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Interrogation of GH109 glycosidase specificity and activity through a synthetic gene library Huff, Drew

Abstract

Glycoside Hydrolases (GHs) or glycosidases play a major role in carbohydrate catabolism, catalyzing the hydrolysis and cleavage of glycosidic bonds. In the Carbohydrate Active enZyme database (CAZy), several classes of enzymes including GHs have been classified into families based on sequence similarity. To date, the CAZy GH family 109 consists solely of exo α-N-acetylgalactosaminidase (nagalase) enzymes that are exclusively found in bacteria. α-GalNAc residues are primarily found in two major contexts in biology. The first is that many O-linked glycans of glycoproteins initiate with an α-linked GalNAc residue that is covalently bound to serine or threonine. The second are the A-antigens found at various sites in the human body, most notably on A-type red blood cells (RBCs), which are defined by a terminal α-GalNAc residue. GH109 enzymes have been studied and exploited based on their ability to convert erythrocyte A-antigens into H-antigens as a means to produce universal donor RBCs. Clinical uptake of this enzymatic conversion of RBC A-antigens has been limited by enzyme competence. Despite GH109 being the best known nagalase-containing family, very few of the glycosidases in the family have been characterized. To better understand GH109 family diversity and search for more efficient enzymes for generating universal blood, a synthetic gene library encoding 11 phylogenetically distinct GH109s was established. This thesis describes the recombinant expression of each enzyme in E. coli, subsequent protein purification, and characterization. Monosaccharide specificity, ability to convert RBC A-antigens, and activity on glycosylated proteins and peptides was assessed for each enzyme in addition to pH optima, cofactor dependence, and kinetic parameters. This project has provided a greater perspective on the CAZy GH109 family, and identified GH109 glycosidases that hydrolyze α-GalNAc residues from mucin and fetuin-A glycans, as well as four enzymes that convert RBC A-antigens to H-antigens.

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Attribution-NonCommercial-NoDerivatives 4.0 International