基于代谢标记和基因编辑技术的核蛋白质新型O-GalNAc糖基化修饰及催化机制研究

O-GalNAc糖基化是蛋白质最普遍的糖基化修饰之一，主要发生在细胞膜蛋白和分泌蛋白上，然而2017年我们最早发现核蛋白上存在O-GalNAc糖基化修饰，最新的国际研究也证实这一现象的存在。但是核蛋白的O-GalNAc糖基化是否具有普遍性，而且这种新型糖基化是由何种糖基转移酶催化调控等基本科学问题有待回答。为此我们在前期研究中，建立了运用糖苷供体异构酶突变细胞结合糖代谢标记技术特异性标记细胞和分离核内O-GalNAc糖蛋白的方法，同时通过生物信息学分析筛选到三种核定位的O-GalNAc糖基转移酶类似基因。本项目拟在上述基础上通过大规模质谱检测细胞内O-GalNAc糖基化核蛋白，验证O-GalNAc糖基化在细胞核内修饰的普遍性，并通过生化和细胞学手段研究新发现的核内O-GalNAc糖基转移酶类似基因的酶活特性。本研究将拓展人们对核蛋白新型O-GalNAc糖基化修饰的理解并提供实验理论依据。

O-GalNAc glycosylation is one of the most abundant post-translational modifications in proteome, and is mainly modified on transmembrane and secreted proteins. In 2017, we have identified and reported that some nuclear proteins could also be O-GalNAc glycosylated. Recently several studies did confirm our identification. However, many issues are still unsolved including what and how many nuclear proteins could be O-GalNAc glycosylated in cells, whether there are novel O-GalNAc glycosyltransferases in the nucleus. In our preliminary study, we combined the metabolic glycan labeling and the UDP-GalNAc 4-epimerase (GALE) mutant cells to specifically label and detect the O-GalNAc glycosylation in cells. We also identified three potential nuclear O-GalNAc glycosyltransferase genes in the human genome using bioinformatics analysis. In this research proposal, we plan to enrich and identify the O-GalNAc glycoproteins from the nucleus of different human cell lines, and analyze the transferase activities of the three potential nuclear O-GalNAc glycosyltransferases both in vivo and in vitro. This research will provide new insights into the recently discovered nuclear O-GalNAc glycosylation in human cells.