Metabolic cross-talk allows labeling of O-linked β- <i>N</i> -acetylglucosamine-modified proteins via the <i>N</i> -acetylgalactosamine salvage pathway
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Machine scores (provisional)
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- Teacher spread
- 0.259 · how far apart the two teachers sit on this one work
- Validation status
score_only:v0-immature-baseline· verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it
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.
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The record
- Venue
- Proceedings of the National Academy of Sciences
- Topic
- Glycosylation and Glycoproteins Research
- Field
- Biochemistry, Genetics and Molecular Biology
- Canadian institutions
- —
- Funders
- National Institute of General Medical SciencesNational Cancer InstituteNational Institutes of HealthSimon Fraser UniversityHoward Hughes Medical InstituteNational Defense Science and Engineering GraduateLife Sciences Research FoundationU.S. Department of Defense
- Keywords
- Bioorthogonal chemistryBiochemistryN-AcetylglucosamineChemistryMetabolic pathwayProteomicsEnzymeCovalent bondNucleotide sugarGlycosyltransferaseAcetylglucosamineGlycosylationClick chemistryCombinatorial chemistry
- Has abstract in OpenAlex
- yes