用中国仓鼠卵巢细胞构建内切-β-N-乙酰氨基葡糖苷酶筛选系统

Construction of A Screening System for Endo-β-N-Acetylglucosaminidase Using Chinese Hamster Ovary Cell

摘要:

糖苷水解酶85家族的内切-β-N-乙酰氨基葡糖苷酶具有糖基转移酶活性，利用该类酶，可以合成特定结构N-糖链修饰的糖基化复合物。为构建糖基转移酶筛选系统，将N-乙酰氨基葡萄糖转移酶的跨膜定位区域与绿色荧光蛋白融合表达，成功把带有天冬酰胺连接糖基化位点的单体增强绿色荧光蛋白定位到中国仓鼠卵巢细胞的高尔基体中。流式细胞仪分析显示，融合了定位区域后，绿色荧光蛋白仍可以发出荧光，且在引入糖基化位点后，细胞荧光强度约降低10倍。经衣霉素处理，细胞糖基化被抑制，同时荧光强度减弱。此细胞株荧光强度对于糖基化水平的依赖性表明其在糖基转移酶的进化筛选方面的应用潜力。

Abstract:

Endo-β-N-Acetylglucosaminidase belonging to glycoside hydrolase family 85（ENGase） exhibits transglycosylation activity，which is a useful tool for the synthesis of glycoconjugates with a defied structure of N-glycan. In the present study，We constructed a cell-based screening system to obtain mutant ENGase. As a substrate for ENGase，we expressed monomer enhanced green fluorescent protein（mEGFP） with N-glycan in the Golgi of Chinese hamster ovary cells. Cells expressing N-glycosylated mEGFP provided 10 times lower fluorescence than those expressing mEGFP. Inhibition of protein glycosylation by tunicamycin caused further decrease of fluorescence from the N-glycosylated mEGFP. Our results suggest that this cell line can be used for the high throughput screening of mutant ENGase.