Characterization of cellular oligosaccharides from normal and cystic fibrotic fibroblasts using sequential endoglycosidase digestions.
Characterization of cellular oligosaccharides from normal and cystic fibrotic fibroblasts using sequential endoglycosidase digestions.
复制标题
使用连续内切糖苷酶消化表征正常和囊性纤维化成纤维细胞的细胞寡糖。
DOI：
10.1016/0003-9861(86)90032-9
复制
发表时间：
1986
影响因子：
3.9
通讯作者：
Maley,F
中科院分区：
文献类型：
--
作者：
Bozon,D;Tarentino,AL;Trimble,RB;Maley,F
开发了一种从[2-3H]甘露糖或[6-3H]岩藻糖标记的细胞糖蛋白获取详细寡糖谱的方法。首先根据类别分离寡糖，使用内切-β-N-乙酰氨基葡萄糖苷酶 H (Endo H) 释放高甘露糖种类，然后使用肽-N4-(N-乙酰基-β-氨基葡萄糖基)天冬酰胺酰胺酶 (PNGase F)，提供完整的复杂寡糖链阵列。随后根据 QAE-Sephadex 上的净负电荷对高甘露糖和复合寡糖进行分级。 TSK HW-40(S) 上的高分辨率凝胶过滤将中性高甘露糖群体解析为 Man9-5N-乙酰氨基葡萄糖类型的物质。用神经氨酸酶对复杂链进行去唾液酸化，使得这些寡糖能够解析成其相应的无唾液酸双触角、三触角和四触角种类。使用这些技术分析来自正常和囊性纤维化细胞的成纤维细胞的糖基化模式的差异。超过 95% 的 [2-3H] 甘露糖标记的糖蛋白对联合糖苷酶消化敏感，但在高甘露糖或复合寡糖中没有观察到差异。尽管如此，本研究开发的方法为研究不同细胞类型和同一类型变体的寡糖提供了一种重要的新方法。如使用加工抑制剂苦豆素所示，细胞糖蛋白诱导的代谢变化证明了该程序在研究与糖蛋白结构和酶特异性相关的问题方面的多功能性。因此，通过采用该方法的变体，可以通过随后用Endo H释放二糖岩藻糖基-N-乙酰葡糖胺来确认岩藻糖在PNGase F释放的杂合寡糖的核心中的位置。
A method was developed for obtaining detailed oligosaccharide profiles from [2-3H]mannose-or [6-3H]fucose-labeled cellular glycoproteins. The oligosaccharides were segregated first according to class, usingendo-β-N-acetylglucosaminidase H (Endo H) to release the high mannose species, and then with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase (PNGase F), which provided a complete array of complex oligosaccharide chains. The high mannose and complex oligosaccharides were fractionated subsequently according to net negative charge on QAE-Sephadex. High resolution gel filtration on TSK HW-40(S) resolved the neutral high mannose population into species of the type Man9-5N-acetylglucosamine. Desialyation of the complex chains with neuraminidase allowed resolution of these oligosaccharides into their corresponding asialo bi-, tri-, and tetraantennary species. Fibroblasts from normal and cystic fibrosis cells were analyzed for differences in their glycosylation patterns using these techniques. Over 95% of the [2-3H]mannose-labeled glycoproteins were susceptible to the combined glycosidase digestions, but no difference in either the high mannose or complex oligosaccharides were observed. Nonetheless, the methodology developed in this study provides an important new approach for investigating oligosaccharides of different cell types and variants of the same type. Metabolic changes induced in cellular glycoproteins, as illustrated by use of the processing inhibitor swainsonine, demonstrated the versatility of this procedure for investigating questions relating to glycoprotein structure and enzyme specificity. Thus, by employing a variation of this method, it was possible to confirm the location of fucose in the core of PNGase F-released hybrid oligosaccharides by the subsequent release with Endo H of the disaccharide, fucosyl-N-acetylglucosamine.