Abstract
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 importance 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-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and 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.