Wayne State University Wayne State University Dissertations 1-1-2015 Biochemical And Structural Characterization Of The orC e Subunits Of Gpi Transamidase Dilani G. Gamage Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Chemistry Commons Recommended Citation G. Gamage, Dilani, "Biochemical And Structural Characterization Of The orC e Subunits Of Gpi Transamidase" (2015). Wayne State University Dissertations. Paper 1132. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF THE CORE SUBUNITS OF GPI TRANSAMIDASE by DILANI G GAMAGE DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2015 MAJOR: CHEMISTRY (Biochemistry) Approved by: Advisor Date © COPYRIGHT BY DILANI G GAMAGE 2015 All Rights Reserved DEDICATION To my loving parents K. M. Rathnaweera and G. G. Gunasena My aunt D. C. Rathnaweera My loving husband N. G. Wijesundara My dissertation advisor Prof T. L. Hendrickson ii ACKNOWLEDGMENTS I would like to acknowledge my advisor, Professor Tamara L. Hendrickson for the constant support and advise given to me during last five years. Her mentorship, guidance and believing in me to be a good research scientist, allowed me be who I am today. With her enormous scientific knowledge and generosity I was able to learn a number of new skills and techniques. The excellent atmosphere she created in our lab reduced our working stress. Staying far away from our motherland, Sri Lanka, she once plays the role of a mother and a dear friend in many occasions. Her encouragement to build up self confident in me not only helped to achieve my targets in science, but also helped me to handle difficult situations in my life. With deepest gratitude, I sincerely thank you, Prof Tamara L. Hendrickson for giving me five blessed years at Wayne State University. My sincere gratitude goes to my thesis committee, Professor Christine S. Chow, Professor Sarah Trimpin and Professor Rafael A. Fridman for the invaluable support and comments given throughout my Ph.D. period. I would like to thank my committee for being supportive and understanding me throughout these time to achieve my goals. Unless the time and efforts you have put together, I won’t be able to fulfill my desires on time. A numerous support was given by my talented colleagues, Dr. Yug Varma, Ms. Megan Ehrenworth, Dr. Keng-Ming Chang, Dr. Sandamali Ekanayaka, Dr. Liangjun Zhao, Dr. Gayathri Silva, Shirin Fathma, Nilesh Joshi, Sairaman Seetharaman, Travis Ness, Udumbara Rathnayaka, Whitney Wood, iii Suwiman Subka and Aaron Sieg. I am thankful to all of them sharing the scientific knowledge and creating a friendly environment inside and outside the lab. I also like to thank Professor Trimpin, Professor Plum, Professor Bhagwat, Professor Feig, professor Ahn and Professor Chow for allowing me to share some of the valuable instruments from your labs. I also would like to acknowledge members from the department of chemistry, for helping me in many ways. Also I’d like to thank to Wayne State University for providing funding and facilities to pursue my Ph.D. My greatest gratitude goes to all of my Sri Lankan friends being with me during life’s challenges and frustrations and remembering me the value of friendship specially in a country far away from the home land. I would like to extend my deepest gratitude to my parents and my aunt for the sacrifices and efforts you have done on me to be who I’m today. Also I’m thankful to my loving brother and sister with their family for the help and support given to me throughout my life. Last but not least I am utterly grateful to my loving husband for the love, kindness and the support given to me throughout these years. I’m really grateful for your constant encouragement on my research and personal life. It’s your belief in me that built myself confident. iv TABLE OF CONTENTS Dedication…. ......................................................................................................... ii Acknowledgments ................................................................................................. iii List of Tables ....................................................................................................... xii List of Figures ..................................................................................................... xiii List of Abbreviations ........................................................................................... xvi Chapter 1 Introduction: GPI transamidase and GPI anchored proteins: Oncogenes and biomarkers for cancer ................................................ 1 1.1 Introduction ...................................................................................................... 1 1.2 The Gpi anchor: A substrate for cancer .......................................................... 4 1.3 Protein substrates for GPI-T ............................................................................ 7 1.4 The GPI trasnamidase complex .................................................................... 13 1.4.1 The PIG-K (Gpi8) subunit ..................................................................... 18 1.4.2 The GPAA1 (Gaa1) subunit ................................................................. 21 1.4.3 The PIG-T (Gpi16) subunit ................................................................... 23 1.4.4 The PIG-S (Gpi17) subunit ................................................................... 25 1.4.5 The PIG-U (Gab1) subunit ................................................................... 25 1.4.6 The TTA1 and TTA2 subunits .............................................................. 27 1.5 GPI-T and cancer .......................................................................................... 28 1.5.1 PIG-U and cancer ................................................................................ 29 1.5.2 PIG-T and cancer ................................................................................. 31 1.5.3 GPAA1 and cancer .............................................................................. 33 v 1.5.4 PIG-K and cancer ................................................................................. 34 1.5.5 PIG-S and cancer ................................................................................. 35 1.6 How does GPI-T subunit overexpression lead to cancer? ............................. 35 1.7 Functions of GPI anchored proteins relevant to tumergenosis ...................... 41 1.7.1 Urokinase plasminogen activated receptor (uPAR) ............................. 42 1.7.2 Glypican-3 ............................................................................................ 43 1.7.3 Folate binding receptor ........................................................................ 44 1.7.4 Prostasin ............................................................................................. 45 1.8 Dissertation research ..................................................................................... 45 Chapter 2: Gpi823-306 dimerization: Effects of N-linked glycosylation and analysis of the predicted dimer interface…………... ......................... 48 2.1 Introduction .................................................................................................... 48 2.2 Results ........................................................................................................... 50 2.2.1 Purification optimization of yeast Gpi81-306 overexpressed in yeast.......................................................................... .......................... 50 2.2.2 Homo-dimerization of yeast Gpi823-306 overexpressed in yeast versus E. coli. .................................................................................... …54 2.2.3 The effect of N-linked glycosylation on dimerization of Gpi823-306 ......... 57 2.2.4 Predicted dimerization interface analysis using single point mutations . 64 2.3 Discussion ..................................................................................................... 68 2.4 Materials and methods .................................................................................. 70 2.4.1 Buffers and solutions. ............................................................................ 70 2.4.2 Over-expression and purification of yeast Gpi823-306 from E. coli. ......... 70 2.4.3 Overexpression and purification of yeast Gpi823-306 from yeast. ........... 73 2.4.4 Analysis of the oligimerization state of Gpi81-306 and its mutants by native PAGE and western blot. ........................................................... 74 vi 2.4.5 Analysis of Gpi81-306 protein secretion to media in yeast. ...................... 74 2.4.6 Removal of glycans using EndoH. ........................................................ 75 2.4.7 Evaluation of dimers using size exclusion chromatography (SEC) ....... 75 2.4.8 Use of ESI/IMS/MS to evaluate dimer formation by Gpi81-306. .............. 76 2.4.9 In-gel trypsin digestion fro protein confirmation. .................................... 76 Chapter 3 Co-expression and characterization of the soluble domains of the core subunits of GPI-T ..............................................................................