲ࡦࡄࠢ⾰ߪㅢᏱ㧘᭽‫ߩࡓ࡯ࠜࡈࠦ࡝ࠣߥޘ‬ᷙว‛ߣߒߡሽ࿷ߒߡ߅ࠅ 22)㧘࠲ࡦࡄࠢ਄ߩ୘೎ߩ♧㎮ర㉂⚛㧔DHFR㧕ߦኻߔࠆࡔ࠻࠻࡟ࠠ࠮࡯࠻ (MTX) ߩ⚿ว (KD<1nM)26)ࠍᵴ↪ߒߚ‫ޕ‬᭽‫♧ࠧ࡝ࠝߥޘ‬-MTX ࠦࡦᯏ⢻ࠍ⸃ᨆߔࠆߎߣߪ࿎㔍ߢ޽ࠆ‫ޕ‬ℂ⺰⊛ߦߪൻቇวᚑ
ߦࠃߞߡဋ৻ߥ♧࠲ࡦࡄࠢ⾰ࠍଏ⛎ߔࠆߎߣߪน⢻ߛ߇㧘ࠫࡘࠥ࡯࠻(30)ߩଏ⛎ߪ㧘ࠣ࡝ࠦࠪ࡞ࠕࡒࡦ(26)ࠍ಴⊒‛♧࠲ࡦࡄࠢ⾰ߩోวᚑߪㄭᐕߩ㜞࡟ࡌ࡞ߥ♖ኒ᦭ᯏวᚑ
ߩജࠍ߽ߞߡߒߡ߽㕖Ᏹߦ࿎㔍ߥ⺖㗴ߢ޽ࠆ 23)‫৻ޕ‬ᣇ㧘♧㎮ࠍ㕖ᄤὼဳߩ⚿ว᭽ᑼߢᄤὼ࠲ࡦࡄࠢ⾰਄ߦዉ౉ߔ
ࠆੱᎿ♧࠲ࡦࡄࠢ⾰24)ߪ㧘⸃ᨆ࠷࡯࡞ߣߒߡߩ♧࠲ࡦࡄ⾰ߣߒߡࠣ࡝ࠪࡦ࡝ࡦࠞ࡯ࠍ੺ߒߡ MTX ࠍዉ౉ߔࠆߎ
ߣߢ㆐ᚑߒߚ‫ ߽ࠇߕ޿ߪࠄࠇߎޕ‬DHFR ߦኻߔࠆᒝ޿ⷫ

๺ᕈࠍ଻ᜬߒߡ߅ࠅ㧘ੱᎿ♧࠲ࡦࡄࠢ⾰(31)ࠍਈ߃ߚ 5)‫ޕ‬ ߐࠄߦࠕࡊࡠ࡯࠴ A ߣ B ࠍ⚵ߺวࠊߖࠆߎߣߢ㧘⇣ߥࠢ⾰ߩଏ⛎ߣ޿߁ⷰὐ߆ࠄߪ㧘ߪࠆ߆ߦ⃻ታ⊛ߢ޽ࠆ‫ޕ‬ࠆ߰ߚߟߩ♧㎮ࠍ᦭ߔࠆੱᎿ♧࠲ࡦࡄࠢ⾰ߩഃ⵾ࠍ⹜ߺ ᦨ߽⋥ធ⊛ߥࠕࡊࡠ࡯࠴ߪࠪࠬ࠹ࠗࡦᱷၮߩ․⇣ߥ෻ᔕᕈࠍ೑↪ߒߚࠤࡒࠞ࡞࡜ࠗࠥ࡯࡚ࠪࡦ 25)ߢ޽ࠆ㧔ࠬࠠߚ‫ޕ‬152 ⇟ߩࠕࡒࡁ㉄ߦ৻ࠞᚲߩࠪࠬ࠹ࠗࡦᱷၮࠍ᦭ߔ

