課題番号：2007A1887，使用ビームライン：BL40B2
   㔍᳓ṁᕈ⮎೷⚿วߦ઻߁࡝ࡐࠞ࡝ࡦဳࡊࡠࠬ࠲ࠣ࡜ࡦࠫࡦ Dวᚑ㉂⚛㧔L-PGDS㧕ߩ᭴ㅧᄌൻ
Conformational Changes Induced in Lipocalin-type Prostaglandin DSynthase (L-PGDS) by Binding of Lipophilic Drugs.
ታ㛎⽿છ⠪㧦ੇ 㓉ᄢ㒋ᐭ┙ᄢቇᄢቇ㒮 ↢๮ⅣႺ⑼ቇ⎇ⓥ⑼ ᔕ↪↢๮⑼ቇኾ᡹
X ✢ዊⷺᢔੂᴺࠍ↪޿ߡ㧘ࠫࠕ࠯ࡄࡓ(DZP)߅ࠃ߮ NBQX ߩ㧞⒳㘃ߩ⇹᳓ᕈ⮎೷ߩ⚿วߦ઻߁㧘࡝ࡐࠞ࡝ࡦဳࡊࡠࠬ࠲ࠣ࡜ࡦࠫࡦ D วᚑ㉂⚛(L-PGDS)ߩࠦࡦࡈࠜࡔ࡯࡚ࠪࡦᄌൻࠍ⺞ᩏߒߚ‫⚿ߩߘޕ‬ᨐ㧘
L-PGDS㧘L-PGDS/DZP ⶄว૕㧘L-PGDS/NBQX ⶄว૕ߩᘠᕈඨᓘߪߘࠇߙࠇ㧘19.4 Å㧘18.2 Å㧘߅ࠃ
߮ 17.3 Å ߣߥࠅ㧘⇹᳓ᕈ⮎೷ߩ⚿วߦ઻޿㧘L-PGDS ಽሶ߇ࠦࡦࡄࠢ࠻ߦߥࠆߎߣ߇್᣿ߒߚ‫ޕ‬L-PGDSߩߎߩࠃ߁ߥᨵエᕈߪ㧘⇹᳓ᕈૐಽሶߦኻߔࠆ L-PGDS ߩ‫ޟ‬㕖ㆬᛯᕈ‫ޕࠆ޽ߢߩ߽ߔ␜ࠍޠ‬We measured small-angle x-ray scattering (SAXS) of lipocalin-type prostaglandin D synthase (L-PGDS)
to clarify their conformational changes induced by binding of small lipophilic drugs, such as diazepam(DZP) and NBQX. The radius of gyration was estimated to be 19.4 Å for L-PGDS, 18.2 Å for
L-PGDS/DZP, and 17.3 Å for L-PGDS/NBQX complexes. These results indicated that L-PGDS becamecompact after binding of these drugs and such structural flexibility of the L-PGDS molecule wasresponsible for the broad ligand selectivity of L-PGDS.
ࠠ࡯ࡢ࡯࠼㧦X ✢ዊⷺᢔੂ㧘࡝ࡐࠞ࡝ࡦဳࡊࡠࠬ࠲ࠣ࡜ࡦࠫࡦ D วᚑ㉂⚛㧘⇹᳓ᕈ⮎೷㧘㕖ㆬᛯᕈ⢛᥊ߣ⎇ⓥ⋡⊛㧦࡝ࡐࠞ࡝ࡦဳࡊࡠࠬ࠲ࠣ࡜ߩ⚿วⷫ๺ᕈࠍ⺞ߴࠆߚ߼ߦ㧘L-PGDSౝ࿃ᕈ

ࡦࠫࡦDวᚑ㉂⚛(L-PGDS)ߪ㧘⣖⣄㜑ᶧਛߦ࠻࡝ࡊ࠻ࡈࠔࡦⰯశᶖశ૞↪ߩ᷹ቯࠍⴕ޿㧘ᄙߊሽ࿷ߒ㧘⇹᳓ᕈ࡝ࠟࡦ࠼ャㅍⰮ⊕⾰⟲ߢ⸃㔌ቯᢙ(K d )߇, 70 - 140 nMߢ޽ࠆߎߣࠍ⷗

