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Development of chemical tools for studying two unusual forms of protein O-glycosylation

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Thesis type
(Thesis) Ph.D.
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The development of tools, techniques and methods has been important to the evolution of glycobiology and the elucidation of the function of many forms of protein glycosylation. O-GlcNAcylation of nucleocytoplasmic proteins and O-glucosylation of Notch are two unusual forms of protein O-glycosylation that require further investigation. The O-GlcNAc modification of proteins is a dynamic process, catalyzed by O-GlcNAc transferase, which installs the O-GlcNAc modification, and O-GlcNAcase, a glycosidase that cleaves O-GlcNAc from proteins. O-GlcNAc modification of proteins has been implicated in various disease states such as diabetes and Alzheimer’s disease. Unfortunately, many of the studies that link O-GlcNAcylation of proteins to these disease states use inhibitors of O-GlcNAcase that also affect functionally-related enzymes. In this thesis, an O-GlcNAcase selective inhibitor termed NButGT was developed as a tool to selectively study the biological role of O-GlcNAcase. A transition state analysis of two potent inhibitors of O-GlcNAcase was also undertaken to elucidate the likeness of these inhibitors to the transition state of the substrate during O-GlcNAcase catalyzed cleavage of O-GlcNAc. This study will influence the design of future generations of O-GlcNAcase inhibitors. O-Glucosylation, the second unusual form of protein O-glycosylation that was investigated in this thesis, modifies Notch a key transmembrane protein of the developmentally essential Notch signalling pathway. O-Glucosylation of Notch and elongation to a trisaccharide plays a role in Notch structure and function. Complete characterization of this trisaccharide is necessary to further our understanding of the role that it plays in the Notch signaling pathway. Di- and trisaccharide standards were synthesized, an α-xylosidase that could cleave the trisaccharide was identified, and a new capillary electrophoresis method for identifying this O-glycan on proteins was developed. These new tools enabled us to unequivocally assign the structure of this trisaccharide as XXG. After establishing the identity of the trisaccharide modifying Notch, we incorporated the trisaccharide into a peptide for use as an antigen to raise polyclonal antibodies. This antigen along with the other new chemical tools and methodologies that were developed will prove valuable in future work directed towards clarifying the roles of these non-canonical forms of O-glycosylation.
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Thesis advisor: Vocadlo, David
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