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      Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation

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          Abstract

          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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          Author and article information

          Journal
          0212271
          5787
          Methods Enzymol
          Meth. Enzymol.
          Methods in enzymology
          0076-6879
          1557-7988
          14 March 2018
          07 August 2017
          2018
          01 January 2019
          : 598
          : 101-135
          Affiliations
          [* ]Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, United States
          Author notes
          [1 ]Corrresponding author: lhw@ 123456caltech.edu
          Article
          PMC5886303 PMC5886303 5886303 nihpa943751
          10.1016/bs.mie.2017.06.009
          5886303
          29306432
          4f22b05a-8dc7-40a6-b165-50f6a4019731
          History
          Categories
          Article

          post-translational modification,protein glycosylation,chemoenzymatic labeling, O-linked β-N-acetylglucosamine (O-GlcNAc),copper-catalyzed azide-alkyne cycloaddition (CuAAC),O-GlcNAcylation

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