࡯ࡓ㧞㧘ࠕࡊࡠ࡯࠴ A㧕‫ߩߎޕ‬ᣇᴺ߇ⶄ㔀ߥࠝ࡝ࠧ♧ߦㆡ

࠼(27A)ࠍಣℂߔࠆߣ G1M9-DHFR(32A)ࠍਈ߃ߚ‫ߩߘޕ‬ࠆ E. coli. DHFR ߣ Glc1Man9GlcNAc2 ࡛࡯࠼ࠕ࠮࠻ࠕࡒ

↪น⢻߆ߤ߁߆ࠍታ⸽ߔࠆߚ߼ߦ㧘὇㉄⣕᳓⚛㉂⚛
(CA)ߦኻߔࠆ㜞ࡑࡦࡁ࡯ࠬ♧㎮ߩዉ౉ࠍᬌ⸛ߒߚ‫ޕ‬CA

ᓟ㧘ࠝ࡝ࠧ♧-MTX ࠍ૞↪ߐߖࠆߣ㧘⋡⊛ߣߔࠆ⇣ߥࠆ߰ߚߟߩ♧㎮ࠍ᦭ߔࠆ DHFR (33)ߩഃ⵾ߦᚑഞߒߚ 27)‫ޕ‬ࠬࠠ࡯ࡓ㧞 ੱᎿ♧࠲ࡦࡄࠢ⾰ߩഃ⵾ Reagents and conditions : (a) sat. aq. NH4HCO3, 40°C (b) [CH2(I)CO]2O, NaHCO3,
DMF, 0°C (74%, 2 steps) (c) FmocGlyCl, NaHCO3, dioxane-H2O(1:1), 0°C (d) Piperidine, DMF (~80%, 3 steps) (e) MTX(αtBu),
DMT-MM, MeOH-H2O (1:1) (f) CF3COOH (~65%, 2 steps) (g) CA, 5M Guanidine, 4°C, 12h (h) DHFR, 2M Guanidine, 4°C, 4h, (i)DHFR, rt, < 5min (j) 35A, rt, < 5min.




㧙㧙

ࠇߥ޿᭴ㅧ߇ᔅⷐߢ޽ࠆ‫ߢߎߘޕ‬ᚒ‫ ߩࠕࠦߪޘ‬3 ♧᭴ㅧ ࠞ࡞࡟࠹ࠖࠠࡘ࡝ࡦߩ㒖ኂ೷ 4c)ߦ㧞ᧄߩ Glc1Man3 ♧㎮ࠍዉ౉ߒߚൻว‛ 34 ࠍ⸳⸘ߒߚ ዊ⢩૕♧࠲ࡦࡄࠢ⾰ຠ⾰▤ℂߦ߅޿ߡ㧘CNX߅ࠃ߮ߘ㧔࿑㧟a㧕‫ߩߎޕ‬ൻว‛ߪ 2 ▎ᚲߩ CRT ⹺⼂᭴ㅧࠍ᦭ߔࠆ

ߩนṁᕈࡎࡕࡠࠣCRTߪ࡟ࠢ࠴ࡦᵴᕈࠍ߽ߟಽሶࠪࡖࡍ
ࡠࡦߣߒߡᄙߊߩ♧㎮↢‛ቇ⠪ߩ⥝๧ࠍᒁ߈ߟߌߡ޿ࠆ‫ޕ‬
ߎࠇࠄߪGlc1Man9GlcNAc2♧࠲ࡦࡄࠢ⾰ߩGlc1Man3♧28)㎮᭴ㅧ㧔Aࠕ࡯ࡓ㧕ࠍ․ߦᒝߊ⹺⼂ߒߡ޿ࠆ ‫ޕ‬ᚒ‫ߪޘ‬

ߎߣߢ CRT ߣߩⷫ๺ᕈࠍ⁓޿㧘߆ߟ㕖ㆶరᧃ┵ߦࡑࡦࡁ࡯ࠬᱷၮࠍᜬߚߥ޿ߚ߼㧘ER-Man’ase ߦࠃࠆಽ⸃ࠍฃߌߥ޿‫ ߦࠄߐޕ‬ER-Glc’ase II ߩᵴᕈߦ㜞ࡑࡦࡁ࡯ࠬ♧㎮ਛ೨ ㅀ ߩ 㜞 ࡑ ࡦ ࡁ ࡯ ࠬ ♧ ㎮ ว ᚑ ࠍ ၮ ⋚ ߣ ߒ ߡ ER ౝ ߢߩ B㧘C ࠕ࡯ࡓ㧔ಽጘဳࡑࡦࡁࠝ࡝ࠧ♧㧕᭴ㅧ߇ᔅ㗇ߣ߅ࠃ߮วᚑࠍ⹜ߺߚ‫ߪߦ߼ߚߩߎޕ‬CNX/CRTߩ⹺⼂᭴ㅧ߇ᦼᓙߐࠇߚ‫ޕ‬วᚑߒߚ Glc2Man7GlcNAc2 (34)ߪ CRT ߦኻ

CNX/CRTߩ⹺⼂⚻〝ࠍ․⇣⊛ߦ㒖ኂߔࠆൻว‛ߩ⸳⸘ࠍ᦭ߒ㧘߆ߟઁߩ♧㎮ࡊࡠ࠮࠶ࠪࡦࠣ㉂⚛ߦࠃߞߡಽ⸃ߐ

⸒ࠊࠇߡ޿ࠆߚ߼㧘Glc’ase II ߦࠃࠆಽ⸃߽ฃߌߥ޿ߎߣ
ߒߡ Glc1Man9GlcNAc2 ߣหߓ⚿วࠝ࡯࠳࡯(KB~106M-1)

ߢ⹺⼂ߐࠇ㧔࿑㧟a㧕㧘߆ߟ Glc’ase II ߩಽ⸃ߪోߊฃߌߥ
޿ߎߣ߇⏕⹺ߐࠇߚ㧔࿑㧟b㧕4c)‫ߩߎߚ߹ޕ‬ൻว‛ 34 ߪታ㓙ߦ CRT ߩࠪࡖࡍࡠࡦᵴᕈࠍ㒖ኂߔࠆߎߣ߽᣿ࠄ߆
ߦߥߞߚ‫ᧄࠅࠃߦࠇߎޕ‬ൻว‛ߪ CNX/CRT ․⇣⊛ߥ㒖ኂ೷ߣߒߡዊ⢩૕♧࠲ࡦࡄࠢ⾰ຠ⾰▤ℂᯏ᭴ࠍ⺞▵ߢ߈ࠆน⢻ᕈ߇␜ໂߐࠇߚ‫ޕ‬ ࡈࠜ࡯࡞࠺ࠖࡦࠣ࠮ࡦࠨ࡯‫ޟ‬7))6‫ߩޠ‬วᚑၮ⾰ 6b)
 UGGT ߪዊ⢩૕♧࠲ࡦࡄࠢ⾰ຠ⾰▤ℂߩਛᔃߢ޽ࠆ

