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dc.contributor.advisorWang, Lai-Xien_US
dc.contributor.authorTong, Xinen_US
dc.date.accessioned2019-09-27T05:37:46Z
dc.date.available2019-09-27T05:37:46Z
dc.date.issued2019en_US
dc.identifierhttps://doi.org/10.13016/b3dg-wc5w
dc.identifier.urihttp://hdl.handle.net/1903/25019
dc.description.abstractMonoclonal antibodies (mAbs) comprise a rapidly growing class of therapeutics with great potential in treating infections, inflammations, cancers, and autoimmunities. The glycosylation of an antibody determines its functional efficacy and structural integrity, but stringent control of the intrinsically heterogeneous N- glycans on an antibody remains a formidable challenge. Recent development of a chemoenzymatic glycosylation remodeling strategy using a family of carbohydrate- modifying enzymes called endoglycosidases is emerging as an attractive method for producing homogeneous antibody glycoforms. The success of this method depends on the discovery of efficient endoglycosidases and glycosynthase mutants. Here, we describe mutagenesis studies on several endoglycosidases and their applications in expanding the current chemoenzymatic glycoengineering strategy to different antibodies. Five projects related to this effort are described here. First, glycosynthase variants of Endo-S were generated by mutagenesis to provide enhanced transglycosylation activities and diminished tendency to hydrolyze the product. Second, mutational studies on another endoglycosidase, Endo-S2, identified novel glycosynthase variants with broader substrate specificity and higher catalytic efficiency than Endo-S mutants. This work also provided the first kinetic studies for Endo-S and Endo-S2 mutants, with important mechanistic implications. Third, the unique properties of Endo-S allowed us to develop an alternative glycan remodeling strategy to synthesize several antibody glycoforms that were not easily accessible using the conventional glycoengineering approaches. This new method can be applied in a facile, one-pot fashion to modify antibody glycosylation without the need for purifying intermediates or switching enzymes. Fourth, to further expand the toolbox for antibody glycoengineering, the substrate specificity of a newly discovered endoglycosidase Endo-CC was characterized. The highly flexible selectivity of this enzyme for protein framework substrates paved the way for glycoengineering of additional antibody isotypes, particularly IgE, as a single N-glycan was found to be indispensable for the biological functions. Finally, remodeling of IgE glycosylation with alternative glycan structures was achieved for the first time by endoglycosidases. These novel IgE glycoforms displayed distinct binding properties for IgE receptors and revealed important new aspects of the structure-function relationship of IgE antibody glycosylation. Together, these studies should facilitate the development of novel antibody-based therapeutics that are optimized in their glycosylation patterns.en_US
dc.language.isoenen_US
dc.titleEXPLORING ENDOGLYCOSIDASES FOR ANTIBODY GLYCOENGINEERINGen_US
dc.typeDissertationen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.contributor.departmentBiochemistryen_US
dc.subject.pqcontrolledBiochemistryen_US


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