Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag)

Abstract

Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting evidence indicates that the modification of proteins by O-linked Nacetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2,219 intact O-linked glycopeptides across 1,045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie in close proximity to or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.

Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes

“…A degree of internal tag fragmentation was observed with ETD, although this was not seen with all precursor ions. 45 A maximum of 0.1 min chromatographic retention time shift between differentially deuterated isotopes was observed by nanoflow liquid chromatography. This was negligible for isotopic pattern matching due to implementation of spectral bundling (Figure S4).…”

T cell development and the physiological role of O‐GlcNAc

“…Together, these studies have identified transcription factors both upstream and downstream of IL-2 that require O-GlcNAc modification for proper function. Interestingly, glycoproteomics approaches used to identify O-GlcNAc modified proteins in activated T cells have uncovered an enrichment for transcription factors [41,42] and RNA metabolism in general [41]. Furthermore, cMyc, which acts downstream of IL-2 and is required for glucose and glutamine intake, requires O-GlcNAc modification for expression [34].…”

Selective protein O-GlcNAcylation in cells by a proximity-directed O-GlcNAc transferase

“…We next evaluated the proximity-directing ability of nGFP (13) in HEK293T cells co-expressing with GFP-Flag-JunB-EPEA, a transcription factor carrying multiple O-GlcNAc sites (15), as the target protein. Immunoprecipitation of GFP-Flag-JunB-EPEA and probing for O-GlcNAc revealed an increase in O-GlcNAc levels on the target protein that was dependent on the co-transfected nGFP(13) ( Figure 2C).…”

“…In contrast to most PTMs, O-GlcNAc is installed and removed by only two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which modify over 3,000 protein substrates ( Figure 1) (11)(12)(13)(14)(15). O-GlcNAc is critical to cellular function as deletion of OGT in mice is embryonic lethal (16), deletion of OGA leads to perinatal death (17), and the conditional deletion of OGT in numerous cell types leads to senescence and apoptosis (18).…”