Stephen G Withers

Stephen G Withers
University of British Columbia - Vancouver | UBC · Department of Chemistry

BSc, PhD

About

746
Publications
60,566
Reads
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33,189
Citations
Citations since 2016
146 Research Items
9772 Citations
201620172018201920202021202205001,0001,500
201620172018201920202021202205001,0001,500
201620172018201920202021202205001,0001,500
201620172018201920202021202205001,0001,500
Additional affiliations
August 1982 - October 2015
University of British Columbia - Vancouver
Position
  • Professor (Full)

Publications

Publications (746)
Preprint
Sialic acids are key mediators of cell function, particularly with regards to cellular interactions with the surrounding environment. Reagents that modulate the display of specific sialyl glycoforms at the cell surface would be useful biochemical tools and potentially allow for therapeutic intervention in numerous challenging chronic diseases. Whil...
Article
Gaucher disease is a lysosomal storage disorder caused by mutations which destabilize the native folded form of GCase, triggering degradation and ultimately resulting in low enzyme activity. Pharmacological chaperones (PCs) which stabilize mutant GCase have been used to increase lysosomal activity through improving trafficking efficiency. By engine...
Article
Gaucher disease is a lysosomal storage disorder caused by mutations which destabilize the native folded form of GCase, triggering degradation and ultimately resulting in low enzyme activity. Pharmacological chaperones (PCs) which stabilize mutant GCase have been used to increase lysosomal activity through improving trafficking efficiency. By engine...
Article
Full-text available
Azido sugars have found frequent use as probes of biological systems in approaches ranging from cell surface metabolic labeling to activity-based proteomic profiling of glycosidases. However, little attention is typically paid to how well azide-substituted sugars represent the parent molecule, despite the substantial difference in size and structur...
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The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally cha...
Article
The 1013–1014 microorganisms present in the human gut (collectively known as the human gut microbiota) dedicate substantial percentages of their genomes to the degradation and uptake of carbohydrates, indicating the importance of this class of molecules. Carbohydrates function not only as a carbon source for these bacteria but also as a means of at...
Article
Donor organ allocation is dependent on ABO matching, restricting the opportunity for some patients to receive a life-saving transplant. The enzymes FpGalNAc deacetylase and FpGalactosaminidase, used in combination, have been described to effectively convert group A (ABO-A) red blood cells (RBCs) to group O (ABO-O). Here, we study the safety and pre...
Article
The prospect of producing human-like glycoproteins in bacteria is becoming attractive as an alternative to already-established but costly mammalian cell expression systems. We previously described an E. coli expression platform that uses a dual-plasmid approach to produce simple mucin type O-glycoproteins: one plasmid encoding the target protein an...
Article
Propionibacterium acnes, though generally considered part of the normal flora of human skin, is an opportunistic pathogen associated with acne vulgaris as well as other diseases, including endocarditis, endophthalmitis and prosthetic joint infections. Its virulence potential is also supported by knowledge gained from its sequenced genome. Indeed, a...
Article
β-N-Acetylhexosaminidases (HexNAcases) are versatile biocatalysts that cleave terminal N-acetylhexosamine units from various glycoconjugates. Established strategies to generate glycoside-forming versions of the wild type enzymes rely on the mutation of their catalytic residues; however, successful examples of synthetically useful HexNAcase mutants...
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Supplementing influenza vaccines with recombinant neuraminidase (rNA) antigens remains a promising approach for improving the suboptimal vaccine efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced using the baculov...
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Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage con...
Preprint
Full-text available
Supplementing influenza vaccines with recombinant neuraminidase (rNA) remains a promising approach for improving the suboptimal efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced from different construct designs u...
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A set of cyclopentanoid α-galactosidase ligands was prepared from a partially protected ω-eno-aldose via a reliable (2 + 3)-cycloaddition protocol with slightly modified conditions. The obtained N-benzylisoxazolidine ring was selectively opened and the configuration of the hydroxymethylgroup was inverted. Consecutive deprotection provided an aminoc...
Article
Purpose This study explores feasibility and safety of using enzymes (FpGalNAc deacetylase & FpGalNase) to remove ABO-A antigens from donor lungs during ex vivo lung perfusion (EVLP). The enzymes have been reported to efficiently convert ABO-A blood to O (Withers, Nat Microbiol, 2019). Methods Human lungs (ABO-A1, n=5), declined for transplant, wer...
Article
N-Glycosylation is a fundamental protein modification found in both eukaryotes and archaea. Despite lacking N-glycans, many commensal and pathogenic bacteria have developed mechanisms to degrade these isoforms for a variety of functions, including nutrient acquisition and evasion of the immune system. Although much is known about many of the enzyme...
Article
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Background The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity...
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The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimall...
Article
As part of a search for a substrate for droplet-based microfluidic screening assay of -N-acetylgalactosaminidases, spectral and physical characteristics of a series of coumarin derivatives were measured. From among these a new coumarin-based fluorophore, Jericho Blue, was selected as having optimal characteristics for our screen. A reliable method...
Article
Gaucher’s disease, the most prevalent lysosomal storage disorder, is caused by missense mutation of the GBA gene, ultimately resulting in deficient GCase activity, hence the excessive build-up of cellular glucosylceramide....
Article
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Alpha-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities. Efforts to find better amylase mutants, such as through high-throughput screening, would be greatly aided by access to precise and robust active site titrating agents for quantitation of active mutants in c...
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Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CH...
Article
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The scope of a series of N-alkylated iminosugar based inhibitors in the d-gluco as well as d-xylo configuration towards their interaction with human lysosomal β-glucocerebrosidase has been evaluated. A versatile synthetic toolbox has been developed for the synthesis of N-alkylated iminosugar scaffolds conjugated to a variety of terminal groups via...
