Analysis of N- and O-Glycosylation of Lysosomal Glycoproteins.
Analysis of N- and O-Glycosylation of Lysosomal Glycoproteins.
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溶酶体糖蛋白的 N-和 O-糖基化分析。
DOI：
10.1007/978-1-4939-6934-0_3
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Vagin,Olga
中科院分区：
文献类型：
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作者：
Tokhtaeva,Elmira;Mareninova,OlgaA;Gukovskaya,AnnaS;Vagin,Olga
绝大多数溶酶体蛋白质是高度糖基化的。本方案描述了分析目标溶酶体蛋白中N-和O-连接聚糖的方法。该方法基于使用去糖基化酶、内切糖苷酶和外切糖苷酶。内切糖苷酶催化寡糖中内部键的裂解，而外切糖苷酶从聚糖中除去末端碳水化合物。不同类型的碳水化合物残基或链可以通过特定的糖苷酶去除。用糖苷酶去除寡糖增加蛋白质的电泳迁移率。这种流动性的增加取决于被去除的碳水化合物链的大小和数量。因此，用特异性糖苷酶处理溶酶体蛋白，然后对目标蛋白进行蛋白质印迹分析，提供了一种通过比较处理前后的凝胶迁移率来确定蛋白质中存在哪种类型的聚糖的方法。
The vast majority of lysosomal proteins are heavily glycosylated. The present protocol describes the method of analyzingN- andO-linked glycans in lysosomal proteins of interest. The method is based on using deglycosylating enzymes, endoglycosidases, and exoglycosidases. Endoglycosidases catalyze the cleavage of an internal bond in an oligosaccharide, while exoglycosidases remove terminal carbohydrates from glycans. Different types of carbohydrate residues or chains can be removed by specific glycosidases. Removing oligosaccharides with glycosidases increases the electrophoretic mobility of a protein. This increase in mobility depends on the size and number of removed carbohydrate chains. Therefore, the treatment of lysosomal proteins with specific glycosidases followed by a western blot analysis of a protein of interest provides a way to determine which types of glycans are present in the protein by comparing the gel mobility before and after treatment.