IDENTIFICATION OF PROTEIN’S COMPLEMENTARY TO THE AUTOANTIGEN PROTEINASE 3 AND THEIR INVOLVEMENT IN THE PATHOGENESIS OF AUTOIMMUNE DISEASE David James Bautz A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics Chapel Hill 2008 Approved by: Ronald J. Falk, MD Gloria A. Preston, PhD Alex Tropsha, PhD Brian Kuhlman, PhD Stephen G. Chaney, PhD ©2008 David James Bautz ALL RIGHTS RESERVED ii ABSTRACT David James Bautz: Identification of Protein’s Complementary to the Autoantigen Proteinase 3 and Their Involvement in the Pathogenesis of Autoimmune Disease (Under the direction of Ronald Falk, M.D., Gloria Preston, Ph.D. and Alex Tropsha, Ph.D.) Previous work by our research group showed that PR3-ANCA patients had an antibody response to a recombinant complementary-PR3 protein encoded by the antisense strand of the PR3 mRNA. To follow up on this work, we sought to determine whether the patients also had a T cell response to this recombinant complementary-PR3 protein and whether a protein reactive with those antibodies could be identified in vivo. + Chapter 2 of the dissertation describes the identification of CD4 TH1 cells that proliferate in response to a complementary-PR3 peptide. This proliferation was seen by both a CFSE assay as well as by interferon-γ production in an ELISPOT assay. Those patients who had a T cell response to complementary-PR3 peptide also had antibodies to the complementary-PR3 protein. We next sought to determine if complementary-PR3 proteins could be identified from patient plasmapheresis material. Chapter 3 of this dissertation describes the identification of two complementary-PR3 proteins, human plasminogen and Protein F, a protein from pseudomonas. These proteins reacted with an antibody raised to a peptide encoded by the antisense RNA of the PR3 gene. As complementary proteins are known to interact, iii plasminogen was shown to be a substrate of PR3, indicative of interaction between the two proteins. Lastly, the anti-complementary PR3 antibodies also bound to normal human leukocytes, cells that are known to bind plasminogen on their surface. Chapter 4 describes the identification of anti-plasminogen autoantibodies in PR3- ANCA positive patients. These antibodies were purified using a complementary-PR3 peptide column, indicating that the anti-cPR3 and anti-plasminogen antibodies are the same. The anti-plasminogen antibodies bound a surface-exposed loop on plasminogen’s catalytic domain. Two in vitro assays confirmed the antibodies affect on plasminogen activity. Serological screening of sera indicated that the anti-plasminogen autoantibodies were more prevalent in those PR3-ANCA patients with a clinical history of venous thrombotic events. By designing an experimental approach that considered protein complementarity, a previously unknown autoantigen and its pathogenic autoantibodies were identified. Consideration of complementary proteins can be used to discover other, and perhaps proximal, autoantigens and autoantibodies in other autoimmune diseases. iv For my beautiful wife Kari, whose unending support and sacrifice made this possible. v ACKNOWLEDGEMENTS The work presented here would not have been possible without the aid and assistance of a host of individuals. I would first and foremost like to thank my wife, Kari, for agreeing to the idea of moving to North Carolina and me taking a 60% pay cut to come back to school. I’m not sure many people would agree to that, but I am very thankful that she did. I’m grateful to my mother for her continuous love, support and encouragement. She’s always been eager to hear about what I’ve been up to in the lab. I would like to thank Dr. Barry Lentz for admitting me to the Biophysics Program, which allowed me to investigate a wide array of laboratories to perform my dissertation work in. I never would have ended up working on this project if Dr. Alex Tropsha had not set up a meeting to introduce me to Drs. Ron Falk and Gloria Preston, and for that I am very grateful. It has been a real pleasure to work with Drs. Falk and Preston due to their outstanding commitment to quality science but also because of the laid back and down to earth nature which they go about it. I can’t imagine too many other graduate students having the opportunities to work with such a diverse and talented group of scientists as are assembled here at the UNC Kidney Center, and I consider myself extremely fortunate to have been given this opportunity. I’d also like to thank the other members of my dissertation committee, Drs. Brian Kuhlman, Stephen Chaney, and Christoph Borchers for asking probing questions and offering insightful advice about the project. I’d like to thank each of the following individuals for helping me out in their own unique ways: vi As the other graduate student in the lab, Dr. Josh Astern could appreciate what life was like as a grad student and it was nice to have someone in the lab going through the same experiences. Josh was also quite adept at providing comic relief to lighten the mood of the lab and there were certainly times when that was needed and appreciated. Not having to worry about ordering supplies allowed me time to work on my various projects, and for that I am indebted to Nirmal Khandoobai since he took care of all supply ordering and restocking, along with so many other laboratory duties. I also thank Nirmal for making my trip to Cancun for the 2007 ANCA meeting as fun as possible. I’d like to thank Julie Hamra for identifying patients coming into the clinic, collecting all of our patient samples, and relaying pertinent patient information. The data generated from this project was made all the more important because of the detailed patient information collated by Dr. Sophia Lionaki. I am very grateful to her for going through stacks and stacks of patient charts, calculating untold numbers of BVAS scores, and preparing tables on patient data This project involved doing untold number of ELISA’s, and I’d like to thank Dr. JJ Yang for all of her help with those. In addition, JJ was adept at essentially every assay done in this lab and was always willing and eager to train and I thank her for taking time to teach me all those various laboratory techniques. I’m indebted to Dr. Peter Hewins for always being willing to lend a hand in any way, shape or form. I consider myself very fortunate to have been able to work with such an intelligent scientist. vii Having enough recombinant protein available for all the assays performed in these experiments was crucial, and I’d like to thank Frances Belmonte and Therese Tulley for their aid and assistance in making sure there was enough protein available at all times. I’d like to thank both Dr. Susan Hogan and Hyunsook Chin for their work on the statistical analysis. Epitope mapping of the anti-plasminogen antibodies was an important piece of data generated during this project, and I’d like to thank Dr. Viorel Mocanu and Dr. Carol Parker for their assistance in epitope mapping of antibody samples using mass spectrometry. I was especially appreciative of their willingness to explain how the epitope mapping worked and to let me sit in on some of those experiments. Assaying antibodies directed against plasminogen was something that our research group had never done before, thus I am indebted to Dr. Alisa Wolberg for providing ideas for assays to test activity of anti-plasminogen autoantibodies and for training me in those asssays. Lastly, I’d like to thank my daughter Kira. She has brought more joy to my life than I thought possible and watching her grow up is the greatest biology experiment I’ll ever be a part of. viii TABLE OF CONTENTS LIST OF TABLES ...............................................................................................................xiii LIST OF FIGURES ............................................................................................................. xiv ABBREVIATIONS............................................................................................................... xv SYMBOLS............................................................................................................................ xix CHAPTER I ............................................................................................................................ 1 AUTOIMMUNE DISEASE ......................................................................................................... 2 SMALL-VESSEL VASCULITIS.................................................................................................. 3 WEGENER’S GRANULOMATOSIS............................................................................................. 4 ANCA ................................................................................................................................... 5 PROTEINASE 3........................................................................................................................ 7 COMPLEMENTARY PROTEINS ................................................................................................. 8 IDIOTYPIC NETWORK THEORY ............................................................................................. 12 IDIOTYPES AND AUTOIMMUNITY ......................................................................................... 13 COMPLEMENTARY PROTEINS AND THE IDIOTYPIC NETWORK .............................................