Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells Peder J. Lund, Joshua E. Elias and Mark M. Davis This information is current as J Immunol published online 21 September 2016 of October 3, 2021. http://www.jimmunol.org/content/early/2016/09/20/jimmun ol.1502031 Supplementary http://www.jimmunol.org/content/suppl/2016/09/20/jimmunol.150203 Downloaded from Material 1.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published September 21, 2016, doi:10.4049/jimmunol.1502031 The Journal of Immunology Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells Peder J. Lund,*,† Joshua E. Elias,‡ and Mark M. Davis†,x,{ T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional impor- tance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAcproteinsinhumanTcells.Manyofthe identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and Downloaded from RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells. The Journal of Immunology, 2016, 197: 000–000. he activation of specific T lymphocytes by Ag is a critical expression of new genes that are necessary for T cell effector step in the initiation of many immune responses. The function. In addition to tyrosine phosphorylation, a variety of http://www.jimmunol.org/ T binding of a TCR to its specific peptide-MHC ligand other posttranslational modifications are involved in the regulation triggers an intracellular signaling cascade, culminating in the ac- of T cell activation, a process that must be strictly controlled to tivation of T cell effector functions and the formation of long-lived permit appropriate responses against foreign Ags while preventing memory cells. Similar to many other cell signaling pathways, the inappropriate responses to self-antigens. Canonical examples regulation of T cell activation is mediated largely through the include serine/threonine phosphorylation, lysine methylation and regulation of protein posttranslational modifications. For instance, acetylation, lysine ubiquitylation, and cysteine palmitoylation, upon TCR ligation, lymphocyte-specific protein tyrosine kinase which can influence transcription factor activation, chromatin (LCK) phosphorylates the cytoplasmic tails of CD3, leading to the accessibility, protein stability, and membrane localization, by guest on October 3, 2021 recruitment of ZAP-70, which potentiates the signaling cascade respectively (3–19). through phosphorylation of additional downstream substrates In contrast to some of these modifications, relatively little is (1, 2). Eventually, these early signaling events translate into the known about the proteins targeted for glycosylation by O-linked N-acetylglucosamine (O-GlcNAc) during T cell activation and its effect on protein function. O-GlcNAc is the only known form of *Interdepartmental Program in Immunology, Stanford University, Stanford, CA intracellular glycosylation, which involves the reversible attach- 94305; †Department of Microbiology and Immunology, Stanford University, Stan- ford, CA 94305; ‡Department of Chemical and Systems Biology, Stanford University, ment of a GlcNAc monosaccharide to serine and threonine resi- Stanford, CA 94305; xStanford Institute for Immunity, Transplantation, and Infection, { dues (20–22). Remarkably, the opposing actions of just two Stanford University, Stanford, CA 94305; and Howard Hughes Medical Institute, enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase Stanford University, Stanford, CA 94305 (OGA), mediate the addition and removal of O-GlcNAc from the ORCIDs: 0000-0002-2146-8724 (P.J.L.); 0000-0002-8063-3259 (J.E.E.). hundreds of protein substrates identified to date, including tran- Received for publication September 15, 2015. Accepted for publication July 22, 2016. scription factors, epigenetic regulators, and kinases (20). Mount- ing evidence suggests that O-GlcNAc functions in the regulation This work was supported by National Institutes of Health Grants AI090019 and AI057229, the Howard Hughes Medical Institute, a George D. Smith Stanford Grad- of protein activity much like phosphorylation and other modifi- uate Fellowship, and a National Science Foundation Graduate Research Fellowship. cations. Although the molecular mechanisms remain mostly un- The Stanford Neuroscience Microscopy Service is supported by National Institutes of Health Grant NS069375. resolved, O-GlcNAc has clear biological significance because deletion of the OGT or OGA gene in mice leads to embryonic Address correspondence and reprint requests to Dr. Mark M. Davis, Beckman Center, Room B221, 279 Campus Drive, Stanford University, Stanford, CA lethality (23) or perinatal death (24), respectively. Furthermore, 94305. E-mail address: [email protected] conditional deletion of OGT results in cellular senescence and The online version of this article contains supplemental material. apoptosis in numerous cell types, including T cells (25). Consid- Abbreviations used in this article: Ac4GalNAz, tetraacetylated N-azidoacetylgalactosamine; ering that abnormal O-GlcNAc levels, possibly stemming from Ac-5SGlcNAc, 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-a-D-glucopyranose; alterations in metabolism (26–29), may contribute to the pathol- BEMAD, b-elimination followed by Michael addition of DTT; CIP, calf intestinal alkaline phosphatase; 2-DG, 2-deoxyglucose; OGA, O-GlcNAcase; O-GlcNAc, ogy of several diseases, including cancer, diabetes, and neuro- O-linked N-acetylglucosamine; OGT, O-GlcNAc transferase; PEG, polyethylene degeneration (29–36), a greater understanding of O-GlcNAc glycol; PIP3, phosphatidylinositol-3,4,5,-trisphosphate. biology in T cells could help to explain the impact of metabolic This article is distributed under The American Association of Immunologists, Inc., health on the function of the immune system. Reuse Terms and Conditions for Author Choice articles. Although previous studies suggested a role for O-GlcNAc in Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 T cell activation (37–39), they focused on immortalized cell lines, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502031 2 ANALYSIS OF O-GlcNAc IN ACTIVATED T CELLS making the relevance to primary T cells unclear. Furthermore, inhibitor). IGEPAL CA-630 (Sigma-Aldrich) was used as a substitute for only a selected few proteins were surveyed. In this study, we NP-40 in buffers in some cases. aimed to investigate the O-GlcNAc modification in primary hu- For subcellular fractionation, cells were washed once in cold PBS and then resuspended in isotonic lysis buffer (10 mM Tris [pH 8], 2 mM MgCl , man T cells so as to be as physiological as possible and to obtain a 2 3 mM CaCl2, 300 mM sucrose). NP-40 was added to a final concentration broader perspective of what proteins are modified using a pro- of 0.5%, and plasma membrane lysis was allowed to proceed for 3 min on teomics approach. In this article, we show that activation of pri- ice. Nuclei were pelleted for 5 min at 500 3 g at 4˚C. The supernatant, mary T cells directly ex vivo leads to increased staining for representing the crude cytosolic fraction, was supplemented with protease inhibitors and frozen at 280˚C. Nuclei were washed once in isotonic lysis O-GlcNAc, which is accompanied by changes in OGT localization buffer and resuspended in extraction buffer (10 mM HEPES [pH 7.9], and splicing. Suppressing O-GlcNAc modification through inhi- 0.75 mM MgCl2, 500 mM NaCl, 0.1 mM EDTA, 12.5% glycerol, 2.5 U/ml bition of OGT activity