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Glycomic Analysis of Biomedically Important O-Glycoconjugates Mohd Nazri Ismail

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Glycomic Analysis of Biomedically Important O-Glycoconjugates Mohd Nazri Ismail Glycomic Analysis of Biomedically Important O-glycoconjugates A thesis submitted for the Degree of Doctor of Philosophy of Imperial College London Submitted by Mohd Nazri Ismail Imperial College London Division of Molecular Biosciences Department of Life Sciences Faculty of Natural Sciences South Kensington London SW7 2AZ United Kingdom 2 Declaration I hereby declare that the work presented in this thesis has not been previously or concurrently submitted for any other degree, diploma or other qualification at any other university, and it is the result of my own independent research unless otherwise indicated. Mohd Nazri Ismail 3 Acknowledgements This PhD work would not have been possible without the support of many people. I would like to express my deepest gratitude to my supervisor, Prof. Anne Dell who was abundantly helpful and offered invaluable assistance, support and guidance. Deepest appreciation is also due to my second supervisor, Dr. Stuart Haslam, without whose knowledge and assistance this thesis would not have been successful. Special thanks also to all former and current group members, Prof. Howard Morris, Dr. Maria Panico, Dr. Berangere Tissot, Dr. Mark Sutton-Smith, Dr. Aristotelis Antonopoulos, Alana Trollope, Rebecca Harrison, Kevin Canis, Dr. Poh-Choo Pang, Dr. Paul Hitchen, Dr. Rositsa Karamanska, David Damerell, Dr. Simon Parry, Dr. Alessio Ceroni and Dr. Simon North, for sharing knowledge and invaluable assistance. Not forgetting to my friends who always been there and making my life journey in this wonderful but challenging city of London much smoother. It was an honour for me to collaborate with some of the best scientists in this field, including Prof Jamey Marth, Dr. Erica Stone, Prof Minoru Fukuda, Dr. Celso Reis, Ana Magalhaes and Dr. Frederic Bard. I would also like to convey my appreciation to the Malaysian Ministry of Higher Education and the Universiti Sains Malaysia for providing the financial means throughout my study. Lastly, I wish to express my love and gratitude to my beloved families; for their understanding & endless love, through the duration of my study. Alhamdulillah - ﺗﺮﻳﻤﺎ ﻛﺎﺳﻴﻪ - Thank you 4 Abstract It has been established that all cells carry an array of glycans attached to proteins and lipids that are crucial in the interaction between cells and the surrounding matrix. Proteins are mainly glycosylated on asparagines (N-glycosylation) and serine or threonine residues (O- glycosylation). Compared to N-glycans, O-glycans offer a higher degree of structural ambiguity due to the existence of several types and cores. This is believed to contribute to the relative lack of knowledge on these molecules. Therefore, improvement to the current methodologies of structural studies is a prerequisite to complement the immense functional findings of O- glycoconjugates in biological systems. This thesis discusses the structural characterisation, regulation and biological roles of O-glycans. The overall aim was to optimise O-glycomic mass spectrometric analysis to help illuminate the phenotypic findings from our collaborators in three separate but related projects. The methodologies utilised involving MALDI-TOF/TOF-MS, GC- EI-MS, ESI-QTOF-MS and MALDI-QIT-TOF-MS. The first project investigated the effects of core 2 GlcNAc transferase (C2GnT) deficiency in mice. This enzyme exists in three isoforms which are expressed differently in different tissues. Analysis of the single knockout of each of these isoenzymes as well as the triple knockouts has allowed the investigation of their unique and overlapping functions. The outcomes of this study include characterisation of alterations of not just mucin-type O-glycans but also O- mannose glycan, which could be associated with several organ lesions and system failures. The second project focused on the gastric mucosa of mice with deficiency in α1,2-fucosyltranferase (FuT2). This enzyme plays an important role in decorating the mucosal mucins with ABH-blood group and Lewis antigens that are known to interact with various gut flora including the pathogen Helicobacter pylori. It has been shown that the binding of H. pylori via BabA adhesins was significantly impaired with the loss of H antigens and Lewis y on O-glycans. The third project investigated the regulation of mucin-type O-glycosylation. The protein Src has been recognised to play an essential role in the localisation of ppGalNAc transferases, the initiating enzyme of O- glycosylation, in the endoplasmic reticulum and Golgi apparatus. Therefore, it could be inferred that Src influences the regulation of protein O-glycosylation. The NIH3T3 and NBT-II cell lines with different levels of Src or different localisation of ppGalNAcT-2 have been analysed in order to identify the changes on the structures of O-glycans and the relative abundances of cores 1 and 2. Valuable information has been gathered which could lead to further investigative work to better understand the role of Src in the regulation of protein O-glycosylation. 