Asparagine-linked glycoprotein biosynthesis in rat epididymis. Presence of a mannosidase II-like enzyme.
Asparagine-linked glycoprotein biosynthesis in rat epididymis. Presence of a mannosidase II-like enzyme.
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大鼠附睾中天冬酰胺连接的糖蛋白生物合成。
DOI：
10.1042/bj2770213
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Tulsiani,DR
中科院分区：
文献类型：
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作者：
Skudlarek,MD;Orgebin-Crist,MC;Tulsiani,DR
来自该实验室的使用对硝基苯基α-D-甘露糖苷（p-NPM）作为底物的先前研究未提供大鼠附睾中存在甘露糖苷酶II的证据[Skudlarek & Orgebin-Crist（1988）J. Reprod.生育84，611-617]。然而，在苦马豆素（一种甘露糖苷酶II的抑制剂）存在下培养的大鼠附睾上皮细胞产生异常加工的N-连接的糖蛋白，其含有杂交型寡糖而不是复合型寡糖[Tulsiani，Skudlarek & Orgebin-Crist（1990）Biol. Reprod. 43，130-138]，这一结果为大鼠附睾中存在甘露糖苷酶II样酶提供了间接证据。在这里描述的研究中，我们提出了证据，这种加工酶在大鼠附睾高尔基膜的发生。这种酶是一个完整的高尔基体膜组件。与肝甘露糖苷酶II一样，附睾酶从GlcNAcMan 5GlcNAc切割α 1，3-和α 1，6-连接的甘露糖残基。然而，与肝甘露糖苷酶II不同，附睾酶对合成底物p-NPM没有活性。附睾甘露糖苷酶与肝抗（甘露糖苷酶II）抗体交叉反应，结果表明这两种酶具有共同的抗原位点。在SDS/PAGE上解析肝脏和附睾高尔基体膜后的免疫印迹研究表明，肝脏甘露糖苷酶II解析为Mr 120，000和122，000的双峰，而在附睾高尔基体膜中仅观察到Mr 120，000条带。免疫印迹的高尔基体丰富的馏分，在非变性条件下解决，显示出不同的模式的电荷和/或大小异构体从两个组织。这些研究证明了具有相似功能的加工酶的组织特异性差异。
Previous studies from this laboratory using p-nitrophenyl alpha-D-mannoside (p-NPM) as substrate provided no evidence for the presence of mannosidase II in the rat epididymis [Skudlarek & Orgebin-Crist (1988) J. Reprod. Fertil. 84, 611-617]. However, rat epididymal epithelial cells cultured in the presence of swainsonine, an inhibitor of mannosidase II, produce abnormally processed N-linked glycoproteins containing hybrid-type oligosaccharides instead of complex-type [Tulsiani, Skudlarek & Orgebin-Crist (1990) Biol. Reprod. 43, 130-138], a result providing indirect evidence for the presence of mannosidase II-like enzyme in rat epididymis. In the studies described here, we present evidence for the occurrence of this processing enzyme in rat epididymal Golgi membranes. This enzyme is an integral Golgi membrane component. Like liver mannosidase II, the epididymal enzyme cleaves alpha 1,3- and alpha 1,6-linked mannosyl residues from GlcNAcMan5GlcNAc. However, unlike liver mannosidase II, the epididymal enzyme shows no activity towards the synthetic substrate, p-NPM. The epididymal mannosidase cross-reacts with liver anti-(mannosidase II) antibody, a result suggesting that the two enzymes share a common antigenic site(s). Immunoblotting studies following resolution of liver and epididymal Golgi membranes on SDS/PAGE show that, whereas the liver mannosidase II was resolved as a doublet of Mr 120,000 and 122,000, only the Mr 120,000 band was observed in the epididymal Golgi membranes. Immunoblotting of the Golgi-rich fractions, resolved under non-denaturing conditions, showed different patterns of charge and/or size isomers from the two tissues. These studies demonstrate tissue-specific differences in processing enzymes with similar function.