Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation.
Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation.
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DOI：
10.1016/bs.mie.2017.06.009
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发表时间：
2018
影响因子：
--
通讯作者：
Hsieh-Wilson LC
中科院分区：
文献类型：
--
作者：
Thompson JW;Griffin ME;Hsieh-Wilson LC
在蛋白质的丝氨酸/苏氨酸残基上添加 O-连接 β-N-乙酰氨基葡萄糖 (O-GlcNAc) 是所有多细胞生物中普遍存在的翻译后修饰。与磷酸化一样，O-GlcNAc 糖基化 (O-GlcNAcylation) 是可诱导的，可调节多种生理和病理过程。然而，由于检测和量化修饰的难度，了解 O-GlcNAc 修饰的多种功能通常具有挑战性。因此，研究 O-GlcNAcNA 酰化的稳健方法对于阐明其在调节单个蛋白质、复杂细胞过程和疾病中的关键作用至关重要。在本章中，我们描述了一组化学酶标记方法，以（1）检测感兴趣蛋白质上的 O-GlcNAcNAc 化，（2）监测感兴趣蛋白质上 O-GlcNAcNAc 总水平及其化学计量的变化，以及（3）能够将 O-GlcNAc 映射到蛋白质内的特定丝氨酸/苏氨酸残基，以促进功能研究。首先，我们概述了多用途突变型半乳糖基转移酶 (Y289L GalT) 的表达和纯化程序。然后，我们描述了使用 Y289L GalT 用功能手柄 N-叠氮乙酰半乳糖胺 (GalNAz) 修饰 O-GlcNAc 残基。最后，我们讨论了铜催化叠氮-炔环加成“点击”反应将各种含炔化学探针附着到 GalNAz 上的几种应用，并演示了如何使用 O-GlcNAc 修饰蛋白的这种功能化来实现上述 (1) – (3)。总的来说，这些方法利用市售试剂和标准蛋白质分析工具，将有助于增进我们对 O-GlcNAc 化的多样化和重要功能的理解。
The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous post-translational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multi-use mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition “click” reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1) – (3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation.
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影响因子：
64.5
作者：
Pekkurnaz G;Trinidad JC;Wang X;Kong D;Schwarz TL
通讯作者：
Schwarz TL
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
影响因子：
15
作者：
Hang, HC;Yu, C;Bertozzi, CR
通讯作者：
Bertozzi, CR
影响因子：
3.8
作者：
Ma J;Hart GW
通讯作者：
Hart GW
硫醇-炔自由基反应通过炔基-L-赖氨酸类似物的遗传掺入介导位点特异性蛋白质标记。
影响因子：
3.2
作者：
Li, Yiming;Pan, Man;Guo, Qingxiang
通讯作者：
Guo, Qingxiang