Medizinische Hochschule Hannover Institut für Transfusionsmedizin THE MOLECULAR BASIS OF MICROPOLYMORPHISM AT RESIDUE 156 AND ITS FUNCTIONAL ROLE IN PEPTIDE SELECTION BY HLA-B*35 ALLOTYPES INAUGURAL DISSERTATION zur Erlangung des Grades einer Doktorin oder eines Doctors der Naturwissenschaften -Doctor rerum naturalium- (Dr. rer. nat.) vorgelegt von Ms. Trishna Manandhar aus Kathmandu, Nepal Hannover, 2014 Dedicated to my mother i Medizinische Hochschule Hannover Institut für Transfusionsmedizin THE MOLECULAR BASIS OF MICROPOLYMORPHISM AT RESIDUE 156 AND ITS FUNCTIONAL ROLE IN PEPTIDE SELECTION BY HLA-B*35 ALLOTYPES INAUGURAL DISSERTATION zur Erlangung des Grades einer Doktorin oder eines Doctors der Naturwissenschaften -Doctor rerum naturalium- (Dr. rer. nat.) vorgelegt von Ms. Trishna Manandhar aus Kathmandu, Nepal Hannover, 2014 ii Angenommen vom Senat der Medizinischen Hochschule Hannover am 18.06.2015 Gedruckt mit der Genehmigung der Medizinischen Hochschule Hannover Präsident: Prof. Dr. med. Christopher Baum Betreuer: Prof. Dr. med. Rainer Blasczyk Kobetreuer: Prof. Dr. rer. nat. Andreas Krueger 1: Gutachter: Prof. Dr. med. Rainer Blasczyk 2: Gutachter: Prof. Dr. rer. nat. Andreas Krueger 3: Gutachter: Prof.„in Dr. phil. nat. Dr. med. Ulrike Köhl Tag der mündlichen Prüfung vor der Prüfungskommission: 18.06.2015 Prof.„in Dr. rer. nat. Christine Falk Prof. Dr. med. Rainer Blasczyk Prof. Dr. rer. nat. Andreas Krueger Prof.„in Dr. phil. nat. Dr. med. Ulrike Köhl iii Wissenschaftliche Betreuung Prof. Dr. med. Rainer Blasczyk Dr. rer. nat. Christina Bade-Doeding Institut für Transfusionsmedizin Institut für Transfusionsmedizin Medizinische Hochschule Hannover Medizinische Hochschule Hannover Wissenschaftliche Zweitbetreuung Prof. Dr. rer. nat. Andreas Krueger Institut für Immunologie Medizinische Hochschule Hannover 1. Erst-Gutachterin / Gutachter Prof. Dr. med. Rainer Blasczyk Dr. rer. nat. Christina Bade-Doeding Institut für Transfusionsmedizin Institut für Transfusionsmedizin Medizinische Hochschule Hannover Medizinische Hochschule Hannover 2. Gutachterliche Stellungnahme durch Prof. Dr. rer. nat. Andreas Krueger Institut für Immunologie Medizinische Hochschule Hannover 3. Gutachterin / Gutachter Prof.‘in Dr. phil. nat. Dr. med. Ulrike Köhl Instituts für Zelltherapeutika mit dem Cellular Therapy Centre (CTC) Und der GMP Development Unit (GMPDU) Medizinische Hochschule Hannover Tag der mündlichen Prüfung: 18.06.2015 iv ERKLÄRUNG Hiermit erkläre ich, dass ich die Dissertation (The molecular basis of micropolymorphism at residue 156 and its functional role in peptide selection by HLA-B*35 allotypes) selbstständig verfasst habe. Ich habe keine entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten (Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar entgeltliche Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. Ich habe die Dissertation an folgenden Institutionen angefertigt: Institut für Transfusionsmedizin, Medizinische Hochschule Hannover. Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen ähnlichen Zweck zur Beurteilung eingereicht. Ich versichere, dass ich die vorstehenden Angaben nach bestem Wissen vollständig und der Wahrheit entsprechend gemacht habe. Ort, Datum: Unterschrift: v ACKNOWLEDGMENTS This dissertation would not have been possible without the guidance and the help of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. First and foremost, I would like to express my sincere gratitude and heartfelt appreciation to my respected supervisor, Dr. Christina Bade-Doeding, for her for whole-hearted support and for being a constant source of inspiration. I would like to thank for all her contributions of time, ideas, and funding to make my Ph.D. experience productive and motivating. It gives me immense pleasure to appreciate my esteemed supervisor, Prof. Dr. Rainer Blasczyk, for his tremendous support, patient assistance and valuable time to help me during entire period of this research work and to prepare this dissertation as accurate and as useful as possible. I sincerely like to express my deepest thanks to my co-supervisor, Prof. Dr. Andreas Krueger, for his cooperation and valuable suggestions throughout the project. In my later work of molecular modeling, I am particularly indebted to Dr. Trevor Huyton for making me able to understand the structural implications of HLA micropolymorphisms. The enthusiasm he has for science and research has always been motivational for me. This work would not have been possible without the constructive ideas and kind efforts of Ms. Soumya Badrinath and Mr. Thomas Kraemer. I am grateful for their unfailing personal and professional support