Interrogation of GH109 glycosidase specificity and activity through a synthetic gene library
department or school: Science, Faculty of
department or school: Chemistry, Department of
data provider: DSpace
degree campus: UBCV
creator: Huff, Drew
issued: 2020-03-04T16:44:51Z
issued: 2020
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: 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-antig…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/73679
note: Interrogation of GH109 Glycosidase Specificity and Activity through a Synthetic Gene Library by Drew Huff B.Sc., Pacific Lutheran University, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February 2020 © Drew Huff, 2020 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Interrogation of GH109 Glycosidase Specificity and Activity through a Synthetic Gene Library submitted by Drew Huff in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Examining Committee: Stephen Withers, Chemistry and Biochemistry Supervisor Martin Tanner, Chemistry Supervisory Committee Member Lawrence McIntosh, Chemistry and Biochemistry and Molecular Biology Supervisory Committee Member Momose Takamasa, Chemistry and Physics and Astronomy Additional Examiner iii 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-…
has type: Thesis/Dissertation
date issued: 2020-05
is shown at: 10.14288/1.0388863
language: eng
degree discipline: Chemistry
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/73679