CNX/CRT ࠨࠗࠢ࡞ߦ߅޿ߡࡈࠜ࡯࡞࠺ࠖࡦࠣ࠮ࡦࠨ࡯
ߩᓎഀࠍᨐߚߒߡ޿ࠆ 11)‫ᧄޕ‬㉂⚛ߪࠣ࡞ࠦ࡯ࠬࠍᜬߚߥ
޿㜞ࡑࡦࡁ࡯ࠬ♧࠲ࡦࡄࠢ⾰ (Man9-7GlcNAc2) ߩ࠲ࡦࡄࠢ㜞ᰴ᭴ㅧࠍ⼂೎ߒ㧘ࡈࠜ࡯࡞࠺ࠖࡦࠣᧂቢᚑߩ߽ߩߩߺࠍ ࠣ࡞ࠦࠪ࡞ൻߒߡ CNX/CRT ߩ࡝ ࠟࡦ࠼ ♧㎮(Glc1Man9-7GlcNAc2)ࠍౣ↢ߔࠆߎߣߢ ER ౝߦ⇐߼߅ߊᯏ⢻ࠍᜬߞߡ޿ࠆ‫ߩߘޕ‬ၮ⾰․⇣ᕈߪ㕖Ᏹߦ࠺࡝ࠤ࡯
࠻ߢ㧘ࡈࠜ࡯࡞࠼ߒߚ♧࠲ࡦࡄࠢ⾰߿ቢోߦࡒࠬࡈࠜ࡯
࡞࠼ߒߚ߽ߩߪၮ⾰ߣߒߡ⹺⼂ߐࠇߥ޿ 29)‫ޕ‬UGGT ߩᯏ⢻⸃ᨆߪ㕖Ᏹߦ㊀ⷐߢ޽ࠅ㧘ᣧᕆߥ⸃᣿߇ᦸ߹ࠇߡ޿ࠆ
ߦ߽߆߆ࠊࠄߕ㧘ߎߩࠃ߁ߥ․ᱶߥၮ⾰․⇣ᕈߩߚ߼ߦ
⹦⚦ߥᬌ⸛߇ㅴࠎߢ޿ߥ޿ߩ߇⃻⁁ߢ޽ࠆ‫ޕ‬ ᦨㄭ㧘ᚒ‫ߪޘ‬೨ㅀߩ Man9GlcNAc2-MTX ࠦࡦࠫࡘࠥ࡯
࠻(30E)߇ UGGT ߩ⦟ᅢߥၮ⾰ߣߥࠆߎߣࠍ⊒⷗ߒߚ㧔࿑㧠a㧕5b)‫ߜࠊߥߔޕ‬㧘ࡊࡠࡇ࡞ၮࠍࠕࠣ࡝ࠦࡦߣߒߡ߽ߟ
Man9GlcNAc2 (22a)ߪ UGGT ⸃ᨆߦ᳢↪ߐࠇߡ޿ࠆࡕ࠺
࡞♧࠲ࡦࡄࠢ⾰‫ޟ‬ᄌᕈ࠴ࡠࠣࡠࡉ࡝ࡦ(dNTg)‫ߦޠ‬Ყߴߡૐ޿ Glc ォ⒖⢻ߦ⇐߹ߞߡ޿ࠆ৻ᣇ㧘MTX ࠦࡦࠫࡘࠥ࡯
࿑㧟 ࠞ࡞࡟࠹ࠖࠠࡘ࡝ࡦߩวᚑ㒖ኂ೷(a) The structureof CRT inhibitor 34 and binding constant for CRT-33
interaction. (b) MALDI-TOF MS spectra for the time courseof glucosidase II mediated Glc-trimming reactions to 34 and
Glc1Man9GlcNAc2 (22a).

