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Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3141-6. doi: 10.1073/pnas.1010045108. Epub 2011 Feb 7.

Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway.

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  • 1Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.

Abstract

Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

PMID:
21300897
[PubMed - indexed for MEDLINE]
PMCID:
PMC3044403
Free PMC Article

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