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

Michael Boyce, Isaac S. Carrico, Anjali S. Ganguli, Seok Ho Yu, Matthew J. Hangauer, Sarah C. Hubbard, Jennifer J. Kohler, Carolyn R. Bertozzi

Research output: Contribution to journalArticle

158 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)3141-3146
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number8
DOIs
StatePublished - Feb 22 2011

ASJC Scopus subject areas

  • General

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