࠻(30E)ߪ UGGT ߦኻߒߡ dNTg ߣห╬એ਄ߩ Glc ォ⒖⢻ࠍ᦭ߒ㧘ዊಽሶࠕࠣ࡝ࠦࡦဳߩೋߩታ↪⊛ߥၮ⾰ߢ޽ࠆߎߣ߇ಽ߆ߞߚ‫ޕ‬ᄌᕈ࠴ࡠࠣࡠࡉ࡝ࡦࠍၮ⾰ߣߒߚ⸃ᨆ
ߢߪ㧘࠲ࡦࡄࠢ⴫㕙ߦ⚂ 8 ᧄሽ࿷ߔࠆ㜞ࡑࡦࡁ࡯ࠬ♧㎮߇ᷙว‛ߢ޽ࠆߚ߼㧘ಽሶ࡟ࡌ࡞ߢߩ⸃ᨆߦᡰ㓚ࠍ߈ߚ㧙㧙

Glc1Man8(B)GlcNAc2 (19a)ߪ Glc1Man9GlcNAc2 ࠃࠅⷫ๺ᕈ߇ૐ޿ߎߣ߆ࠄ㧘CRT ߣߩ⚿วߦ߅޿ߡ GlcMan3᭴ㅧߦട߃ߡ Man5 ಽጘ᭴ㅧ߇ੑᰴ⊛ߦ㑐ਈߒߡ޿ࠆߎ
ߣ߇᣿ࠄ߆ߣߥߞߚ‫ޕ‬ ߹ߚ㧘Glc’ase II ߦኻߔࠆ⠴ᕈ߇ᦼᓙߐࠇࠆࡈ࠶⚛⟎឵ဳ㘃✼૕ 36 ߪ㧘CRT ߣߩ⚿ว߇᷹ⷰߐࠇߚ߽ߩߩ㧘

Glc1Man3 ᭴ㅧࠃࠅ߽ⷫ๺ᕈ߇ૐ޿ߣ޿߁⚿ᨐ߇ᓧࠄࠇ

ߡ޿ࠆ 4b)‫ޕ‬࿑㧡 *0/4 ߦࠃࠆ %46 ߣ )NE/CP)NE0#E ߩ⋧੕૞↪
⸃ᨆ (a) Anomeric region of 1D 1H NMR spectra acquiredof Glc1Man9GlcNAc2 (18a) in 2H2O, 10 mMTris-HCl buffer,
10 mM CaCl2 at pH 7.3. CRT was added to the sample
stepwise; the final ratio of 18a and CRT was 1:1. (B)Anomeric region of 1D 1H NMR spectra acquired of 18a +18a’ (1:1) without and with 0.5 equiv of CRT.

ࡄࠢ⾰ߩߺߦኻߒߡࠪࡖࡍࡠࡦ૞↪ࠍⴕ߁ߎߣ߇␜ߐࠇߚ‫ޕ‬ ߐࠄߦ CRT ߩ♧㎮⹺⼂᭽ᑼߦߟ޿ߡ⠨ኤߔࠆߣ㧘CRT
ߪ Glc1Man9GlcNAc2 ߩㇱಽ᭴ㅧߢ޽ࠆ Glc1Man3(35)ߣ

ᒝߊ⚿วߔࠆߎߣ߆ࠄ㧘CRT ߪ㕖ㆶరᧃ┵ߩ GlcMan3࿑㧢 +6% ߦࠃࠆ %46 ߣวᚑ♧㎮ߩ⋧੕૞↪⸃ᨆ(a) Titration was conducted by injecting 6 µl of
Glc1Man9GlcNAc2 (300 µM in MOPs buffer) every 3 min
into buffer solution (MOPs) containing CRT (28 µM). Molar
heat values plotted as a function of the molar ratio([Glc1Man9GlcNAc2]/[CRT]). The solid line represents thebest-fit binding isotherm. The data were fitted using a single
site model. (b) Binding constants of carbohydrate-CRT
interactions were summarized.

᭴ㅧࠍਥߦ⹺⼂ߒߡ޿ࠆߣ⠨߃ࠄࠇࠆ 28)‫ߚ߹ޕ‬㧘GlcMan3᭴ ㅧ ࠍ 㧞 ଔ ߦ ߒ ߚ 㕖 ᄤ ὼ ဳ ߩ 344c) ߿ ᄤ ὼ ဳ ߩ㧙㧙ⴕ޿㧘ߘߩ♧ォ⒖↢ᚑ‛ߩ᭴ㅧࠍ HPLC㧘MS㧘NMR ࠍߒߡ޿ߚ‫ޕ‬ᚒ‫ߪޘ‬᭴ㅧ⏕ታߥวᚑ♧㎮ߩ․ᓽࠍᵴ߆ߒ㧘
Man9GlcNAc2-MTX㧔30E㧕ࠍၮ⾰ߣߒߡ UGGT ෻ᔕࠍ↪޿ߡೋ߼ߡൻቇ⊛ߦ᳿ቯߒߚ㧔࿑㧠b, c, d, e㧕5b)‫ޕ‬࿑㧠 วᚑၮ⾰ߦኻߔࠆ 7))6ߦࠃࠆࠣ࡞ࠦࠪ࡞ൻ෻ᔕ (a) Time course of [3H]-Glc uptake in UGGT reactions for 30E, 22a,denatured thyroglobulin (DNTg) and native thyroglobulin (NTg) using UDP[3H]-Glc as a donor substrate. (b) HPLC analysis[TSK-GEL Amide-80 column (0.46 φx 25 cm), 1.0 mL/min at 40 °C, solvent (CH3CN and 3% AcOH-Et3N, pH 7.3, 65:35 to 50:50)
with a linear gradient in 50 min] of the UGGT-glucosylation of 30E. (c) MALDI-TOF MS spectra of the HPLC peak I (30E) and II(corresponds to Glc1Man9GlcNAc-MTX (30A)). (d) 1H NMR spectra of synthetic 30A and the resulting UGGT product II in D2O.
HDO signal was suppressed by DANTE pre-saturation. (e) Differential NOE spectra (trace through the G:C1H resonance) of 30A
and II.