to this project. Special thanks goes to Mr. Alexander Celik for helping me with the FACS analysis. I take this opportunity to thank Ms. Heike Kunze-Schumacher, Ms. Tanja Schwarz and Mr. Lars Haack for their excellent technical assistance. I would like to thank the tremendously helpful and supportive contributions of all the group leaders and staffs of Institute for Transfusion Medicine (MHH) for their cooperation during the thesis work. Special thanks go to Ms. Claudia Van Deventer for making all necessary arrangements during my Ph.D. I gratefully acknowledge the funding sources that made my Ph.D. work possible. It is an honor for me to thank German Academic Exchange Service (DAAD) for granting me the scholarship and providing the financial support during my stay in Germany. I would like to vi acknowledge Integrated Research and Treatment Center Transplantation (IFB-Tx) for providing the funds to this project. I would like to acknowledge TOPLAB GmbH for providing MALDI-TOF-MS and nano-LC ultra 2D HPLC-MS/MS services. I would like to thank my family and friends for providing me a huge support through my Ph.D. experiences. Special thanks go to Mr. Suman Bajracharya for sustained but silent encouragement during tough times in my Ph.D. pursuit. I would like to thank my friends Ms. Zhang Haijiao, Ms. Anja Baigger, Ms. Yuliia Yuzefovych, Dr. Dharmendra Gupta and Dr. Sonal Prasad for motivating me throughout my research. Last but not the least, I would like to express my deepest thanks to my parents and grandparents who encouraged me in all my pursuits. Ort, Datum: Unterschrift: vii TABLE OF CONTENTS List of Tables ................................................................................................... x List of Figures ................................................................................................ xi List of Abbreviations ..................................................................................... xii List of Appendices ....................................................................................... xvi ABSTRACT ................................................................................................... 1 ZUSAMMENFASSUNG .............................................................................. 3 1 INTRODUCTION............................................................................... 5 1.1 Overview of the immune system ................................................ 5 1.2 Introduction to Human Leukocyte Antigen (HLA) .................... 6 1.2.1 Discovery of HLA ........................................................................... 6 1.2.2 Nomenclature of HLA ..................................................................... 7 1.2.3 Genomic organization of HLA ........................................................ 8 1.2.4 HLA polymorphism ......................................................................... 9 1.3 Structure of HLA class I molecules.......................................... 11 1.4 Roles of HLA class I molecules ............................................... 12 1.4.1 Presentation of self and non-self peptides ..................................... 12 1.4.2 T cell recognition ........................................................................... 13 1.4.3 HLA and transplantation ............................................................... 15 1.5 HLA class I antigen presentation pathway ............................... 16 1.5.1 Peptide loading complex ............................................................... 16 1.5.2 Dynamics of the HLA class I antigen presentation pathway ........ 16 1.5.2.1 Generation of antigenic peptides in the cytosol............................. 16 1.5.2.2 Translocation of peptides to the ER .............................................. 17 1.5.2.3 Customised peptide trimming by ERAAP..................................... 19 1.5.2.4 Folding of HLA class I molecules ................................................. 19 1.5.2.5 Peptide loading and surface expression ......................................... 20 1.6 Down-regulation of HLA class I presentation ......................... 21 1.7 HLA class I polymorphism and TPN dependence ................... 22 1.8 Effect of polymorphism at AA position 156 within the PBR .. 23 1.9 Peptide prediction tools ............................................................ 25 1.10 Aim of the study ....................................................................... 26 2. OBJECTIVES ................................................................................... 28 viii 3 MATERIALS AND METHODS ..................................................... 29