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

Publication ,  Journal Article
Boyce, M; Carrico, IS; Ganguli, AS; Yu, S-H; Hangauer, MJ; Hubbard, SC; Kohler, JJ; Bertozzi, CR
Published in: Proc Natl Acad Sci U S A
February 22, 2011

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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Published In

Proc Natl Acad Sci U S A

DOI

EISSN

1091-6490

Publication Date

February 22, 2011

Volume

108

Issue

8

Start / End Page

3141 / 3146

Location

United States

Related Subject Headings

  • Receptor Cross-Talk
  • Protein Processing, Post-Translational
  • Methods
  • Metabolic Networks and Pathways
  • Humans
  • Glycosylation
  • Chromatography, Affinity
  • Cell Line
  • Affinity Labels
  • Acetylglucosamine
 

Citation

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Boyce, M., Carrico, I. S., Ganguli, A. S., Yu, S.-H., Hangauer, M. J., Hubbard, S. C., … Bertozzi, C. R. (2011). Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci U S A, 108(8), 3141–3146. https://doi.org/10.1073/pnas.1010045108
Boyce, Michael, Isaac S. Carrico, Anjali S. Ganguli, Seok-Ho Yu, Matthew J. Hangauer, Sarah C. Hubbard, Jennifer J. Kohler, and Carolyn R. Bertozzi. “Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway.Proc Natl Acad Sci U S A 108, no. 8 (February 22, 2011): 3141–46. https://doi.org/10.1073/pnas.1010045108.
Boyce M, Carrico IS, Ganguli AS, Yu S-H, Hangauer MJ, Hubbard SC, et al. Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3141–6.
Boyce, Michael, et al. “Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway.Proc Natl Acad Sci U S A, vol. 108, no. 8, Feb. 2011, pp. 3141–46. Pubmed, doi:10.1073/pnas.1010045108.
Boyce M, Carrico IS, Ganguli AS, Yu S-H, Hangauer MJ, Hubbard SC, Kohler JJ, Bertozzi CR. Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3141–3146.
Journal cover image

Published In

Proc Natl Acad Sci U S A

DOI

EISSN

1091-6490

Publication Date

February 22, 2011

Volume

108

Issue

8

Start / End Page

3141 / 3146

Location

United States

Related Subject Headings

  • Receptor Cross-Talk
  • Protein Processing, Post-Translational
  • Methods
  • Metabolic Networks and Pathways
  • Humans
  • Glycosylation
  • Chromatography, Affinity
  • Cell Line
  • Affinity Labels
  • Acetylglucosamine