Development of chemical tools for the study of intracellular glycosylation

Author: 
Date created: 
2020-06-04
Identifier: 
etd21031
Keywords: 
O-GlcNAc
Chemical probes
High-throughput screening
Glycosylation
Assay development
Abstract: 

Intracellular protein O-glycosylation is an important post-translational modification in mammalian cells with critical regulatory functions relating to transcription, stress response, cell signalling, and cell cycle control. This process is controlled by two enzymes: O-GlcNAc transferase (OGT), which catalyzes the addition of a single O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of proteins, and O-GlcNAcase (OGA), which hydrolyzes the glycosidic linkage on proteins. While notable progress has been made in the design and application of chemical tools for the study of OGA, methods and tool compounds to detect and modulate the activity of OGT remain limited. In this thesis, I describe the development of a fluorescent glycosyl donor analogue which is tolerated by OGT and transferred to peptides and proteins. This substrate was exploited to develop a convenient and direct in vitro activity assay, enabling the study of OGT catalysis in the presence of various substrates and inhibitors. After optimizing the assay for high-throughput screening, a collection of small molecule libraries encompassing approximately 64,000 compounds was screened, leading to the identification of a novel and selective inhibitor of OGT. This assay was further applied to the functional analysis of peptide-based inhibitors of OGT that were discovered through phage display and in vitro mRNA display technologies. These efforts enabled the development of chemical tools with potent nanomolar affinity for OGT. The in vitro assay was also used for the study of novel glycosylation activity by OGT towards cysteine-containing acceptor substrates, permitting a detailed kinetic analysis of this recently discovered phenomenon. Finally, I describe the biochemical characterization of human Hexosaminidase D (HexD), an intracellular glycoside hydrolase of unknown physiological function, and use this information in the design of potential selective inhibitors. The methods, results, and insights gained from this research should prove useful in advancing our understanding of intracellular glycosylation and for furthering the application of this knowledge for translational therapeutic benefit.

Document type: 
Thesis
Rights: 
This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
File(s): 
Supervisor(s): 
David Vocadlo
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) Ph.D.
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