㧠㧚วᚑ♧㎮ࡊࡠ࡯ࡉࠍ↪޿ߚ♧㎮⹺⼂࠲ࡦࡄࠢ⾰♧(18a)ߩ⋧੕૞↪⸃ᨆࠍⴕߞߚ 3a)‫ޕ‬࿑㧡ߦ␜ߒߚࠃ߁ߦ㧘
CRT ߩᷝടߦࠃߞߡ♧㎮↱᧪ߩࡇ࡯ࠢ߇ోߡ CRT Ớᐲଐߩቯ㊂⊛ߥ♧㎮․⇣ᕈ⸃ᨆሽ⊛ߦᶖᄬߒߚ‫ߪࠇߎޕ‬㧘♧㎮ߩ⷗߆ߌ਄ߩಽሶ㊂߇Ⴧ ᚒ‫ߪޘ‬㧘೨ㅀߩࠃ߁ߦዊ⢩૕♧࠲ࡦࡄࠢ⾰ຠ⾰▤ℂߦᄢߒߚὑߛߣ⠨߃ࠄࠇ㧘CRT ߣ♧㎮߇ᒝߊ⚿วߒߡ޿ࠆ

㑐ࠊࠆ᭽‫♧ߥޘ‬㎮ൻว‛ࠍ♽⛔⊛ߦวᚑߒߚ‫ߢ߹ࠇߎޕ‬ߎߣࠍ␜ߒߡ޿ࠆ‫ޕ‬18a ߣߘߩβ-Glc ⇣ᕈ૕ 18a’ߩ 1:1 ᷙวᚑ♧㎮ࠍ↪޿ߚ♧࠲ࡦࡄࠢຠ⾰▤ℂᯏ᭴ߩ✂⟜⊛ߥ⸃ว‛ߦ CRT ࠍട߃ߚ㓙ߦߪ 18a ߩߺ߇ࡉࡠ࡯࠼ൻߒߚ੐

ᨆߩႎ๔ߪ⊝ήߢ޽ࠅ㧘ᚒ‫ߩ⥄⁛ߪޘ‬วᚑ♧㎮ၮ⾰ࠍಽ߆ࠄ㧘CRT ߪ Glc1Man9GlcNAc2 ♧㎮ߩ Glc ᱷၮࠍ෩ኒߦ

ሶࡊࡠ࡯ࡉߣߒߡߎߩ⺖㗴ߦขࠅ⚵ࠎߢ޿ࠆ‫⃻ޕ‬࿷ߩߣ⹺⼂ߒߡ޿ࠆߣ⠨߃ࠄࠇࠆ‫ޕ‬ߎࠈ㧘ಽሶࠪࡖࡍࡠࡦ‫ޟ‬CRT‫ޠ‬㧘࡙ࡆࠠ࠴ࡦ࡝ࠟ࡯࠯‫ޟ‬Fbs1‫ޠ‬㧘 ᰴߦ㧘วᚑߒߚ㜞ࡑࡦࡁ࡯ࠬဳ♧㎮෸߮ߘߩ㘃✼૕ߣࡈࠜ࡯࡞࠺ࠖࡦࠣ࠮ࡦࠨ࡯‫ޟ‬UGGT‫♧ߩޠ‬㎮․⇣ᕈ⸃ᨆߦ

CRT ߣߩ⋧੕૞↪ࠍ╬᷷Ṣቯࠞࡠ࡝ࡔ࠻࡝࡯(ITC)ߦࠃࠅ

߅޿ߡ㗼⪺ߥᚑᨐࠍ᜼ߍߡ޿ࠆߩߢ㧘ᧄ㗄ߢߪߘࠇࠄߦ

ቯ㊂⊛ߦ⸃ᨆߒߚ 30)‫ޕ‬Glc1Man9GlcNAc2 ♧㎮(18a)ߪ CRT
ߦ ᒝ ߊ ⚿ ว ߒ ߚ ߇ (KB = 5.2 x 106 M-1) 㧘 ห ߓ ᧦ ઙ ߢߟ޿ߡ⹦⚦ࠍ⺑᣿ߔࠆ‫ޕ‬

Glc2Man9GlcNAc2 (17a)෸߮ Man9GlcNAc2 ဳ♧㎮ (22a) ࡟ࠢ࠴ࡦ᭽ಽሶࠪࡖࡍࡠࡦ‫ޠࡦ࡝ࡘࠠࠖ࠹࡟࡞ࠞޟ‬ߩ♧㎮․⇣ᕈ 3a)4b)4c)30)
 CRT ߪ㧘Glc1Man9GlcNAc2 ♧㎮ࠍ⹺⼂ߔࠆߣ⠨߃ࠄࠇߡ޿ࠆ‫ޕ‬ᚒ‫ߪޘ‬㧘߹ߕೋ߼ߦ H NMR ࠍ↪޿ߡ CRT ߣ 121