Article
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The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophili...
Article
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Arabinoxylan is mainly found in the hemicellulosic fractions of rice straw, corn cobs, and rice husk. Converting arabinoxylan into (arabino)xylo-oligosaccharides as added-value products that can be applied in food, feed, and cosmetics presents a sustainable and economic alternative for the biorefinery industries. Efficient and profitable AX degrada...
Article
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Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid β-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent β-d-galactosi...
Article
Purpose We explored the feasibility and safety of using recently developed enzymes (FpGalNAc deacetylase and FpGalNase) to remove histo-blood type antigens from donor lungs during ex vivo lung perfusion (EVLP). The enzymes have been reported to efficiently convert Type A whole blood to O (Withers, Nature Microbiology, 2019). Methods Human type A r...
Article
Full-text available
Transfusion of blood, or more commonly red blood cells (RBCs), is integral to healthcare systems worldwide, but requires careful matching of blood types to avoid serious adverse consequences. Of the four main blood types, A, B, AB, and O, only O can be given to any patient. This universal donor O-type blood is crucial for emergency situations where...
Article
The protective immunological effects of the influenza neuraminidase (NA) surface protein are of renewed interest but NA content in vaccines remains unstandardized and methods to easily and reliably quantify NA content are unsatisfactory. We describe the use of a recently developed fluorometric titration reagent, TR1, to efficiently quantify the tot...
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Simplified analogues of the potent human amylase inhibitor Montbretin A were synthesised and shown to bind tightly, Ki = 60 and 70 nM, with improved specificity over medically relevant glycosidases,...
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Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-through...
Article
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Alpha-linked N-acetyl-galactosamine (α-GalNAc) is most notably found at the nonreducing terminus of the blood type–determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans. However, despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few α-N-acetylga...
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Access to efficient enzymes that can convert A and B type red blood cells to ‘universal’ donor O would greatly increase the supply of blood for transfusions. Here we report the functional metagenomic screening of the human gut microbiome for enzymes that can remove the cognate A and B type sugar antigens. Among the genes encoded in our library of 1...
Article
Polysialyltransferases (polySTs) are glycosyltransferases that synthesize polymers of sialic acid found in vertebrates and some bacterial pathogens. Bacterial polySTs have utility in the modification of therapeutic proteins to improve serum half-life, and the potential for tissue engineering. PolySTs are membrane-associated proteins and as recombin...
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The generation of new biocatalysts for plant biomass degradation and glycan synthesis has typically relied on the characterization and investigation of one or a few enzymes at a time. By coupling functional metagenomic screening and high-throughput functional characterization, we can progress beyond the current scale of catalyst discovery and provi...
Article
An influenza A(H1N1)pdm09 and an influenza B virus were passaged in 3-fluoro(eq)-4-guanidino difluoro sialic acid (3Feq4Gu DFSA), an inhibitor of the influenza neuraminidase (NA) to determine whether resistant variants could be selected. 3Feq4Gu DFSA is a mechanism-based inhibitor, forming a covalent link to Y406 in the NA active site. Given its si...
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Modified 1,5-dideoxy-1,5-imino-d-xylitol analogues with different substitution patterns involving position C-1 and/or the ring nitrogen were prepared, which were designed to serve as precursors for the preparation of iminoxylitol-based ligands and tools for the elucidation and modulation of human lysosomal β-glucocerebrosidase. Biological evaluatio...
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The reaction mechanism of glycoside hydrolases belonging to family 1 (GH1) of carbohydrate-active enzymes classification, hydrolysing beta-O-glycosidic bonds, is well characterised. This family includes several thousands of enzymes with more than 20 different EC numbers depending on the sugar glycone recog-nised as substrate. Most GH1 b-glycosidase...
Article
From 1,2;3,4-di-O-isopropylidene-d-galactopyranose, a series of highly functionalized branched-chain cyclopentanes was easily available. The initial partially protected cyclopentane tetraol is a versatile central intermediate and was exploited as subject to various highly regio- and stereoselective structural alterations with a view to prepare sele...
Article
Glycoside phosphorylases (GPs) catalyze the reversible phosphorolysis of glycosidic bonds, releasing sugar 1-phosphates. To identify a greater range of these under-appreciated enzymes, we have developed a high-throughput functional screening method based on molybdenum blue formation. In a proof-of-principle screen focused on cellulose-degrading GPs...
Article
The construction of large, phylogenomically diverse libraries of synthetic genes from a sequence-based family allows rapid evaluation of the substrate specificity encoded within the gene products and identification of rare catalytic capabilities. For carbohydrate-active enzymes such as glycosidases this opens up approaches to search for enzymes tha...
Article
Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing β-3-deoxy-D-manno-oct-2-ulosonic acid (β-Kdo). The β-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of gl...
Article
We have developed an Escherichia coli strain for the in vivo production of O-glycosylated proteins. This was achieved using a dual plasmid approach: one encoding a therapeutic protein target, and a second encoding the enzymatic machinery required for O-glycosylation. The latter plasmid encodes human polypeptide N-acetylgalactosaminyl transferase as...
Article
T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H‐T4L follows a double displacement mechanism with His26 serv...
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Thioglycosides are hydrolase‐resistant mimics of O‐linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. Traditional chemical syntheses of thioglycoside analogues of O‐GlcNAc‐modified peptides and proteins require lengthy, multi‐step approaches. We report the development of efficient, enzym...
Article
Full-text available
Thioglycosides are hydrolase‐resistant mimics of O‐linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. Traditional chemical syntheses of thioglycoside analogues of O‐GlcNAc‐modified peptides and proteins require lengthy, multi‐step approaches. We report the development of efficient, enzym...