The modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, is necessary for T cell activation. A mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive. To advance insight into the role of O-GlcNAc in T cell activation, we used Isotope Targeted Glycoproteomics (IsoTaG) to map glycosites in resting and activated primary human T cells. IsoTaG involves metabolic labeling of the glycoproteome combined with chemical enrichment and isotopic recoding of the glycopeptides for targeted mass-independent mass spectrometry assignment of the intact glycopeptide.  Our findings provide a quantitative characterization of 2219 intact O-linked glycopeptides across 1045 glycoproteins. We employed gel shift assays based on conjugating mass tags to O-GlcNAc groups to validate our results. Approximately half of the identified O-GlcNAc sites lie near or coincide with a known phosphorylation site, offering the intriguing possibility of cross-talk between O-GlcNAc and phosphorylation signaling pathways. Systematic evaluation of the role of O-GlcNAc in T cell activation and cross-talk mechanisms in cells requires a general method to control glycosylation on a target protein.  We developed a proximity-directed O-GlcNAc transferase to selectively increase O-GlcNAc occupancy on specific proteins and characterize the molecular role of O-GlcNAc during T cell activation.  These approaches will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation and cellular signaling through posrt-translational modifications in general.

Mapping and manipulating O-GlcNAc during T cell activation

256th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.