ߣ CRT ߣߩ⋧੕૞↪ߪ᷹ⷰߐࠇߥ߆ߞߚ‫⚿ߩߎޕ‬ᨐߪ㧘⃻࿷⠨߃ࠄࠇߡ޿ࠆ CRT ߩᯏ⢻߿♧㎮ㆬᛯᕈߣ⦟ߊ৻⥌ߒߡ޿ࠆ‫ߜࠊߥߔޕ‬㧘CRT ߪ㜞ࡑࡦࡁ࡯ࠬဳ♧㎮ߩ Glc
ᱷၮߩ࠻࡝ࡒࡦࠣ⁁ᘒࠍ෩ኒߦ⷗ಽߌ㧘Glc1 ဳߩ♧࠲ࡦ㧙㧙 ࡙ࡆࠠ࠴ࡦ࡝ࠟ࡯࠯‫(ޟ‬DU‫♧ߩޠ‬㎮․⇣ᕈ 6a)(NeuAcα2-3Galβ1-4GlcNAcβ)߇ዉ౉ߐࠇߚߚ߼ߢ޽ࠆߣ ࠲ࡦࡄࠢ⾰ߩ࡙ࡆࠠ࠴ࡦൻߪಽ⸃ⵝ⟎ࡊࡠ࠹ࠕ࠰࡯ࡓ

⺑᣿ߢ߈ࠆ‫ޕ‬

߳ߩ⒖ⴕࠪࠣ࠽࡞ߢ޽ࠅ㧘࡙ࡆࠠ࠴ࡦൻ㉂⚛ߪಽ⸃߳ะ
߆߁࠲ࡦࡄࠢ⾰ߩขᝥㆬᛯࠍⴕߞߡ޿ࠆ‫ޕ‬SCFⶄว૕ߪ㧘
Skp1㧘Cullin1㧘෸߮F-box࠲ࡦࡄࠢ⾰߆ࠄᚑࠆ࡙ࡆࠠ࠴ࡦ
ൻ㉂⚛ߢ޽ࠅ㧘ၮ⾰⹺⼂ࠍⴕ߁࡙࠾࠶࠻F-box࠲ࡦࡄࠢ⾰ߩᄙ᭽ᕈߦࠃࠅ᭽‫ࡦ࠴ࠠࡆ࡙ߩ⾰ࠢࡄࡦ࠲ߥޘ‬ൻࠍⴕߞߡ޿ࠆ‫߽ߢ߆ߥޕ‬ㄭᐕ⊒⷗ߐࠇߚF-box࠲ࡦࡄࠢ⾰Fbs1
෸߮Fbs2ߪ㧘㜞ࡑࡦࡁ࡯ࠬဳ♧㎮ࠍ⹺⼂ߔࠆߣ޿߁ߎࠇ
߹ߢߦߥ޿ᯏ⢻ࠍᜬߞߡ޿ࠆ15a)‫ࠄ߆ߣߎߩߎޕ‬㧘SCFFbs
ⶄว૕ߪ㧘♧㎮ࠍ⹺⼂ߔࠆ࡙ࡆࠠ࠴ࡦൻ㉂⚛ߣߒߡᯏ⢻ߒ㧘ዊ⢩૕ߢᱜᏱߥࡈࠜ࡯࡞࠺ࠖࡦࠣߦ⥋ࠄߥ߆ߞߚ♧࠲ࡦࡄࠢ⾰ߩಽ⸃ߦ㑐ਈߒߡ޿ࠆߣ⠨߃ࠄࠇߡ޿ࠆ15)‫ޕ‬ Fbs1ߩ♧㎮⹺⼂ᯏ᭴ߦߟ޿ߡߪ㧘㜞ࡑࡦࡁ࡯ࠬဳ♧㎮ߩ ㆶ ర ᧃ ┵ ߦ ሽ ࿷ ߔ ࠆ ࠠ ࠻ ࡆ ࠝ ࡯ ࠬ ᭴ ㅧ(GlcNAcb1-4GlcNAc)ߣFbs1ߩ♧㎮⚿ว㗔ၞߣߩX✢෸߮

NMRߦࠃࠆ⚿ว᭴ㅧ⸃ᨆ߇ὑߐࠇ㧘♧㎮ߩㆶరᧃ┵஥ࠍᒝߊ⹺⼂ߔࠆߎߣ߇ಽ߆ߞߡ޿ࠆ31)‫ޕ‬ᚒ‫ߪޘ‬㧘Fbs1ߩ♧㎮⹺⼂ᯏ᭴ࠍࠃࠅ⹦⚦ߦᬌ⸛ߔࠆߚ߼㧘วᚑߒߚ৻ㅪߩ
♧㎮ࠍ↪޿ߡ㧘ITCߦࠃࠆቯ㊂⊛ߥ⋧੕૞↪⸃ᨆࠍⴕߞ
ߚ㧔࿑㧣㧕‫⚿ߩߘޕ‬ᨐ㧘Fbs1ߣߩ⚿วߪ㜞ࡑࡦࡁ࡯ࠬဳߩ
ࠦࠕㇱಽߢ޽ࠆ㧡♧᭴ㅧMan3GlcNAc2 (37)߇ᦨ߽ᒝߊ

