Proteoglycan
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硫酸転移酵素

(First version published:2001年5月15日)

硫酸化グリコサミノグリカンはくり返し二糖単位の構造により、コンドロイチン硫酸 (CS)、デルマタン硫酸 (DS)、ケラタン硫酸 (KS)、ヘパラン硫酸 (HS)、ヘパリンに区分される。各グリコサミノグリカン糖鎖の骨格はエピメラーゼ、硫酸転移酵素による修飾を受けて多様な構造を作りあげている(図1)。グリコサミノグリカンの多様な構造は種々の生理的な機能と関連している。硫酸転移酵素は硫酸基の供与体である活性硫酸(3'-phosphoadenosine 5'-phosphosulfate, 略してPAPS)から繰り返し二糖の糖残基の特定の位置に硫酸基を転移し、グリコサミノグリカンの多様構造を作るうえで鍵となる役割を果たしている。今までにグリコサミノグリカン硫酸転移酵素の単離と遺伝子クローニングはほぼ完成された。


図1
Figure

硫酸転移酵素の特異性
グリコサミノグリカンの硫酸化を行う硫酸転移酵素の種類と特異性を表1に示す。表1では全ての硫酸転移酵素についてヒトの相同分子種を示したが、各遺伝子ファミリーで最初に均一精製またはクローニングされた種の酵素についても示した。硫酸転移酵素は、硫酸基が転移される糖残基中の位置を厳密に認識しているだけでなく、さらに特定の糖鎖配列を認識することがある。GalNAc4S-6STは糖鎖内部だけでなく非還元末端に存在するGalNAc(4S)-GlcA(2S) -GalNAc(6S)という配列のGalNAc(4S)残基を効率よく硫酸化する。この活性はGalNAc(4S)に隣接するGlcA(2S)により著しく活性化される 22。大部分を占めるIdoAに加えて少量のGlcAを含む脱硫酸化DSをC4ST-1の基質とするとき、C4ST-1は微量含まれるGlcAの還元側のGalNAc残基を選択的に硫酸化する 5。UA2OSTはCSとDSのどちらも硫酸化するが、CSを基質とするときはGalNAc(4S)-GlcA-GalNAc(6S)という配列のGlcAの2位に硫酸基を転移する 37。HS2STはGlcNSに隣接したIdoAの2位を硫酸化するが、GlcNS(6S)に隣接したIdoAを硫酸化しない 34。HS6STのアイソフォームは基質糖鎖のGlcNS残基に隣接するウロン酸の構造を識別し、HS6ST-1はIdoAを、HS6ST-2はGlcAを好み、HS6ST-3は中間的な性質を示す40。GlcNAc6ST-1とC-GlcNAc6STはKSの非還元末端のGlcNAc残基の6位に硫酸基を転移する8。これらの観察は、種々の組織における硫酸転移酵素の発現パターンが、その組織で合成されるグリコサミノグリカンの特定の配列形成に寄与していることを示す。

硫酸転移酵素遺伝子
 今までにクローニングされたグリコサミノグリカン硫酸転移酵素は、アミノ酸配列の相同性から、C6ST/KSGal6ST/GlcNAc6STグループ、C4ST/D4STグループ、GalNAc4S-6STグループ、HS2ST/UA2OSTグループ、NDSTグループ、HS6STグループ、HS3STグループの7つに分類が可能である(図2)。NDSTグループ、HS3STグループ、GalNAc4S-6ST間には弱い相同性が見られる。C6STグループの酵素(C6ST-2, KSGal6ST, GlcNAc6ST-1)及びC4STグループの酵素(GalNAc4ST-1, GalNAc4ST-2, HNK-1ST)は、糖蛋白質糖鎖の硫酸化活性を示す。このことはグリコサミノグリカンと糖蛋白質糖鎖の硫酸化を行う硫酸転移酵素が共通の遺伝子から進化したことを示唆している。HS2ST, UA2OST, GalNAc4S-6STにはアイソフォームは知られていない。HS2ST KOマウスでHSのIdoAの2硫酸化が全く起きないので、HSのウロン酸の2硫酸化は単一のHS2ST により行われると考えられる 75


