Employing Exoglycosidase And Transferase Enzymes in Capillary Nanogel Electrophoresis for the Determination of N-glycan Linkages and Enzyme Michaelis-Menten Constants.
Date of Graduation
Eberly College of Arts and Sciences
As a post translational modification protein glycosylation plays a crucial role in protein signaling, binding, kinetics and folding. In disease diagnosis and prognosis, monitoring glycosylation has been identified as a biomarker. Sialylation and sialic acid linkage in N-glycans are markers of cancers including liver, pancreatic and kidney cancer. Quantification of sialic acid linkage is analytically challenging because of the diverse linkages and the presence of heterogenous branching. A capillary electrophoresis method is reported that integrates a unique combination of enzymes and lectins to modify sialylated asparagine-linked glycans (N-glycans) in real time in the capillary so that N-glycan structures containing α2–6-linked sialic acid are easily separated, detected, and quantified. N-glycans were sequentially cleaved by the enzymes at the head of the separation capillary so that the presence of α2–6-linked sialic acids corresponded to a shift in the analyte migration time in a manner that enabled interpretation of the N-glycan structure. Complex N-glycans from α-1-acid glycoprotein were analyzed using this approach, revealing that a limited number of α2–6-linked sialic acids were present with biantennary, triantennary, and tetraantennary N-glycans of α-1-acid glycoprotein (AGP) generally containing 0 or 1 α2–6-linked sialic acid. The capillary electrophoresis method quantified the sialic acid linkages using nanoliter volumes of enzyme and lectins. Conversion with enzyme was in real time and incubation and separation occurred in less than 40 minutes.
In biotherapeutics glycosylation of IgG is a critical attribute, and modification of the glycosylation will influence the IgG efficacy. Glycosyltransferases have been employed to modify this glycosylation. Capillary nanogel electrophoresis was utilized to create discrete regions for an online galactosyltransferase reaction and subsequent separation of substrate and product. The β1-4 galactosyltransferase enzyme, donor, and co-factor were patterned in the capillary. The substrate was driven through these zones and converted to galactosylated products which were separated and identified. The degree of glycosylation was discernable. The method was applied in establishing the Michaelis-Menten value, KM of the enzyme. Additionally, the method was adapted to transfer galactose residues to protein. The applicability of the method for real-time online modification of whole protein was demonstrated with the Herceptin glycoprotein. The method demonstrated the applicability of capillary electrophoresis to characterize glycosyltransferases. This method is compatible with low enzyme or substrate volumes and is fast and automated.
Bwanali, Lloyd, "Employing Exoglycosidase And Transferase Enzymes in Capillary Nanogel Electrophoresis for the Determination of N-glycan Linkages and Enzyme Michaelis-Menten Constants." (2021). Graduate Theses, Dissertations, and Problem Reports. 8273.