(KB = 8.5 x 10 M )㧘ᧄ᧪Fbs1ߩၮ⾰ߣ⠨߃ࠄࠇߡ޿ࠆ㜞
5-1ࡑࡦࡁ࡯ࠬဳߩ22b(KB = 3.3 x 105 M-1)߿23b(KB = 3.2 x

105 M-1)ࠃࠅ߽ᒝߊ⹺⼂ߐࠇࠆߎߣ߇᣿ࠄ߆ߣߥߞߚ6a)‫ޕ‬࿑ 㧣  (DU ߣ ว ᚑ ♧ ㎮ ߩ ⋧ ੕ ૞ ↪ ⸃ ᨆ  (a) Binding
constants for the binding of synthetic oligosaccharides to Fbs1.
(b) Proposed structure of Man3GlcNAc2 in complex withFbs1
ߎߩߎߣ߆ࠄ㧘Man3GlcNAc2᭴ㅧ߇⹺⼂ߦᔅⷐߥ㎛㛽ᩰߢ޽ࠅ㧘ᄖ஥ߩManᱷၮߪ┙૕⊛ߦਇ೑ߦ௛ߊߣផኤߢ

 ࡈࠜ࡯࡞࠺ࠖࡦࠣ࠮ࡦࠨ࡯㉂⚛‫ޟ‬7))6‫♧ߩޠ‬㎮․߈ࠆ‫ߦࠄߐޕ‬㧘α1-6 Manᱷၮࠍ᦭ߔࠆ㧠♧(39)ߪ㜞ࡑࡦࡁ࡯ࠬဳ♧㎮22b߿23bߣห╬ߦFbs1ߣ⚿วߔࠆ(KB = 3.3

x 10 M )߇㧘α1-3 Manߩ㧠♧(40) ߢߪ⪺ߒߊ⚿ว߇ૐਅ
5-1

ߒߚ(KB = 0.63 x 10 M )ߎߣ߆ࠄ㧘α1-6 Manᱷၮ߇Fbs1
5-1

ߦࠃࠆ♧㎮⹺⼂ߦᒝߊ㑐ਈߒߡ޿ࠆߎߣ߇␜ߐࠇߚ‫߹ޕ‬ߚ㧘ㆶరᧃ┵஥ߩ♧ᱷၮ߇ᰳᅤߒߚMan8GlcNAc1♧㎮(38)߿㧘ㆶరᧃ┵ߩࠠ࠻ࡆࠝ࡯ࠬ᭴ㅧߩߺߢߪ⚿ว߇᷹ⷰߐࠇߥ߆ߞߚ‫⚿ߩࠄࠇߎޕ‬ᨐ߆ࠄ㧘ࠠ࠻ࡆࠝ࡯ࠬ᭴ㅧ
ߣα1-6 Manᱷၮ߇දᄼ⊛ߦFbs1ߣ⋧੕૞↪ߒߡ޿ࠆߣ⠨⇣ᕈ 6b) ᚒ‫߇ޘ‬㐿⊒ߒߚೋߩวᚑၮ⾰ߢ޽ࠆ♧-MTX ࠍ↪޿ߡ
UGGT ߩ♧ォ⒖෻ᔕࠍⴕ޿㧘෻ᔕㆊ⒟ࠍ HPLC ߦߡቯ㊂⊛ߦ⸃ᨆߒߚ㧔࿑㧤㧕6b)‫ޕ‬೨ㅀߩ 30E-H ߅ࠃ߮೎ㅜวᚑ
ߒߚ Man8(B)GlcNAc1-MTX(41)ࠍၮ⾰ߣߒ㧘ฦ‫ߦޘ‬ኻߔࠆ UGGT ᵴᕈࠍᲧセߒߚߣߎࠈ㧘ߘߩ♧㎮․⇣ᕈߪ
Man9GlcNAc2>Man8(B)GlcNAc2=Man8(C)GlcNAc2>Man

7GlcNAc2>>>Man8(B)GlcNAc1=0 ߣߥߞߚ㧔࿑㧤a㧕‫ߐޕ‬ࠄߦ୘‫ ߩޘ‬Km㧘Vmax ࠍ᳞߼㧘UGGT ߩ෻ᔕᕈߦ㑐ߒߡೋ߃ࠄࠇࠆ‫ޕ‬߼ߡቯ㊂⊛ߥ⸃ᨆ⚿ᨐࠍ␜ߔߎߣ߇ߢ߈ߚ㧔࿑㧤b㧕‫ޕ‬ᚒ‫ޘ‬ X✢᭴ㅧ⸃ᨆ߆ࠄᓧࠄࠇߚࠠ࠻ࡆࠝ࡯ࠬߣFbs1ߩ⚿วߩ⸃ᨆߦࠃߞߡᓧࠄࠇߚ♧㎮․⇣ᕈߪ㧘એ೨ߦ᭴ㅧߦኻߒߡMan3ᱷၮࠍട߃㧘᭴ㅧᦨㆡൻࠍⴕߞߚ‫ޕ‬Man9-7GlcNAc2 ♧㎮ߩᷙว‛ߢ޽ࠆᄌᕈ࠴ࡠࠣࡠࡉ࡝

