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

Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked ß-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.

Metabolic labeling of glycans with azido sugars for visualization and glycoproteomics.

The staggering complexity of glycans renders their analysis extraordinarily difficult, particularly in living systems. A recently developed technology, termed metabolic oligosaccharide engineering, enables glycan labeling with probes for visualization in cells and living animals, and enrichment of specific glycoconjugate types for proteomic analysis. This technology involves metabolic labeling of glycans with a specifically reactive, abiotic functional group, the azide. Azido sugars are fed to cells and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged, either ex vivo or in vivo, using one of two azide-specific chemistries: the Staudinger ligation, or the strain-promoted [3+2] cycloaddition. These reactions can be used to tag glycans with imaging probes or epitope tags, thus enabling the visualization or enrichment of glycoconjugates. Applications to noninvasive imaging and glycoproteomic analyses are discussed.