Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O-GlcNAcylation via regulation of enzymatic activity. O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O-GlcNAcylation via regulation of enzymatic activity. O-GlcNAcylation, the addition of a single sugar (β-N-acetylglucosamine) to serine and threonine residues on intracellular domains of proteins, is a regulated, reversible post-translational modification. The O-GlcNAcylation state of proteins is responsive to numerous cellular stimuli, including nutrient levels and stress. The addition of this post-translational modification is catalyzed by a single enzyme known as uridine diphosphate-N-acetylglucosamine:peptide β-N-acetylglucosaminyltransferase, referred to as O-GlcNAc-transferase (OGT) 1The abbreviations used are: OGTO-GlcNAc-transferaseDSSPDefine Secondary Structure of ProteinsECDelectron capture dissociationETDelectron transfer dissociationemPAIexponentially modified protein abundance indexFDRfalse discovery rateHCDhigher-energy C-trap dissociationLWAClectin weak affinity chromatographyOGAO-glycoprotein 2-acetamido-2-deoxy-β-d-glucopyranosidase, referred to as O-GlcNAcaseO-GlcNAcO-linked N-acetylglucosaminePTMpost-translational modificationTPRtetratricopeptide repeatWGAwheat germ agglutinin. 1The abbreviations used are: OGTO-GlcNAc-transferaseDSSPDefine Secondary Structure of ProteinsECDelectron capture dissociationETDelectron transfer dissociationemPAIexponentially modified protein abundance indexFDRfalse discovery rateHCDhigher-energy C-trap dissociationLWAClectin weak affinity chromatographyOGAO-glycoprotein 2-acetamido-2-deoxy-β-d-glucopyranosidase, referred to as O-GlcNAcaseO-GlcNAcO-linked N-acetylglucosaminePTMpost-translational modificationTPRtetratricopeptide repeatWGAwheat germ agglutinin., and it is removed by a single enzyme known as O-glycoprotein 2-acetamido-2-deoxy-β-d-glucopyranosidase, referred to as O-GlcNAcase (OGA) (1Hanover J.A. Krause M.W. Love D.C. The hexosamine signaling pathway: O-GlcNAc cycling in feast or famine.Biochim. Biophys. Acta. 2010; 1800: 80-95Crossref PubMed Scopus (268) Google Scholar). Both of these enzymes are highly expressed in the brain. The physiological roles of protein O-GlcNAcylation may be particularly important in the central nervous system (2Cole R.N. Hart G.W. Cytosolic O-glycosylation is in PubMed Scopus Google Hart G.W. O-glycosylation of and and of a from PubMed Scopus Google Scholar). is in and it is also with R.N. Hart G.W. of the O-GlcNAc and proteins in PubMed Scopus Google Scholar). of in in to and Hart G.W. a in and PubMed Scopus Google Scholar). O-GlcNAc-transferase Secondary Structure of capture transfer modified protein abundance discovery C-trap weak affinity O-glycoprotein 2-acetamido-2-deoxy-β-d-glucopyranosidase, referred to as O-GlcNAcase O-linked N-acetylglucosamine post-translational modification germ agglutinin. O-GlcNAc-transferase Secondary Structure of capture transfer modified protein abundance discovery C-trap weak affinity O-glycoprotein 2-acetamido-2-deoxy-β-d-glucopyranosidase, referred to as O-GlcNAcase O-linked N-acetylglucosamine post-translational modification germ agglutinin. O-GlcNAcylation serine and threonine is the potential for between the of this and of phosphorylation. proteins been identified as O-GlcNAc Although the of these are also phosphorylated G.W. O-GlcNAcylation and in and PubMed Scopus Google the of this are that the of all cellular proteins are phosphorylated. In the of O-GlcNAc modification within a protein is has numerous proteins that been to be phosphorylated and O-GlcNAcylated, including and protein in G.W. O-GlcNAcylation and in and PubMed Scopus Google Scholar). 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