5 Thesis publications 1. Glycosyltransferase function in core 2-type protein O-glycosylation. Molecular and Cellular Biology. 29: 3770-3782. July 2009. Stone EL, Ismail MN, Lee SH, Luu Y, Ramirez K, Haslam SM, Ho SB, Dell A, Fukuda M, Marth JD. 2. Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa. Glycobiology. 19: 1525-1536. December 2009. Magalhaes A, Gomes J, Ismail MN, Haslam SM, Mendes N, Osorio H, David L, Le Pendu J, Haas R, Dell A, Boren T, Reis CA. 3. Sweet receptors mediate the adhesion of the gastric pathogen Helicobacter pylori: glycoproteomic strategies. Expert Review of Proteomics. 7: 307-310. June 2010. Magalhães A, Ismail MN, Reis CA. 4. Mass spectrometric analysis of mutant mice. In: Minoru F, editors. Methods in Enzymology. Academic Press. Volume 478: p. 27-77. 2010. North SJ, Jang-Lee J, Harrison R, Canis K, Ismail MN, Trollope A, Antonopoulos A, Pang P-C, Grassi P, Al- Chalabi S, Etienne AT, Dell A, Haslam SM. 5. Characterization of mice with targeted deletion of the gene encoding core 2 β1,6-N- acetylglucosaminyltransferase 2. In: Minoru F, editors. Methods in Enzymology. Academic Press. Volume 479: 155-172. 2010. Stone EL, Lee SH, Ismail MN, Fukuda M. 6. High Sensitivity O-glycomic Analysis of Mice Deficient in Core 2 β1,6-N- acetylglucosaminyltransferases. Glycobiology. Accepted: September 2010. Ismail MN, Stone E, Panico M, Lee S, Luu Y, Ramirez K, Ho S, Fukuda M, Marth J, Haslam S, Dell A. 6 Contents DECLARATION ...................................................................................................................................... 2 ACKNOWLEDGEMENTS ....................................................................................................................... 3 ABSTRACT............................................................................................................................................. 4 THESIS PUBLICATIONS ....................................................................................................................... 5 CONTENTS............................................................................................................................................. 6 INDEX OF FIGURES .............................................................................................................................. 9 INDEX OF TABLES .............................................................................................................................. 12 1 INTRODUCTION .......................................................................................................................... 16 1.1 Sweet interactions: biomedically important glycoconjugates .............................................. 16 1.2 Protein glycosylation ............................................................................................................ 19 1.2.1 N-glycosylation ................................................................................................................ 20 1.2.2 O-glycosylation ................................................................................................................ 21 1.2.2.1 β-O-galactose, α-O-fucose, β-O-glucose and α-O-mannose ................................. 22 1.2.2.2 Mucin type O-glycans ............................................................................................. 24 1.2.2.3 Core 2 β1,6-N-acetylglucosaminyltransferase ....................................................... 26 1.2.3 Glycan extension, branching and terminal epitopes ....................................................... 29 1.2.3.1 Lewis antigens ........................................................................................................ 32 1.2.3.2 Functional significance of fucosyltransferases ....................................................... 33 1.2.3.3 Carbohydrate recognition by lectins ....................................................................... 35 1.2.4 Mucins ............................................................................................................................. 40 1.2.5 Helicobacter pylori: a specialist in human stomach colonisation .................................... 44 1.2.6 Regulation of protein O-glycosylation ............................................................................. 46 1.2.7 Glycomics and glycoproteomics ...................................................................................... 52 1.3 Animal models
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