Molecular Evolution of Structure and Specificity of Asparagine-Linked Oligosaccharide Releasing Enzyme

天冬酰胺连接寡糖释放酶结构和特异性的分子进化

• 批准号: 08680662
• 负责人: ITO Kazuo
• 金额: $ 1.47万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680662/
• 关键词: Endo-beta-N-acetylglucosaminidase HS; Asparagine-linked Oligosaccharide; Glycoprotein; Epithelial Cell; Human Oral Cavity Epithelial Cell; Human Salive; Glycobiology; エンド-β-N-アセチルグルコザミニダーゼHS; Endo-beta-N-acetylglucosaminidase HS; Asparagine-linked Oligosaccharide; Glycoprotein; Epithelial Cell; Human Oral Cavity Epithelial Cell; Human Salive; Glycobiology; エンド-β-N-アセチルグルコザミニダーゼHS

An improved purification method was established for an effective purification of endo-beta-N-acetylglucosaminidase (Endo) HS,resulting that Endo HS was purified about 100-fold with activity recovery of about 100% using a chitin column. The purified Endo HS was separated into three multiple forms (Endo HS-I,II and III) with different isoelectric point by HPLC with Mono Q5/5 column. Purity of Endo HS-I and Endo HS-II was 100% and Endo HS-III,about 15%. Total amount of Endo HS-I and Endo Hs-II, about 67 pmole and 240 pmole, respectively was not sufficient for determination of the partial amino acid sequence for cloning of Endo gene. The result indicates that sufficient amount of Endo HS for amino acid sequencing must be obtained from human oral cavity epithelial cell obtained from 5-10 liter of human saliva for ourification. The multiple forms of Endo HS showed a quite similar substrate specificity. Endo HS was specifically released asparagine-linked oligosaccharides of bi, tri and tetrantennary complex types from native glycoproteins and asparagine-oligosaccharides regardless of the existence of the fucose residue at the proximal N-acetylglucosamine and side chains and the sialic acid at the side chains. On the other hand, Endo HS did not act on high-mannose and hybrid type oligosaccharides on glycoproteins. The comparison of the specificity of Endo HS with other enzymes means that Endo HS evolved after appearing the synthetic pathway of complex type oligosaccharides for control of the amount of asparagine-linked oligosaccharides of complex type on animal cell glycoproteins.

建立了一种改进的纯化方法，以有效纯化内抗-beta-n-乙酰葡萄糖酰胺酶（Endo）HS，从而使Endo HS纯化了约100倍，而使用壳蛋白柱的活性回收率约为100％。纯化的内托HS分为三种多种形式（Endo HS-I，II和III），并由Mono Q5/5列的HPLC分为不同的等电点。 Endo HS-I和Endo HS-II的纯度为100％，而Endo HS-III约为15％。 Endo HS-I和Endo HS-II的总量分别为67 pmole和240 pmole，不足以确定偏侧氨基酸序列来克隆内托基因。结果表明，必须从5-10升人类唾液中获得足够数量的氨基酸测序的氨基酸测序。多种形式的内托HS显示出非常相似的底物特异性。 Endo HS专门释放出来自本机糖蛋白和天冬糖糖蛋白和天冬酰胺 - 寡糖的BI，TRI和Tetrantennary复合物的白天连接的寡糖，而不管在近端N-乙酰基果糖氨基链链链以及sialic and Sialec Chains酸中存在Fucose残基。另一方面，Endo HS在糖蛋白上没有作用于高甘露糖和杂化型寡糖。 Endo HS与其他酶的特异性的比较意味着，在出现了复杂型寡糖的合成途径后，Endo HS进化，以控制动物细胞糖蛋白上复杂类型的天冬酰胺连接寡糖的量。