Browsing School, Graduate by Subject "glycoengineering"
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Chemoenzymatic synthesis of homogenous glycopeptides and glycoproteins using bacterial endoglycosidasesChemoenzymatic synthesis of homogenous glycoproteins and glycopeptides using bacterial endoglycosidases is a powerful approach that enables functional and structural studies. This method requires a mutant enzyme, an activated glycan oxazoline donor, and a GlcNAc glycoprotein or glycopeptide acceptor. The following dissertation describes four projects that expand this method. First, we described the purification of homogenous biantennary and triantennary complex type N-glycans from natural sources: sialylglycopeptide from chicken egg yolk and bovine fetuin respectively. The semi-synthesis of biantennary complex type oxazoline from SGP was achieved along with the semi-synthesis of triantennary complex type oxazoline from fetuin in high yields and purity. These glycans were then transferred to Fmoc-Asparagine-GlcNAc-Fuc for further functional studies. Next, we described the substrate specificity of a panel of endoglycosidases using four glycoprotein substrates that have varying N-glycan types attached including high mannose, hybrid, and complex type. Each glycoprotein was treated with PNGaseF to release the all N-glycans and this profile was compared to the N-glycans released by each endoglycosidase. Next, we described the expression of endoglycosidase F3 and we tested its hydrolytic activity on three glycoprotein substrates fibrinogen, IgG, and bovine fetuin. Two glycosynthase mutants were generated using site directed mutagenesis, EndoF3 D165A and D165Q, and the ability of each to transfer SCTox to Fmoc-Asparagine-GlcNAc-Fuc was investigated and compared to the WT enzyme. We were also able to synthesize a homogenous triantennary glycoform of CD52 using our EndoF3 D165A mutant in combination with our newly synthesized triantennary glycan oxazoline. Lastly, we demonstrated the ability of EndoF3 D165A to synthesize homogenous glycoforms of the monoclonal antibody rituximab, including a novel triantennary complex type form in addition to biantennary complex type forms. The binding affinity of these glycoforms of rituximab to Galectin-3 and FcγRIIIa was investigated with surface plasmon resonance. Lastly, we described the EndoS mediated glycoengineering of homogenous glycoforms of rituximab using newly generated EndoS D233A and D233Q glycosynthase mutants. We demonstrated that EndoS could use both fucosylated and afucosylated acceptor substrates making complex type glycoforms with and without fucose. The binding affinity of these glycoengineered rituximab glycoforms to FcγRIIIa was investigated with surface plasmon resonance.