Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

Abstract

O-linked β- N -acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.

A Sweet Embrace: Control of Protein–Protein Interactions by O-Linked β-N-Acetylglucosamine

“…66 The authors’ work established GlcNDAz as a powerful tool for identifying direct, glycosylation-mediated interactions between endogenous proteins in live cells. 66 …”

“…Brief treatment of GlcNDAz-labeled cells with UV light affords the carbene-mediated cross-linking of O-GlcNDAz-modified proteins and any binding partner proteins within ~2–4 Å of the glycan. For additional details, see the text and ref 66.…”

A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics

“…Metabolic labeling to introduce a chemical tag on GlcNAc has also been used in enrichment. These tags include diazirine, azide, or alkyne modification groups added to GlcNAc (25,26). Upon click reaction or photo-crosslinking, GlcNAc peptides were enriched with beads bearing chemical groups that react with these tags (27).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…Metabolic labeling to enable selective photocrosslinking of O-GlcNAc-modified proteins to identify their binding partners (Yu et al, 2012d) Glycopeptides, glycoproteins R-, L-TOF Evaluation of GalNAc analogues as substrates for enzymes of the mammalian GalNAc salvage pathway (Pouilly et al, 2012b) Glycoproteins TOF/TOF (Hart et al, 2011) Human -defensin TOF (sinapinic), peptides…”