Li Feng, Yang Ganglong, Tachikawa Hiroyuki, Shao Kankai, Yang Yan, Gao Xiao-Dong, Nakanishi Hideki
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University.
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo.
J Gen Appl Microbiol. 2021 Apr 16;67(1):33-41. doi: 10.2323/jgam.2020.04.002. Epub 2020 Nov 21.
O-GlcNAc modification mediated by O-GlcNAc transferase (OGT) is a reversible protein modification in which O-GlcNAc moieties are attached to target proteins in the cytosol, nucleus, and mitochondria. O-GlcNAc moieties attached to proteins can be removed by O-GlcNAcase (OGA). The addition of an O-GlcNAc moiety can influence several aspects of protein function, and aberrant O-GlcNAc modification is linked to a number of diseases. While OGT and OGA are conserved across eukaryotic cells, yeasts lack these enzymes. Previously, we reported that protein O-GlcNAc modification occurred in the budding yeast Saccharomyces cerevisiae when OGT was ectopically expressed. Because yeast cells lack OGA, O-GlcNAc moieties are stably attached to target proteins. Thus, the yeast system may be useful for finding novel OST substrates. By proteomic analysis, we identified 468 O-GlcNAcylated proteins in yeast cells expressing human OGT. Among these proteins, 13 have human orthologues that show more than 30% identity to their corresponding yeast orthologue, and possible glycosylation residues are conserved in these human orthologues. In addition, the orthologues have not been reported as substrates of OGT. We verified that some of these human orthologues are O-GlcNAcylated in cultured human cells. These proteins include an ubiquitin-conjugating enzyme, UBE2D1, and an eRF3-similar protein, HBS1L. Thus, the yeast system would be useful to find previously unknown O-GlcNAcylated proteins and regulatory mechanisms.
由O-连接的N-乙酰葡糖胺转移酶（OGT）介导的O-GlcNAc修饰是一种可逆的蛋白质修饰，其中O-GlcNAc部分连接到细胞质、细胞核和线粒体中的靶蛋白上。连接到蛋白质上的O-GlcNAc部分可被O-连接的N-乙酰葡糖胺酶（OGA）去除。O-GlcNAc部分的添加可影响蛋白质功能的多个方面，异常的O-GlcNAc修饰与多种疾病有关。虽然OGT和OGA在真核细胞中是保守的，但酵母缺乏这些酶。此前，我们报道当OGT异位表达时，蛋白质O-GlcNAc修饰发生在出芽酵母酿酒酵母中。由于酵母细胞缺乏OGA，O-GlcNAc部分稳定地连接到靶蛋白上。因此，酵母系统可能有助于发现新的O-GlcNAc底物。通过蛋白质组学分析，我们在表达人OGT的酵母细胞中鉴定出468种O-GlcNAc化蛋白。在这些蛋白质中，有13种具有人类直系同源物，它们与其相应的酵母直系同源物显示出超过30%的同一性，并且这些人类直系同源物中可能的糖基化残基是保守的。此外，这些直系同源物尚未被报道为OGT的底物。我们验证了其中一些人类直系同源物在培养的人类细胞中被O-GlcNAc化。这些蛋白质包括一种泛素结合酶UBE2D1和一种eRF3类似蛋白HBS1L。因此，酵母系统将有助于发现以前未知的O-GlcNAc化蛋白和调控机制。
