title: Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway.
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.
authors.affiliation: Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Boyce
authors.forename: Isaac S
authors.initials: IS
authors.lastname: Carrico
authors.forename: Anjali S
authors.initials: AS
authors.lastname: Ganguli
authors.forename: Seok-Ho
authors.initials: SH
authors.lastname: Yu
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Hangauer
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Hubbard
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Kohler
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism*; D000117:Acetylglucosamine / Q000378:metabolism*; D000345:Affinity Labels; D002460:Cell Line; D002846:Chromatography, Affinity; D006031:Glycosylation; D006801:Humans; D053858:Metabolic Networks and Pathways; D008722:Methods; D011499:Protein Processing, Post-Translational; D020239:Receptor Cross-Talk
chemical_list: D000345:Affinity Labels; D000116:Acetylgalactosamine; D000117:Acetylglucosamine