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 post-translational modification of Ser and Thr residues on cytosolic and nuclear proteins found in all higher eukaryotes. 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 {approx}100 {micro}g 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 on Ser and Thr residues in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located in close proximity to known phosphorylation sites. These findings support the proposed regulatory crosstalk 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-{beta}-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, thus representing the first evidence for extracellular O-GlcNAcylation in mammalian systems by the ER-resident O-GlcNAc transferase (EOGT). We also report a GlcNAc-{beta}-1,3-Fuc-{alpha}-1-O-Thr modification on the EGF-like repeat of the Versican core protein, a novel substrate of Fringe {beta}1,3-N-acetylglucosaminyltransferases.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1042524
- Report Number(s):
- PNNL-SA-84997; PNASA6; 40072; 400412000; TRN: US201212%%810
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, Issue 19; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
BRAIN
CLEAVAGE
DISEASES
MASS SPECTROSCOPY
MEMBRANE PROTEINS
MODIFICATIONS
PEPTIDES
PHOSPHORYLATION
PROTEINS
RESIDUES
SUBSTRATES
TARGETS
TRANSFERASES
O-GlcNAc
CEPC enrichment
mass spectrometry
glycosylation
HCD
CID
ETD
mouse cerebral cortex.
Environmental Molecular Sciences Laboratory