ᓧࠄࠇߚMan3GlcNAc2-Fbs1ⶄว૕ߩࡕ࠺࡞᭴ㅧࠍ␜ߔ

ࡦࠍၮ⾰ߣߒߚ⎇ⓥߦࠃߞߡឭ໒ߐࠇߚ௑ะࠍᡰᜬߔࠆ(࿑㧣b)‫࡞࠺ࡕߩߎޕ‬᭴ㅧߪ㧘α1-6⚿วߒߚManᱷၮ߇߽ߩߢ޽ߞߚ߇㧘Man8GlcNAc2 ਔ⇣ᕈ૕ (30F ߅ࠃ߮

Asp216ߣ⋧੕૞↪ߒߡ߅ࠅ㧘ᚒ‫ߩޘ‬ታ㛎⚿ᨐ߿NMRߦࠃࠆ᭴ㅧ⸃ᨆߣ⦟޿৻⥌߇⷗ࠄࠇࠆ‫ߦࠄߐޕ‬㧘ࠦࡦࡊ࡟࠶

ࠢࠬဳߩ11♧(1)ߩ⚿ว߇ᒙߊߥߞߚ(KB = 0.24 x 105 M-1)

ߩ ߪ 㧘 α1-6 Man ᱷ ၮ ߩ 㧞 ૏ ߦ ፾ 㜞 ޿ 㧟 ♧ ᭴ ㅧ30G)ߩ෻ᔕᕈߩ㆑޿߇ߪߓ߼ߡ᣿ࠄ߆ߦߥߞߚ‫ޕ‬ਔ⇣ᕈ૕ߩ⚻ᤨ⊛ߥ Glc ขࠅㄟߺჇടߦ㑐ߒߡߪ㧘੹࿁ߩ෻ᔕ
᧦ઙߢߪ⷗߆ߌ਄㧘ห╬ߢ޽ߞߚ߇㧘Km㧘Vmax ߦߪ᣿⏕ߥ ㆑ ޿ ߇ ޽ ߞ ߚ ‫ ߚ ߹ ޕ‬Man7GlcNAc2-MTX (30H) ߪ㧙㧙Man9GlcNAc2-MTX (30E)ߣᲧセߒߡ♧ォ⒖ㅦᐲߪㆃ޿
৻ᣇ㧘Km ߪࠃࠅૐߊ㧘UGGT ߦ⹺⼂ߐࠇ߿ߔ޿ߎߣ߇ಽ

ෳ⠨ᢥ₂߆ߞߚ‫ߦࠄߐޕ‬㧘ㆶరᧃ┵ߩ GlcNAc ࠍᶖᄬߒߚ 41 ߪ1) (a)R. A. Dwek, Chem Rev., 96, 683 (1996); (b)W. J. Welch,ⷐߥᓎഀࠍᨐߚߒߡ޿ࠆߎߣ߇᣿ࠄ߆ߦߥߞߚ‫ޕ‬2) (a)A. Helenius, M. Aebi, Annu, Rev. Biochem., 73, 1019

Glc ォ⒖߇ోߊㅴⴕߖߕ㧘ߎߩㇱ૏߇ UGGT ߩ⹺⼂ߦ㊀

Semin. Cell. Dev. Biol., 15, 31 (2004)(2004); (b)R. G. Spiro, Cell. Mol. Life Sci., 61, 1025

(2004); (c)R. Sitia, I. Braakman, Nature, 426, 891 (2003)

3) (a)I. Matsuo, M. Wada, S. Manabe, Y. Yamaguchi, K.
Otake, K. Kato, Y. Ito, J. Am. Chem. Soc., 125, 3402

(2003); (b)I. Matsuo, Y. Ito,Carbohydr. Res., 338, 2168
(2003); (c)I. Matsuo, T. Kashiwagi, K. Totani, Y. Ito,

Tetrahedron Lett., 46, 4197 (2005); (d)I. Matsuo, Y. Ito,
Trends Glycosci. Glycotec., 17, 85 (2005)4) (a)M. Takatani, I. Matsuo, Y. Ito, Carbohydr. Res., 338,

1073 (2003); (b)Y. Ito, S. Hagihara, M. A. Arai, I. Matsuo,

M. Takatani,Glycoconj. J., 21, 257 (2004); (c)M. A. Arai, I.

Matsuo, S. Hagihara, K. Totani, J. Maruyama, K. Kitamoto,Y. Ito, ChemBioChem, 6, 2281 (2005)

5) (a)K. Totani, I. Matsuo, Y. Ito, Bioorg. Med. Chem. Lett.,

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࿑㧤 7))6 ߦࠃࠆࠣ࡞ࠦࠪ࡞ൻ෻ᔕߩቯ㊂⸃ᨆ(a) Relative oligosaccharide specificities of UGGT reactions.
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