Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway.
Published
Journal Article
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.
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Duke Authors
Cited Authors
- Boyce, M; Carrico, IS; Ganguli, AS; Yu, S-H; Hangauer, MJ; Hubbard, SC; Kohler, JJ; Bertozzi, CR
Published Date
- February 22, 2011
Published In
Volume / Issue
- 108 / 8
Start / End Page
- 3141 - 3146
PubMed ID
- 21300897
Pubmed Central ID
- 21300897
Electronic International Standard Serial Number (EISSN)
- 1091-6490
Digital Object Identifier (DOI)
- 10.1073/pnas.1010045108
Language
- eng
Conference Location
- United States