Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets

O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.

[1]  L. Hsieh‐Wilson,et al.  The chemical neurobiology of carbohydrates. , 2008, Chemical reviews.

[2]  Robert J Chalkley,et al.  Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides , 2009, Proceedings of the National Academy of Sciences.

[3]  Richard D Smith,et al.  Recommendations for Mass Spectrometry Data Quality Metrics for Open Access Data (Corollary to the Amsterdam Principles)* , 2011, Molecular & Cellular Proteomics.

[4]  I. Grundke‐Iqbal,et al.  Impaired brain glucose metabolism leads to Alzheimer neurofibrillary degeneration through a decrease in tau O-GlcNAcylation. , 2006, Journal of Alzheimer's disease : JAD.

[5]  S. Marshall,et al.  UDP‐N‐acetylglucosaminyl transferase (OGT) in brain tissue: temperature sensitivity and subcellular distribution of cytosolic and nuclear enzyme , 2003, Journal of neurochemistry.

[6]  Sunghoon Kim,et al.  Hormonal activity of AIMP1/p43 for glucose homeostasis , 2006, Proceedings of the National Academy of Sciences.

[7]  S. Gygi,et al.  An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets , 2005, Nature Biotechnology.

[8]  María Martín,et al.  The Universal Protein Resource (UniProt) in 2010 , 2010 .

[9]  Christian Rosenmund,et al.  Regulation of Rap2A by the Ubiquitin Ligase Nedd4-1 Controls Neurite Development , 2010, Neuron.

[10]  J. Carmichael,et al.  Prostate adenocarcinoma cells release the novel proinflammatory polypeptide EMAP-II in response to stress. , 2000, Cancer research.

[11]  Joshua N. Adkins,et al.  MASIC: A software program for fast quantitation and flexible visualization of chromatographic profiles from detected LC-MS(/MS) features , 2008, Comput. Biol. Chem..

[12]  Sunghoon Kim,et al.  A Cofactor of tRNA Synthetase, p43, Is Secreted to Up-regulate Proinflammatory Genes* , 2001, The Journal of Biological Chemistry.

[13]  Steven P Gygi,et al.  Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations , 2005, Nature Methods.

[14]  Lance Wells,et al.  Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment. , 2011, Journal of proteome research.

[15]  G. Glish,et al.  High amplitude short time excitation: A method to form and detect low mass product ions in a quadrupole ion trap mass spectrometer , 2006, Journal of the American Society for Mass Spectrometry.

[16]  Friedrich W. Herberg,et al.  Neurobeachin: A Protein Kinase A-Anchoring,beige/Chediak-Higashi Protein Homolog Implicated in Neuronal Membrane Traffic , 2000, The Journal of Neuroscience.

[17]  Linda C Hsieh-Wilson,et al.  Chemical approaches to understanding O-GlcNAc glycosylation in the brain. , 2008, Nature chemical biology.

[18]  A. Burlingame,et al.  Polycomb repressive complex 2 is necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem cells , 2011, Proceedings of the National Academy of Sciences.

[19]  G. Hart,et al.  O-linked beta-N-acetylglucosamine (O-GlcNAc): Extensive crosstalk with phosphorylation to regulate signaling and transcription in response to nutrients and stress. , 2010, Biochimica et biophysica acta.

[20]  T. Hunter.  The age of crosstalk: phosphorylation, ubiquitination, and beyond. , 2007, Molecular cell.

[21]  R. Haltiwanger,et al.  Mammalian Notch1 Is Modified with Two Unusual Forms ofO-Linked Glycosylation Found on Epidermal Growth Factor-like Modules* , 2000, The Journal of Biological Chemistry.

[22]  G. Davies,et al.  A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo. , 2008, Nature chemical biology.

[23]  Scott B Ficarro,et al.  Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. , 2007, Nature chemical biology.

[24]  G. Hart,et al.  O-GlcNAc modification in diabetes and Alzheimer's disease. , 2007, Molecular bioSystems.

[25]  K. Furukawa,et al.  O-Linked N-Acetylglucosamine Is Present on the Extracellular Domain of Notch Receptors* , 2008, Journal of Biological Chemistry.

[26]  M. Mattson,et al.  Triple-Transgenic Model of Alzheimer's Disease with Plaques and Tangles Intracellular Aβ and Synaptic Dysfunction , 2003, Neuron.

