Date of Award

Spring 1-1-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Zhongping Tan

Second Advisor

Wei Zhang

Third Advisor

Joel Kaar

Fourth Advisor

David Walba

Fifth Advisor

Tarek Sammakia

Abstract

Protein glycosylation, the covalent attachment of carbohydrates to amino acid side chains of proteins, is a ubiquitous post-translational modification across all branches of life. Due to many factors including the vast structural complexity of glycans and the convoluted processes regulating their construction, protein glycosylation is a significantly understudied phenomenon. In particular, the study of protein O-glycosylation, where the carbohydrate moieties are attached via the oxygen of a serine or threonine residue, is lacking because there exists no well-defined consensus sequence for its occurrence and the enzymatic construction of O-glycosylated proteins in a controlled manner is very difficult. We employed chemical synthesis for the construction of homogeneous and well-defined O-glycoproteins with a large variety of structures and used these synthetic biomolecules to systematically and quantitatively investigate the effects of glycosylation on the biophysical and biological properties of proteins. Our ultimate goal is to develop a set of principles that can be widely applied to the rational engineering of enzymes and therapeutic proteins through glycosylation and other post-translational modifications. The initial focus was on examining the effects of O-glycosylation on the properties of a carbohydrate binding module (CBM) of an industrially important fungal cellulase. We used a wide range of biochemical assays to characterize a library of 51 differently-glycosylated CBM isoforms, and observed strong effects of glycosylation, in a pattern specific manner, on the folding, stability, solubility, chromatographic behavior, binding affinity and specificity of this small domain. In the long term, this project is expected to lead to fungal cellulases with optimal stability, specificity, activity required to achieve efficient saccharification of biomass for biofuels production. We then expanded our methodology to investigate the influence of O-glycosylation on two important therapeutic peptides: insulin and glucagon-like peptide-1 (GLP-1). As with the CBM system, we observed that glycosylation can significantly impact physical and/or functional properties of these molecules. We have identified specific isoforms of both insulin and GLP-1 that have increased stability and unchanged biological functions. It is our hope that further development of the most promising lead candidates for insulin and GLP-1 could lead to better therapies for the treatment of metabolic disorders.

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