Investigation of the role of xylosyltransferases I and II and of the kinase Fam20B in the regulation of proteoglycan synthesis Irfan Shaukat To cite this version: Irfan Shaukat. Investigation of the role of xylosyltransferases I and II and of the kinase Fam20B in the regulation of proteoglycan synthesis. Biochemistry, Molecular Biology. Université de Lorraine, 2015. English. NNT : 2015LORR0267. tel-01754553 HAL Id: tel-01754553 https://hal.univ-lorraine.fr/tel-01754553 Submitted on 9 Jan 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Contact : [email protected] LIENS Code de la Propriété Intellectuelle. articles L 122. 4 Code de la Propriété Intellectuelle. articles L 335.2- L 335.10 http://www.cfcopies.com/V2/leg/leg_droi.php http://www.culture.gouv.fr/culture/infos-pratiques/droits/protection.htm Ecole Doctorale BioSE (Biologie-Santé-Environement) Thèse Présenté et soutenu publiquement pour l’obtention du titre de DOCTEUR DE L’UNIVERSITE DE LORRAINE Mention : 《Science de la Vie et de la Santé》 Par Irfan SHAUKAT Etude du rôle des xylosyltransférases I et II et de la kinase Fam20B dans la régulation de la biosynthèse des protéoglycanes Investigation of the role of xylosyltransferases I and II and of the kinase Fam20B in the regulation of proteoglycan synthesis. Soutenue publiquement le 18 Novembre 2015 Membres de jury : Rapporteur: M. Abderrahman MAFTAH PR, Université de Limoges M. Yanusz WEGROWSKI CR, Université de Reims Examinateur: M. Marc DIEDERICH PR, LBMCC, Luxemburg M. Mohamed OUZZINE DR, Université de Lorraine Directeur de thèse ------------------------------------------------------------------------------------------------------- UMR 7365 CNRS, INGENIERIE MOLECULAIRE ET PHYSIOPATHOLOGIE ARTICULAIRE (IMoPA) 9, Avenue de la Foret de Haye, Biopôle de l’Université de Lorraine, 54500 Vandoeuvre lès Nancy Acknowledgement I would like to express my deepest gratitude to my thesis director Mohamed OUZZINE, Directeur de Recherche, a l’INSERM for accepting me under his kind supervision and his patience and encouragement throughout my research. It was impossible to complete this thesis without his guidance. I am grateful to Pr Abderrahman MAFTAH and Dr. Yanusz WEGROWSKI for accepting the request to be Rapporteur of my thesis and Pr Marc DIEDERICH for accepting to be part of my thesis Jury. I would like to thank Li Dong and Lydia Barré for their help in experiments. I will not forget the nice company of Xavier especially during late hours and weekends. This research work was conducted in the UMR-6375 IMoPA (Ingénierie Moléculaire et Physiopathologie Articulaire), Biopole, Faculty of medicine, University de Lorraine. Therefore, I am grateful to Pr. Jouzeau, Director, UMR-6375 and Pr. Magdalou the previous director of UMR- 7561 for welcoming me in the Lab. I am grateful to all the members of équipe 2, Ingénierie Moléculaire, Cellulaire, Thérapeutique & Glycosyltransférases (MolCelTEG) of UMR-7365 IMoPA, especially Sylvie Fournel-Gigleux, Sandrine Gulberti, Nick Ramalanjaona and Jean-Baptiste Vincourt. I am highly grateful to all my office mates Ann, Caroline, Dima, Mathieu, Mahdia, Mineem, Isabelle, Xiaomeng and Xu for their nice company, humor, friendliness, and valuable suggestions. I would like to like to acknowledge previous PhD students Emilie, Hassan, Jun, Lina, Mustafa, Reine and Suzanne. I would like to thanks all other members of the UMR-6375 IMoPA. Last but not least, I would like to thank my family for their support despite the geographical distance. Irfan SHAUKAT Table of Contents Acknowledgement .......................................................................................................................... 1 Table of Contents ............................................................................................................................ 2 List of Figures ................................................................................................................................. 5 List of tables .................................................................................................................................... 7 List of abbreviations ....................................................................................................................... 8 Chapter 1 ....................................................................................................................................... 10 1. Proteoglycans......................................................................................................................... 11 1.1 Glycosaminoglycans (GAGs) chains ................................................................................. 11 1.2 Classification of the PGs .................................................................................................... 13 1.3 Heparan sulfate proteoglycans (HSPG) ............................................................................. 15 1.3.1 HS synthesis ..................................................................................................................... 15 1.3.2 Syndecans ................................................................................................................... 18 1.3.3 Glypicans .................................................................................................................... 22 1.3.4 Perlecan ...................................................................................................................... 25 1.3.5 Betaglycan/TGFβ type III receptor............................................................................. 26 1.4 Chondroitin sulfate proteoglycans (CSPG) .................................................................... 28 1.4.1 Decorin ......................................................................................................................... 28 1.4.2 Biglycan ..................................................................................................................... 32 1.4.3 CSPG4/NG2 ............................................................................................................... 34 1.4.4 Aggrecan ..................................................................................................................... 35 1.4.5 Versican ...................................................................................................................... 36 Chapter 2 ....................................................................................................................................... 38 2. Biosynthesis of GAG chains.................................................................................................. 39 2.2 Synthesis of the tetrasaccharide linker ........................................................................... 39 2 2.3 Elongation of the CS chains ........................................................................................... 40 2.4 Elongation of the HS Chains .......................................................................................... 41 2.4 Regulation of PG synthesis ................................................................................................ 44 Chapter 3 ....................................................................................................................................... 47 3 Xylosyltransferase ................................................................................................................. 48 3.1 Specificity of the xylosyltransferase ............................................................................. 48 3.2 Structure of the XT-I and XT-II .................................................................................... 49 3.4 Structural/Functional relationship .................................................................................. 50 3.4 Purification of the XT from tissue source .................................................................... 52 3.5 Purification of the XT from cellular source as cDNA cloning ..................................... 52 3.6 Intracellular localization ................................................................................................ 53 3.7 XT activity analysis ...................................................................................................... 54 3.8 XT in health and disease ............................................................................................... 55 3.9 XT-I and XT-II genetic variants.................................................................................... 55 3.10 Regulation of Xylosyltransferase .................................................................................