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 N-acetylglucosamine (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 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie near 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.
Highlights
From the ‡Departments of Chemistry, §Microbiology & Immunology, and ¶Radiology; ʈInterdepartmental Program in Immunology; **Canary Center for Cancer Early Detection; ‡‡Howard Hughes Medical Institute; Stanford University, Stanford, California 94305; §§KAYAK, Cambridge, Massachusetts 02142
Identification of Over 2000 O-GlcNAz Containing Peptides from Human T Cells—Human T cells isolated from healthy blood bank donors were cultured with peracetylated N-azidoacetyl galactosamine (Ac4GalNAz, 40 M) for 50 h to metabolically label O-GlcNAc
Isotope Targeted Glycoproteomics (IsoTaG) enabled the identification of 1000 glycopeptides from a single donor on average, which in aggregate generated a data set containing 2219 unique O-linked glycopeptides from 1045 glycoproteins from human T cells
Summary
From the ‡Departments of Chemistry, §Microbiology & Immunology, and ¶Radiology; ʈInterdepartmental Program in Immunology; **Canary Center for Cancer Early Detection; ‡‡Howard Hughes Medical Institute; Stanford University, Stanford, California 94305; §§KAYAK, Cambridge, Massachusetts 02142. Activation of T cells during an immune response is a prime example of a biological context in which PTMs, such as phosphorylation [1, 2], methylation [3], acetylation [4], ubiquitylation [5, 6], and palmitoylation [7], regulate intracellular signaling pathways. These signaling events culminate in the activation of T cell effector functions and clonal expansion with the subsequent formation of long-lived memory cells. Several studies have correlated these global observations to the functional relevance of O-GlcNAc [23, 26, 28]
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