޽ࠆ࡝ࡐࠞ࡝ࡦࡈࠔࡒ࡝࡯ߦዻߔࠆⰮ⊕⾰ߢ಴ߒ㧘ฦ࡝ࠟࡦ࠼ߣ߽L-PGDSߦኻߒߡ㧘߶߷޽ࠆߣߣ߽ߦ㧘ࡊࡠࠬ࠲ࠣ࡜ࡦࠫࡦH2 (PGH 2 )ห⒟ᐲߩ㜞޿⚿วⷫ๺ᕈࠍᜬߟߎߣࠍ␜ߒߚ㧝㧕‫ޕ‬ࠍၮ⾰ߣߒߡ㧘⣖ౝߦ߅ߌࠆ⌧⌁⺃⊒‛⾰ߢએ਄ߩ⚿ᨐߪ㧘L-PGDSߩ㛳ߊߴ߈᭴ㅧ⊛ᨵエ޽ ࠆ PGD 2 ࠍ ว ᚑ ߔ ࠆ ㉂ ⚛ ߣ ߒ ߡ ߩ ᯏ ⢻ ࠍ ૬ᕈࠍ␜ߒ㧘ઁߩหዻಽሶߦߪߥ޿⇹᳓ᕈ࡝ࠟߖᜬߟᄙᯏ⢻Ⱞ⊕⾰ߢ޽ࠆ‫ޕ‬ᚒ‫ߢ߹ࠇߎߪޘ‬ࡦ࠼ߦኻߔࠆ‫ޟ‬㕖ㆬᛯᕈ‫⸽ߩߣߎࠆߔ᦭ࠍޠ‬ߩ⎇ ⓥߢ㧘SPring-8᡼ ኿శ ᣉ⸳ࠍ૶↪ ߒ㧘⇹᣿ߢ޽ࠆ‫ޕ‬᳓ᕈ࡝ࠟࡦ࠼ߢ޽ࠆࡆ࡝ࡌ࡞ࠫࡦ(BV)㧘ࡆ࡝ ᚒ‫ߪޘ‬㧘L-PGDS߇᦭ߔࠆ࡝ࠟࡦ࠼㕖ㆬᛯᕈ

࡞ࡆࡦ(BR)㧘෸߮࡟࠴ࡁࠗࡦ㉄(RA)ߣL-PGDSࠍ೑↪ߒ㧘㔍᳓ṁᕈߢ޽ࠆ߇ࠁ߃ߦ⮎೷㐿⊒ߩⶄว૕ߦߟ޿ߡ㧘㨄✢ṁᶧᢔੂᴺࠍ↪޿ߚ߇࿎㔍ߢ㧘⮎೷୥⵬߆ࠄ࠼ࡠ࠶ࡊࠕ࠙࠻ߒߡ

᭴ㅧ⸃ᨆࠍⴕ޿㧘L-PGDSߪ⚿วߔࠆ࡝ࠟࡦ࠼޿ߚ⮎ߦ⣉శࠍᒰߡ㧘ߘࠇߙࠇߩ⮎೷᭴ㅧߦߩᄢ߈ߐߦࠃߞߡߘߩ᭴ㅧࠍᄌൻߐߖࠆ⃟ߒㆡߒߚੱᎿⰮ⊕⾰ࠍ⸳⸘࡮㐿⊒ߒ㧘࠼࡜࠶ࠣ࡮޿Ⱞ⊕⾰ߢ޽ࠆߎߣࠍ⷗಴ߒߚ‫৻ޕ‬ᣇ㧘࠺࡝ࡃ࡝࡯࡮ࠪࠬ࠹ࡓ(DDS)ߦ೑↪ߔࠆߎߣL-PGDSߣ⇹᳓ᕈ࡝ࠟࡦ࠼(BV, BR, RA╬)ߣࠍ⋡ᜰߔ‫ޕ‬ታ㛎㧦ṁᶧᢔੂታ㛎ߪBL40B2ߢⴕߞߚ‫ޕ‬න⦡↪޿ߡࠡ࠾ࠛ࡮ࡊࡠ࠶࠻⸃ᨆࠍⴕ޿㧘Ⱞ⊕⾰ൻߐࠇߚX✢ࠍ㓸శࡒ࡜࡯ߦࠃߞߡ㓸శߒߚỚᐲߦኻߔࠆ Rg(C)2 (Rg㧦ᘠᕈඨᓘ)ߩᄌൻࠍᓧ

ᓟ㧘ࠬ࡝࠶࠻ߢᢛᒻߒ㧘⹜ᢱߦ౉኿ߒߚ‫ޕ‬X

ߚ(࿑㧞)‫ޕ‬Ớᐲ࠯ࡠߢᓧࠄࠇߚ L-PGDS ߩᘠ✢ߩᵄ㐳ߪ1.0Έࠍ↪޿ߚ‫ޕ‬ᬌ಴ེߪ㧘ࡆ࡯ࡓᕈඨᓘߪ㧘19.4Έߢ޽ࠅ㧘L-PGDS/DZP㧘߅ࠃ࡜ࠗࡦߦ⸳⟎ߐࠇߡ޿ࠆ⥄േ⺒ߺขࠅဳࠗ