[27]  K. Furukawa,et al.  O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1. , 2012, Biochemical and biophysical research communications.

[28]  M. Missler,et al.  Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses , 2009, The Journal of physiology.

[29]  H. Hang,et al.  Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1 , 2011, Proceedings of the National Academy of Sciences.

[30]  Baris E. Suzek,et al.  The Universal Protein Resource (UniProt) in 2010 , 2009, Nucleic Acids Res..

[31]  Jonathan C Trinidad,et al.  O-Linked N-Acetylglucosamine Proteomics of Postsynaptic Density Preparations Using Lectin Weak Affinity Chromatography and Mass Spectrometry*S , 2006, Molecular & Cellular Proteomics.

[32]  Peter R Baker,et al.  Modification Site Localization Scoring Integrated into a Search Engine* , 2011, Molecular & Cellular Proteomics.

[33]  R. Rampal,et al.  Notch signaling in normal and disease States: possible therapies related to glycosylation. , 2007, Current molecular medicine.

[34]  M. Wolfert,et al.  Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. , 2010, Nature chemical biology.

[35]  Henriette van Praag,et al.  When neurogenesis encounters aging and disease , 2010, Trends in Neurosciences.

[36]  Yang Wang,et al.  Fringe is a glycosyltransferase that modifies Notch , 2000, Nature.

[37]  B K Hayes,et al.  Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry. , 1996, Analytical biochemistry.

[38]  G. Hart,et al.  Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. , 2011, Annual review of biochemistry.

[39]  S. Cohen,et al.  Glycosyltransferase activity of Fringe modulates Notch–Delta interactions , 2000, Nature.

[40]  G. Hart,et al.  Nucleocytoplasmic O-glycosylation: O-GlcNAc and functional proteomics. , 2001, Biochimie.

[41]  T. Lefebvre,et al.  Protein ubiquitination is modulated by O‐GlcNAc glycosylation , 2008, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[42]  E. Suzuki,et al.  O-linked-N-acetylglucosamine on extracellular protein domains mediates epithelial cell-matrix interactions. , 2011, Nature communications.

[43]  Jianhua Shi,et al.  Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer's disease. , 2009, Brain : a journal of neurology.

[44]  Richard D. Smith,et al.  Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput. , 2009, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[45]  R. Haltiwanger,et al.  Fringe benefits: functional and structural impacts of O-glycosylation on the extracellular domain of Notch receptors. , 2011, Current opinion in structural biology.

[46]  J. Shabanowitz,et al.  Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis , 2010, Science Signaling.

[47]  Sunghoon Kim,et al.  The novel cytokine p43 stimulates dermal fibroblast proliferation and wound repair. , 2005, The American journal of pathology.

[48]  S. Hoyer.  Causes and consequences of disturbances of cerebral glucose metabolism in sporadic Alzheimer disease: therapeutic implications. , 2004, Advances in experimental medicine and biology.

[49]  J. Kornhauser,et al.  PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation , 2004, Proteomics.

[50]  Kumaran Kandasamy,et al.  Systematic evaluation of alternating CID and ETD fragmentation for phosphorylated peptides , 2011, Proteomics.

[51]  J. Shabanowitz,et al.  Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry* , 2009, Molecular & Cellular Proteomics.

[52]  Steven P Gygi,et al.  A probability-based approach for high-throughput protein phosphorylation analysis and site localization , 2006, Nature Biotechnology.

[53]  Edward L. Huttlin,et al.  A Tissue-Specific Atlas of Mouse Protein Phosphorylation and Expression , 2010, Cell.

[54]  Brad T. Sherman,et al.  Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists , 2008, Nucleic acids research.

[55]  G. Hart,et al.  Dynamic Glycosylation of Nuclear and Cytosolic Proteins , 1997, The Journal of Biological Chemistry.

[56]  G. Hart,et al.  Glycomic Approaches to Study GlcNAcylation: Protein Identification, Site-mapping, and Site-specific O-GlcNAc Quantitation , 2008, Clinical Proteomics.

[57]  David H Perlman,et al.  Software tool for researching annotations of proteins: open-source protein annotation software with data visualization. , 2009, Analytical chemistry.

[58]  P. Stanley,et al.  Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase. , 2001, The Journal of biological chemistry.