図2 Phylogenic tree of GAG sulfotransferases
硫酸転移酵素に結合する糖鎖の機能
精製した硫酸転移酵素の多くはN-glycanase消化により低分子化することから、N-結合糖鎖をもつ糖タンパク質である。特にC6ST-1, C4ST-1, GalNAc4S-6STでは約40%の糖鎖含量を示す。C4ST-1には4個のN-結合糖鎖付着部位があるが、N末端から4番目のN-glycan(N-4)が酵素活性に必要であった。C4ST-1の活性に必要なN-4糖鎖のみをもった酵素は、野生型に比べ37℃で失活しやすいことから、N-4以外の糖鎖は酵素の安定性に寄与していると考えられる 76。C6ST-1に含まれる6個のN-結合糖鎖付着部位のうち、N末端から4番目と6番目のN-glycan(N-4とN-6)が酵素活性に必要であり、N-4糖鎖はGolgiへの局在に必要であった。N末端から5番目のN-glycanはコンドロイチンのGalNAcの6位を硫酸化する活性には影響しないが、ケラタン硫酸のGalの6位を硫酸化する活性に必要であった77

硫酸転移酵素の生物学的機能
個体レベルでの硫酸転移酵素の多彩な生物学的機能は、ノックアウトマウスの作製と解析、遺伝子ノックダウン実験、ヒトの遺伝疾患の解析から明らかにされてきた。現在までに報告されている硫酸転移酵素の機能を表2に示した。ヒトの遺伝疾患である斑状角膜ジストロフィではC-GlcNAc6STが 10、脊椎骨端異形成症ではC6ST-1の欠損が報告されている65。マウスにおけるC4ST-1 51、HS2ST 60, NDST-153-55の欠損は新生児期での致死を、HS6ST-1欠損は後期胎生期での致死を引き起こすことから61、これらの硫酸転移酵素は特定の構造を持ったグリコサミノグリカン合成を通して、正常な生命の維持に必須な役割を果たしていると考えられる。炎症及び肝臓でのNDST-1の機能を解析するため、内皮細胞 56及び肝細胞 57で選択的にNDST-1を欠失する条件KOマウスが作製された。これらのマウスの解析から、好中球の浸潤に内皮細胞のHSが、リポプロテインのクリアランスに肝臓のHSが必要なことが示された。単純ヘルペスウィルス1型(HSV-1)の野生型CHO細胞への感染にHS3ST-3B 47が, HSV-1に部分的に耐性でHSを合成しないgro2C細胞から分離されたsog9細胞への感染にC4ST-178が必要な遺伝子として報告された。HS3ST-3Bにより合成される-IdoA(2S)-GlcN(3S)-という単位を含むヘパラン硫酸がHSV-1の糖タンパク質gDとの、C4ST-1とGalNAc4S-6STとの共同により合成されるCS-EがgCとの結合に必要であると考えられる。

羽渕 脩躬(愛知教育大学)
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Bink RJ, Habuchi H, Lele Z, Dolk E, Joore J, Rauch GJ, Geisler R, Wilson SW, den Hertog J, Kimata K, Zivkovic D, Heparan sulfate 6-o-sulfotransferase is essential for muscle development in zebrafish. J. Biol. Chem. 278, 31118-31127, 2003

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Chen E, Stringer SE, Rusch MA, Selleck SB, Ekker SC, A unique role for 6-O sulfation modification in zebrafish vascular development. Dev. Biol. 284, 364-376, 2005

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Merry CL, Bullock SL, Swan DC, Backen AC, Lyon M, Beddington RS, Wilson VA, Gallagher JT,  The molecular phenotype of heparan sulfate in the Hs2st-/- mutant mouse. J. Biol. Chem. 276, 35429-35434, 2001

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Yusa A, Kitajima K, Habuchi O, N-linked oligosaccharides are required to produce and stabilize the active form of chondroitin 4-sulphotransferase-1. Biochem. J. 388, 115-121, 2005

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Yusa A, Kitajima K, Habuchi O, N-linked oligosaccharides on chondroitin 6-sulfotransferase-1 are required for production of the active enzyme, Golgi localization, and sulfotransferase activity toward keratan sulfate. J. Biol. Chem. 281, 20393-20403, 2006

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Habuchi O. Diversity and functions of glycosaminoglycan sulfotransferases. Biochim. Biophys. Acta, 1474, 115-127, 2000

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Kimata K, Habuchi O, Habuchi H, Watanabe H, Kockout Mice and Proteoglycans, Complehensive Glycoscience Vol. 4, Capter 3.11, Elsevier, 2007

2007年 9月 15日

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