Glycoproteins: Biosynthesis, Structure and Biological Functions
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GLYCOPROTEINS: BIOSYNTHESIS, STRUCTURE AND BIOLOGICAL FUNCTIONS by Wesley F. Zandberg B.Sc. (Hons.), Trinity Western University 2002 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Chemistry © Wesley F. Zandberg 2010 SIMON FRASER UNIVERSITY Fall 2010 All rights reserved. However, in accordance with the Copyright Act of Canada, this work may be reproduced, without authorization, under the conditions for Fair Dealing. Therefore, limited reproduction of this work for the purposes of private study, research, criticism, review and news reporting is likely to be in accordance with the law, particularly if cited appropriately. APPROVAL Name: Wesley F. Zandberg Degree: Ph.D Title of Thesis: Glycoproteins: Biosynthesis, structure and biological functions Examining Committee: Chair: Dr. Hua-Zhong (Hogan) Yu Professor, Department of Chemistry ______________________________________ Dr. B. Mario Pinto Professor, Senior Supervisor ______________________________________ Dr. Vance E. Williams Associate Professor, Committee Member ______________________________________ Dr. Margo M. Moore Professor, Committee Member ______________________________________ Robert N. Young Professor, Internal Examiner ______________________________________ Dr. Harry Schachter Professor Emeritus, Department of Biochemistry, University of Toronto, External examiner ______________________________________ Date Defended/Approved: November 25 2010 ii Declaration of Partial Copyright Licence The author, whose copyright is declared on the title page of this work, has granted to Simon Fraser University the right to lend this thesis, project or extended essay to users of the Simon Fraser University Library, and to make partial or single copies only for such users or in response to a request from the library of any other university, or other educational institution, on its own behalf or for one of its users. The author has further granted permission to Simon Fraser University to keep or make a digital copy for use in its circulating collection (currently available to the public at the “Institutional Repository” link of the SFU Library website <www.lib.sfu.ca> at: <http://ir.lib.sfu.ca/handle/1892/112>) and, without changing the content, to translate the thesis/project or extended essays, if technically possible, to any medium or format for the purpose of preservation of the digital work. The author has further agreed that permission for multiple copying of this work for scholarly purposes may be granted by either the author or the Dean of Graduate Studies. It is understood that copying or publication of this work for financial gain shall not be allowed without the author’s written permission. Permission for public performance, or limited permission for private scholarly use, of any multimedia materials forming part of this work, may have been granted by the author. This information may be found on the separately catalogued multimedia material and in the signed Partial Copyright Licence. While licensing SFU to permit the above uses, the author retains copyright in the thesis, project or extended essays, including the right to change the work for subsequent purposes, including editing and publishing the work in whole or in part, and licensing other parties, as the author may desire. The original Partial Copyright Licence attesting to these terms, and signed by this author, may be found in the original bound copy of this work, retained in the Simon Fraser University Archive. Simon Fraser University Library Burnaby, BC, Canada Last revision: Spring 09 ABSTRACT Glycoproteins are formed by the covalent attachment of monosaccharides or their oligomers to proteins. The intermediates through which glycoproteins are biosynthesized are nucleotide sugars. The research described within this thesis focuses on the use of chemical methods by which the structures of glycans (the carbohydrate portion of a glycoconjugate) can be experimentally manipulated. Most of the glycan-altering compounds discussed herein prevent the normal biosynthesis of glycoproteins either because of their metabolism into non-natural nucleotide sugars or by preventing the normal utilization of nucleotide sugars. The perturbation of glycan structures is one of the main ways in which the biological functions of glycans can be assessed. Three different classes of glycoproteins were investigated, namely, glycoproteins containing asparagine- or serine/threonine-linked glycans, and those containing only serine/threonine- linked N-acetylglucosamine residues. Two thiosugar-containing disaccharide analogues were discovered, in a cell-based screening assay, to significantly impair the biosynthesis of asparagine-linked