Metabolic cross-talk allows labeling of O-linked β- N -acetylglucosamine-modified proteins via the N -acetylgalactosamine salvage pathway
Top Cited Papers
- 7 February 2011
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 108 (8), 3141-3146
- https://doi.org/10.1073/pnas.1010045108
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.Keywords
This publication has 44 references indexed in Scilit:
- Targeted metabolic labeling of yeast N-glycans with unnatural sugarsProceedings of the National Academy of Sciences, 2010
- Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass SpectrometryMolecular & Cellular Proteomics, 2010
- In Vivo Imaging of Caenorhabditis elegans GlycansACS Chemical Biology, 2009
- The hexosamine signaling pathway: O-GlcNAc cycling in feast or famineBiochimica et Biophysica Acta (BBA) - General Subjects, 2009
- A Mitotic GlcNAcylation/Phosphorylation Signaling Complex Alters the Posttranslational State of the Cytoskeletal Protein VimentinMolecular Biology of the Cell, 2008
- Cross-talk between GlcNAcylation and phosphorylation: Site-specific phosphorylation dynamics in response to globally elevated O -GlcNAcProceedings of the National Academy of Sciences of the United States of America, 2008
- In Vivo Imaging of Membrane-Associated Glycans in Developing ZebrafishScience, 2008
- A chemical method for fast and sensitive detection of DNA synthesis in vivoProceedings of the National Academy of Sciences of the United States of America, 2008
- Regulation of a COPII component by cytosolic O‐glycosylation during mitosisFEBS Letters, 2004
- Activation of the Ets Transcription Factor Elf‐1 Requires Phosphorylation and GlycosylationAnnals of the New York Academy of Sciences, 2003