߮ L-PGDS/NBQX ⶄว૕ߪߘࠇߙࠇ㧘18.3Έ㧘ࡔ ࡯ ࠫ ࡦ ࠣ ࡊ ࡟ ࡯ ࠻ (R-AXIS IV ++ system)ࠍ

߅ࠃ߮ 17.4Έߣߥࠅ㧘L-PGDS ߪ⮎೷ߣߩⶄ↪޿ߚ‫ޕ‬ᢔੂ᷹ቯߪ㧘Ⱞ⊕⾰Ớᐲଐሽᕈ㧘ࠦว૕ᒻᚑߦ઻޿㧘ᘠᕈඨᓘࠍᷫዋߐߖࠆߎߣ

ࡦ࠻࡜ࠬ࠻ࡃ࡝ࠛ࡯࡚ࠪࡦᴺߥߤߩฦ⒳᧦ઙ

߇್᣿ߒߚ‫ޕ‬ਅߢⴕߞߚ‫ޕ‬ಽሶ㊂᳿ቯߩߚ߼ߩ࡝ࡈࠔ࡟ࡦ
ࠬߣߒߡ㧘ovalbumin (Mr㧦45,000, Sigma)ࠍ↪

㪊㪏㪇޿ߚ‫(ࡓࡄ࠯ࠕࠫޕ‬DZP㧘Mr㧦284.7)㧘߅ࠃ߮㪊㪍㪇NBQX (Mr:336)ߪ㧘LгPGDSߣ1:1ߩࡕ࡞Ყߢ

㪊㪋㪇

-1-1ᷙวߒ㧘Ớ❗ߒߚᓟ㧘2.5 mg ml , 5.0 mg ml ,-1㩷㪣㪄㪧㪞㪛㪪㩷㪂㪥㪙㪨㪯
㩷㪂㪛㪱㪧

㪊㪉㪇

-1

8.0 mg ml , ߅ࠃ߮12.5 mg ml ߩฦỚᐲߦ⺞㪊㪇㪇ᢛߒ㧘ታ㛎ߦ↪޿ߚ‫ޕ‬

㪇

㪉

㪋

㪍

㪏

㪈㪇

㪈㪉

㪚㩷㩷㩿㫄㪾㪆㫄㫃㪀㪚㫆㫅㪺㪼㫅㫋㫉㪸㫋㫀㫆㫅㩷㫆㪽㩷㫇㫉㫆㫋㪼㫀㫅⚿ᨐߣ⠨ኤ㧦࿑㧝ߦ㧘ṁᶧਛߦ߅ߌࠆL-PGDS㧘߅ ࠃ ߮ L-PGDSߣ DZP(L-PGDS/DZP)㧘 ޽ ࠆ ޿ߪ L-PGDS ߣ NBQX(L-PGDS/NBQX) ⶄ ว ૕ ߩX✢ṁᶧᢔੂࡄ࠲࡯ࡦࠍ␜ߔ‫ޕ‬

Fig.2 Concentration dependence of R g 2 of L-PGDS
with or without NBQX and DZP. Samples used were
L-PGDS (blue circles), L-PGDS/NBQX (red circles),and L-PGDS/DZP (green circles), respectively. Data areexpressed as the mean ± S.D. of 3 independent
experiments.

એ਄ߩ⚿ᨐࠃࠅ㧘L-PGDS ߪ㧘ಽሶ㊂߇ 300
L-PGDS೨ᓟߩ⇹᳓ᕈૐಽሶ⮎೷ߩ⚿วߦ઻޿㧘ߘߩ

L-PGDS/NBQX
L-PGDS/DZPᘠᕈඨᓘ߇㧘1 - 2Έ⒟ᐲࠦࡦࡄࠢ࠻ߦߥࠆⰮ
⊕⾰ߢ޽ࠆߎߣ߇್᣿ߒߚ‫ ߩߎޕ‬L-PGDS ߩᨵエᕈߪ㧘L-PGDS ߩ⇹᳓ᕈૐಽሶߦኻߔࠆ
‫ޟ‬㕖ㆬᛯᕈ‫ࠅ޽ߢߩ߽ߔ␜ࠍޠ‬㧘L-PGDS ࠍ↪޿ߚ᭽‫ߥޘ‬㔍᳓ṁᕈ⮎೷ߦኻߔࠆ DDS ߩFig.1 SAXS profiles of L-PGDS and each L-PGDScomplex. SAXS profiles of L-PGDS (blue dots),L-PGDS/NBQX (red dots), and L-PGDS/DZP (greendots) are shown. The logarithm of scattering intensity isshown as a function of reciprocal vector (S).

น⢻ᕈࠍ␜ໂߔࠆ‫ޕ‬ෳ⠨ᢥ₂1) T. Inui et al., J. Biol. Chem. 278 (2003) 2845.

ߎࠇࠄߩᢔੂᦛ✢ࠃࠅ㧘L-PGDS ߪ⃿⁁Ⱞ⊕⾰ߢ޽ࠆߎߣ߇್᣿ߒߚ‫ߚ߹ޕ‬㧘⮎೷⚿วߦ઻ ޿ ዊ ⷺ 㗔 ၞ (S<0.02Έ -1 )ߦ ᄌ ൻ ߇ ޽ ࠆ ߎ ߣ߇⏕⹺ߐࠇߚ‫ߦࠄߐޕ‬㧘ᓧࠄࠇߚᢔੂᦛ✢ࠍ