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dc.contributor.authorGiddens, John Patrick
dc.date.accessioned2017-06-20T18:14:21Z
dc.date.available2018-01-10T19:37:37Z
dc.date.issued2017
dc.identifier.urihttp://hdl.handle.net/10713/6756
dc.descriptionUniversity of Maryland, Baltimore. Biochemistry. Ph.D. 2017en_US
dc.description.abstractChemoenzymatic 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.en_US
dc.language.isoen_USen_US
dc.subjectchemoenzymatic synthesisen_US
dc.subjectEndoF3en_US
dc.subjectendoglycosidaseen_US
dc.subjectEndoSen_US
dc.subjectglycoengineeringen_US
dc.subjectoxazolineen_US
dc.subject.meshGlycoside Hydrolasesen_US
dc.titleChemoenzymatic synthesis of homogenous glycopeptides and glycoproteins using bacterial endoglycosidasesen_US
dc.typedissertationen_US
dc.contributor.advisorWang, Lai-Xi
dc.description.urinameFull Texten_US
refterms.dateFOA2019-02-19T18:18:14Z


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