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Structure-Function Studies of the Vaccinia Virus Complement Control Protein University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 8-2002 Structure-function studies of the vaccinia virus complement control protein. Scott Alan Smith University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Recommended Citation Smith, Scott Alan, "Structure-function studies of the vaccinia virus complement control protein." (2002). Electronic Theses and Dissertations. Paper 1347. https://doi.org/10.18297/etd/1347 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. STRUCTURE-FUNCTION STUDIES OF THE VACCINIA VIRUS COMPLEMENT CONTROL PROTEIN By Scott Alan Smith A.S. Community College of Allegheny County, 1995 B.S. California University of Pennsylvania, 1997 A Dissertation Submitted to the Faculty of the Graduate School of the University of Louisville in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Department of Microbiology and Immunology University of Louisville Louisville, Kentucky August 2002 STUCTURE-FUNCTION STUDIES OF THE VACCINIA VIRUS COMPLEMENT CONTROL PROTEIN By Scott Alan Smith A.S. Community College of Allegheny County, 1995 B.S. California University of Pennsylvania, 1997 A Dissertation Approved on March 27, 2002 by the following Dissertation Committee: ______________________________________ Dissertation Director ______________________________________ ______________________________________ ______________________________________ ______________________________________ ii DEDICATION I dedicate the work presented in this dissertation to my mother, Toni Smith, who not only gave me quality genes, but also the nurturing and knowledge to exploit them. She has always encouraged me to explore my curiosity, and has taught me to be determined and persistent with my dreams. iii ACKNOWLEDGEMENTS I would first like to thank my advisor, Dr. Girish Kotwal, for all his time, guidance, and endless diligence. I am extremely grateful for his kindness and all the effort he made in ensuring that my goals were achieved. I will never forget the many long nights we spent preparing manuscripts for submission, or the long discussions we have had. I am also indebted to my dissertation committee, Drs. David Justus, Larry Hunt, Jaydev Dholakia, and Robert Stout for their advise, guidance, and thorough review of my dissertation. In addition, I would like to express thanks to my fellow students and lab mates: Jaime Anderson, David Reynolds, Barry Billings, Purushottam Jha, Ming Yan Yang, Zhouning Zhang, and Kristen Keeling, for all their assistance and comradeship over the last four years. Many collaborating labs also need to be acknowledged. I would first like to thank Dr. Paul Barlow and his group from the University of Edinburgh, Scotland, including: Nick Mullins, Krystyna Bromek, Alan Cooper, and John Parkinson for their work with the structure and stability of VCP. Next, I would like to thank Dr. Krishna Murthy and his group from the University of Alabama at Birmingham, including: Gunasekaran Krishnasamy, and Ken Judge, for their work on the crystal structure of VCP and preparation C3/C4. In addition, thanks must be given to my friends and collaborators in South Africa, Drs. Shrihari Arjunwadkar, and David Pugh for their help with the Biacore experiments. iv Finally, and most importantly, I would like to thank the love of my life, Helen, for enduring many long years of only brief encounters and innumerable phone calls and letters. Thank you for your support, encouragement, and never-ending faith in my ability. You are, and will always be my driving and inspirational force. I would also thank my family and friends for their patience while I am so distant. v ABSTRACT Structure-Function Studies of the Vaccinia Virus Complement Control Protein Scott Alan Smith March 27, 2002 The vaccinia virus complement control protein (VCP) is involved in the modulation of the host inflammatory response during vaccinia virus infection. It possesses the ability to inhibit both classical and alternative pathways of complement activation, as well as bind to heparin or heparan sulfate proteoglycans, making it a unique multifunctional protein with therapeutic potential. VCP is able to block complement activity through its ability to bind C3b and C4b. Other very novel activities arise from VCP's ability to bind heparin and heparan sulfate proteoglycans, allowing the protein to attach itself to cell surfaces. The goal of this research was to fully characterize the structure and various functions of VCP. The structural basis for VCP's