U.S. Department of Energy

Pacific Northwest National Laboratory

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

TitleTandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
Publication TypeJournal Article
Year of Publication2012
AuthorsAlfaro JF, Gong C-X, Monroe ME, Aldrich JT, Clauss TRW, Purvine SO, Wang Z, Camp DG, Shabanowitz J, Stanley P, Hart GW, Hunt DF, Yang F, Smith RD
JournalProc Natl Acad Sci U S A
KeywordsAcetylglucosamine, Amino Acid Sequence, Animals, Binding Sites, Brain, Cell Membrane, Cell Nucleus, Cytoplasm, Epidermal Growth Factor, Glycoproteins, Glycosylation, Mice, Molecular Sequence Data, N-Acetylglucosaminyltransferases, Organelles, Peptides, Phosphorylation, Proteome, Proteomics, Tandem Mass Spectrometry
Abstract

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.

DOI10.1073/pnas.1200425109
PubMed ID22517741
PubMed Central IDPMC3358849
Grant List5P41RR018522-10 / RR / NCRR NIH HHS / United States
8 P41 GM103493-10 / GM / NIGMS NIH HHS / United States
AG027429 / AG / NIA NIH HHS / United States
GM 037537 / GM / NIGMS NIH HHS / United States
N01-HV-00240 / HV / NHLBI NIH HHS / United States
P01HL107153 / HL / NHLBI NIH HHS / United States
P41 GM103493 / GM / NIGMS NIH HHS / United States
P41 RR018522 / RR / NCRR NIH HHS / United States
R01 36434 / / PHS HHS / United States
R01 CA42486 / CA / NCI NIH HHS / United States
RR018522 / RR / NCRR NIH HHS / United States
TW008123 / TW / FIC NIH HHS / United States
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