The Metabolic Chemical Reporter 6-Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O-GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O-Glucose.
The Metabolic Chemical Reporter 6-Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O-GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O-Glucose.
复制标题
DOI：
10.1021/jacs.7b13488
复制
发表时间：
2018-06-13
影响因子：
15
通讯作者：
Pratt MR
中科院分区：
文献类型：
--
作者：
Darabedian N;Gao J;Chuh KN;Woo CM;Pratt MR
糖基化的代谢化学报告基因与生物正交反应相结合已为人所知已有二十年，并已被许多不同的研究实验室用于糖复合物的识别和可视化。然而，最近他们开始看到其在研究细胞代谢以及生物合成酶和糖基转移酶对不同糖的耐受性方面的实用性。在这里，我们通过使用代谢化学报告分子 6-叠氮基-6-脱氧葡萄糖 (6AzGlc) 将这一概念更进一步。我们证明，用全 O-乙酰化版本的 6AzGlc 处理哺乳动物细胞可以对多种蛋白质进行稳定标记。值得注意的是，这种标记的模式与 O-GlcNAc 修饰一致，表明 O-GlcNAc 转移酶对其供体糖底物相当混杂。为了证实这种可能性，我们证明 6AzGlc 处理会导致已知 O-GlcNAc 酰化蛋白的标记，UDP-6AzGlc 供体糖确实是在活细胞中产生的，并且重组 OGT 将接受 UDP-6AzGlc 作为体外底物。最后，我们使用蛋白质组学首先鉴定哺乳动物细胞中的几种真正的 6AzGlc 修饰，然后鉴定宿主细胞因子上的内源性 O-葡萄糖修饰。这些结果支持这样的结论：OGT 可以用 N-乙酰氨基葡萄糖和葡萄糖内源性修饰蛋白质，从而提高了细胞内 O-葡萄糖修饰可能是在某些条件或特定组织中广泛存在的修饰的可能性。
Metabolic chemical reporters of glycosylation in combination with bioorthogonal reactions have been known for two decades and have been used by many different research laboratories for the identification and visualization of glycoconjugates. More recently, however, they have begun to see utility for the investigation of cellular metabolism and the tolerance of biosynthetic enzymes and glycosyltransferases to different sugars. Here, we take this concept one step further by using the metabolic chemical reporter 6-azido-6-deoxy-glucose (6AzGlc). We show that treatment of mammalian cells with the per-O-acetylated version of 6AzGlc results in robust labeling of a variety of proteins. Notably, the pattern of this labeling was consistent with O-GlcNAc modifications, suggesting that the enzyme O-GlcNAc transferase is quite promiscuous for its donor sugar substrates. To confirm this possibility, we show that 6AzGlctreatment results in the labeling of known O-GlcNAcylated proteins, that the UDP-6AzGlc donor sugar is indeed produced in living cells, and that recombinant OGT will accept UDP-6AzGlc as a substrate in vitro. Finally, we use proteomics to first identify several bona fide 6AzGlc-modifications in mammalian cells and then an endogenous O-glucose modification on host cell factor. These results support the conclusion that OGT can endogenously modify proteins with both N-acetyl-glucosamine and glucose, raising the possibility that intracellular O-glucose modification may be a widespread modification under certain conditions or in particular tissues.
登录
查看更多内容
影响因子：
--
作者：
Griffin ME;Jensen EH;Mason DE;Jenkins CL;Stone SE;Peters EC;Hsieh-Wilson LC
通讯作者：
Hsieh-Wilson LC
DOI：
10.1083/jcb.201501101
发表时间：
2015-03-30
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bond MR;Hanover JA
通讯作者：
Hanover JA
影响因子：
5.3
作者：
O'Donnell, N;Zachara, NE;Marth, JD
通讯作者：
Marth, JD
影响因子：
7.8
作者：
Chuh, Kelly N.;Pratt, Matthew R.
通讯作者：
Pratt, Matthew R.
代谢聚糖标记过程中全 O 乙酰化非天然单糖诱导的人工半胱氨酸 S-糖基化。
影响因子：
16.6
作者：
Qin, Wei;Qin, Ke;Chen, Xing
通讯作者：
Chen, Xing