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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 8; pp. 3141 - 3146
• Main Authors: Boyce, Michael, Carrico, Isaac S, Ganguli, Anjali S, Yu, Seok-Ho, Hangauer, Matthew J, Hubbard, Sarah C, Kohler, Jennifer J, Bertozzi, Carolyn R
• Format: Journal Article
• Language: English
• Published: National Academy of Sciences 2011
• Subjects: enzymes; glycosylation; humans; proteins; proteomics
• ISSN: 0027-8424; 1091-6490

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.