Abstract
O‐linked N‐acetylglucosamine transferase, OGT, is an essential mammalian enzyme that transfers a single N‐acetylglucosamine (GlcNAc) moiety to specific serine and threonine residues in numerous cellular proteins. Like protein phosphorylation, protein O‐GlcNAcylation is dynamic. However, unlike protein phosphorylation, which involves many highly specific kinases, OGT is the sole nuclear and cytoplasmic glycosyltransferase. Like protein phosphorylation, glycosylation is believed to play signaling and regulatory roles as aberrant glycosylation has been linked to numerous disease states from cancer to diabetes and neurodegeneration. Previous efforts to identify substrates of OGT have largely been based on the enrichment of in vivo samples and subsequent LC‐MS/MS analysis. This work describes an ex vivo approach that promises to offer unique information about structure‐function relationships within OGT. This approach has several advantages over previous methods including the ability to overcome the challenges of target enrichment and identifying low abundance proteins. The development of chemical and biochemical tools to enable an alternative detection method for O‐GlcNAc modified proteins will be described. Using this tool we have been able to identify previously unknown OGT substrates and our ongoing efforts aim to delineate the substrate profiles of various natural and unnatural OGT constructs as well as examine the roles of glycosylation conditions and accessory proteins on the identified substrates.
Published Version
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