ability to bind heparin was investigated using heparin affinity chromatography, surface plasmon resonance analysis, and homology modeling with well-characterized heparin binding proteins. VCP was found to possess two regions involved in heparin binding, one involved in weak binding located at the junction of complement control protein (CCP) modules 1 and 2, and a second involved in strong binding located at the extreme C-terminal tip of the protein. Additional functions, involved in blocking molecular interactions with cells, were also identified and determined to be a result of VCP's ability to bind heparin. vi The structural basis for VCP's ability to bind complement was investigated using the hemolysis assay, surface plasmon resonance analysis, and homology modeling with well-mapped complement regulatory proteins. VCP was found to be the smallest functional unit able to bind and cause factor I cleavage of C3b/C4b, thus inhibiting complement activity. VCP was also shown to possess the ability to simultaneously bind and inhibit complement activation, using its entire exposed surface, while remaining attached to heparin by its C-terminal heparin binding site. The structure of VCP and its structural and functional stability were determined using X-ray crystallography, NMR, DSC, and the hemolysis assay. VCP was found to be an elongated filamentous protein, with defined CCP modular regions with limited intermodular interfacing, existing as a monomer in solution. VCP was found to possess the ability to structurally withstand temperatures in excess of 90oC. In addition, VCP was able to retain functional integrity following exposure to many adverse physical conditions, which would result in irreversible denaturation of most other proteins. Lastly, the in vivo pharmacokinetics of VCP was investigated in rats. VCP was injected as a bolus at different routes and using various concentrations. Continuous and intermittent intravenous injection strategies were also investigated. In all cases, the serum CH50 was monitored over time to determine the half-life of VCP activity in the serum. Traces of intact VCP was detected in urine by SDS-PAGE analysis, giving clues to its excretion and elimination from the body. This research has provided greater understanding of the architectural features of a protein that contribute to heparin and complement binding. In addition, this research may prove important for the future use of VCP as a novel immunomodulating agent, as well as vii future protein-engineering efforts to design a better treatment for many complement mediated diseases, such as: Alzheimer's disease, central nervous system (CNS) injury, systemic lupus erythematosus, and xenograft transplant rejections. viii TABLE OF CONTENTS PAGE DEDICATION……………………………………………………………………… iii ACKNOWLEDGEMENTS…………………………………………………………. iv ABSTRACT………………………………………………………………………… vi LIST OF FIGURES………………………………………………………………….. xiii LIST OF ABBREVIATIONS……………………………………………………… xvii CHAPTER 1. POXVIRUSES: VACCINIA VIRUS Introduction………………………………..………………..…………1 Virus Structure……………………………………………..…………12 Genome Organization……………………….……………..………… 16 Virus Life Cycle……….…………………………………..………... 20 Poxvirus Immunomodulation……………………………..…………. 23 Complement Regulatory Proteins…………………..………... 27 Cytokine Homologues…………………………..……..………27 Cytokine Receptor Homologues (Viroceptors)………...……..28 Chemokine Homologues……………………….…..………… 31 Chemokine Inhibitors……………………………..…………. 31 Serine Protease Inhibitors……………………………...……...32 Vaccinia Virus Complement Control Protein (VCP)………….………35 2. EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF VCP VCP Produced from Natural VV Infection of Mammalian Cell Culture…………………………………………..………………..37 Pichia pastoris Yeast Expression System…………..…….…………. 39 Growth and Affinity Purification…………………….…..…………... 44 BMGY –versus- BMMY………….………………...………. 44 Affinity Purification and Endotoxin Removal…………..…… 45 Assays and Characterization……………………………..…..………..54 Hemolysis Assay and Activity Units………………...……….. 54 Protein Estimation Assay……………………….…..…………57 ix VCP Production for NMR, X-Ray Crystallography, and Antibodies……..………………………………………………..………58 Labeling for NMR………………………………..……………58 Large Scale Purification for X-Ray Crystallography………..…61 Anti-VCP Production, Purification, and Characterization…………………………..……………………61 3. HEPARIN BINDING ACTIVITY OF VCP Introduction to Heparin Binding…………………………………..……67 Materials and Methods………………………………..………..………73 Flow Microfluorimetric Analysis…………………..…..…….…73 Heparin Binding Ability……………………………….………74
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