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

建立了一种改进的纯化方法，对内切- β - n -乙酰氨基葡萄糖苷酶（Endo） HS进行了有效的纯化，利用几丁质柱纯化Endo HS约100倍，活性回收率约为100%。利用Mono Q5/5柱高效液相色谱法将纯化后的远藤HS分离为具有不同等电点的三种多形态（远藤HS- i、II和III）。远藤HS-I和HS-II的纯度为100%，远藤HS-III的纯度约为15%。Endo HS-I和Endo Hs-II的总量分别约为67和240摩尔，不足以确定用于克隆Endo基因的部分氨基酸序列。结果表明，从5 ~ 10 l人唾液中获得的人口腔上皮细胞必须获得足够量的Endo HS以进行氨基酸测序。多种形式的Endo HS表现出相当相似的底物特异性。Endo HS能够从天然糖蛋白和天冬酰胺寡糖中特异性释放出双、三、四价络合物类型的天冬酰胺连接寡糖，而不考虑近端n -乙酰氨基葡萄糖和侧链以及侧链上的唾液酸是否存在聚焦残留物。另一方面，远藤HS对高甘露糖和杂交型低聚糖对糖蛋白没有作用。Endo HS与其他酶的特异性比较表明，Endo HS是在出现了控制动物细胞糖蛋白上复杂型天门酰胺连接寡糖数量的复合型寡糖合成途径后进化而来的。

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.