glycans within treated cells. These compounds, generally called 5-thiomannosides, were extensively characterized in the context of their effects on proprotein convertases, a class of proteases that are responsible for the production of a wide variety of bioactive peptides. The 5-thiomannosides investigated inhibited the production of endorphin in pituitary cells and were used iii to demonstrate the importance of correct glycosylation on the regulated peptide- hormone-targeting inside cells. Furthermore, this class of compounds was found to inhibit another key proprotein convertase involved in cholesterol biosynthesis. Several different techniques were used to investigate the structures of these glycans isolated from treated cells, the most significant of which was capillary electrophoresis (CE). This technique was used to deduce the structures of glycans obtained from cells by comparison with known standards or on the basis of their sensitivity in various enzyme assays. Furthermore, CE methods for the analysis of nucleotide sugars or nucleotides, and carbohydrate-like natural products were developed. These methods were used to develop an assay for an important drug target in the bacterium Mycobacterium tuberculosis and to investigate the biosynthesis of unusual nucleotide sugars within cultured cells. Of particular interest was the biosynthesis of nucleotide sugars containing thiosugar moieties, that is, sugars that contain an endocyclic sulfur atom. iv EXECUTIVE SUMMARY This thesis describes research that was carried out in several areas of glycobiology. Chapter 1 introduces the concept of glycobiology and briefly reviews the subject of glycoproteins, in particular N-linked glycoproteins. The processing of N-glycans by ER glycosidases and the role of these events in glycoprotein folding and quality-control are reviewed since these concepts are investigated in subsequent chapters. Chapter 2 discusses in detail the biosynthesis of glycoproteins focusing especially on (1) N-glycosylation and the origin of the diversity within this class of glycoconjugate and (2) nucleotide sugars, which are the primary glycan precursors through which glycoconjugates are biosynthesized. Both of these subjects have been extensively reviewed before; however, Chapter 2 focuses primarily on a relatively unexplored aspect of these processes which is the link between the availability of the nucleotide sugars and the glycan diversity observed among glycoproteins. The availability of these sugar donors is very often responsive to changes in the basic metabolism of cells, that is, the processes by which cells obtain energy and by which they biosynthesize glycoconjugate building blocks—two processes which are highly integrated. Secondary metabolism, then, would be considered to be the assembly of glycoconjugates, a process that requires both energy and previously made v building blocks. Only recently have analytical techniques become available for the analysis of nucleotide sugar metabolism and this chapter reviews some of these methods. Chapter 3 describes several applications of the use of CE for nucleotide sugar analysis. It begins by describing my work on improving existing methods, especially in terms of lowering analyte detection limits. This improved method was developed into a CE-based in vitro assay of an enzyme that interconverts the nucleotide sugars uridine diphosphate-α-D-galactopyranoside (UDP-Galp) to UDP-α-D-galactofuranoside (UDP-Galf). This enzyme, known as UDP-Galp mutase (UGM), is an important drug target in the bacteria Mycobacterium tuberculosis, the causative agent of tuberculosis. This assay was used to screen several potential inhibitors of UGM and the discovery of one new UGM inhibitor is reported. Chapter 3 also describes the use of CE to analyze the biosynthesis of chemically altered monosaccharides into nucleotide sugars both in vitro and in cultured cells. My work on the biosynthesis of UDP-α-D-N-glycolyglucosamine (UDP-GlcNGc) has important implications for sialic acid metabolism, while my work on the non-natural nucleotide sugar UDP-α-D-5-thio-N-acetylglucosamine (UDP-5-thioGlcNAc) has led to the first inhibitor of an important enzyme known as O-GlcNAc transferase (OGT). The chemoenzymatic synthesis of a new compound, UDP-5-thioGlcNAc, is also be reported in this chapter. Chapter 4 investigates the unusual O-glycosylation of a protein known as Notch in which a glucose (Glc) residue is transferred onto the protein where it is subsequently elongated upon the successive transfer of two xylose (Xyl) vi residues. A structure for the resulting trisaccharide had been proposed by analogy with similar modifications found on other proteins. Chapter 4 reports how a combination of CE and mass spectrometry was used to prove the structure of this