Tokhtaeva Elmira, Mareninova Olga A, Gukovskaya Anna S, Vagin Olga
David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte Ave, Los Angeles, 90095, CA, USA.
VA Greater Los Angeles Healthcare System, 11301 Wilshire Blvd, VAGLAHS/West LA, Building 113, Room 324, Los Angeles, CA, 90073, USA.
Methods Mol Biol. 2017;1594:35-42. doi: 10.1007/978-1-4939-6934-0_3.
The vast majority of lysosomal proteins are heavily glycosylated. The present protocol describes the method of analyzing N- and O-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.
绝大多数溶酶体蛋白都高度糖基化。本方案描述了分析目标溶酶体蛋白中N-连接和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内部键的裂解，而外切糖苷酶从聚糖中去除末端碳水化合物。特定的糖苷酶可以去除不同类型的碳水化合物残基或链。用糖苷酶去除寡糖会增加蛋白质的电泳迁移率。这种迁移率的增加取决于去除的碳水化合物链的大小和数量。因此，用特定的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，通过比较处理前后的凝胶迁移率，提供了一种确定蛋白中存在哪些类型聚糖的方法。
