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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

基本信息

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

项目摘要

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.

建立了有效纯化内切-β-N-乙酰氨基葡萄糖苷酶(Endo) HS的改进纯化方法，使用甲壳素柱将Endo HS纯化约100倍，活性回收率约100%。纯化后的Endo HS经HPLC采用Mono Q5/5柱分离为三种不同等电点的多重晶型（Endo HS-I、II和III）。 Endo HS-I和Endo HS-II的纯度为100%，Endo HS-III的纯度约为15%。 Endo HS-I和Endo Hs-II的总量分别约为67 pmole和240 pmole，不足以确定用于克隆Endo基因的部分氨基酸序列。结果表明，必须从5-10升人类唾液中获得的人类口腔上皮细胞中获得足够量的Endo HS用于氨基酸测序。 Endo HS 的多种形式表现出非常相似的底物特异性。 Endo HS 是从天然糖蛋白和天冬酰胺寡糖中特异性释放出双、三和四触角复合物类型的天冬酰胺连接寡糖，无论近端 N-乙酰葡糖胺和侧链处是否存在岩藻糖残基以及侧链处是否存在唾液酸。另一方面，Endo HS 不作用于糖蛋白上的高甘露糖和杂合型寡糖。 Endo HS与其他酶的特异性比较表明，Endo HS是在出现复合型寡糖合成途径后进化而来的，用于控制动物细胞糖蛋白上天冬酰胺连接的复合型寡糖的量。