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Structural and Functional Analysis of Heparan Sulfate Sulfotransferases

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Structural and Functional Analysis of Heparan Sulfate Sulfotransferases STRUCTURAL AND FUNCTIONAL ANALYSIS OF HEPARAN SULFATE SULFOTRANSFERASES Heather Nicole Bethea 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 Pharmaceutical Sciences (Medicinal Chemistry and Natural Products) Chapel Hill 2010 Approved by: Michael Jarstfer, Ph.D. Jian Liu, Ph.D. Lars Pedersen, Ph.D. Scott Singleton, Ph.D. Qisheng Zhang, Ph.D. ABSTRACT HEATHER BETHEA: Structural and Functional Analysis of Heparan Sulfate Sulfotransferases (Under the direction of Jian Liu, Ph.D.) Heparan sulfate (HS), a major polysaccharide component of the vascular system, is involved in regulating a number of functions of the blood vessel wall including blood coagulation, cell differentiation, and the inflammatory response. The wide range of biological functions makes HS an attractive therapeutic target. The long term goal of our research involves utilizing an enzyme-based approach to develop HS-based therapeutics for treating thrombotic diseases, cancer and excessive inflammatory responses. The biosynthesis of HS involves multiple specialized sulfotransferases such as 2-O-sulfotransferase (2OST) and 6-O-sulfotransferase (6OST), which are essential for preparing HS with activities in regulating vascular development and blood coagulation. The substrate specificity of the HS sulfotransferases controls the sulfation patterns of HS, permitting HS to exhibit a specific function, however, limited knowledge regarding the mechanism of these enzymes has hindered our ability to prepare functionally-specific HS. We aim to understand the mechanism of action of these two enzymes in hopes of developing heparin/HS with improved anticoagulant efficacy. In this dissertation, we present successful crystallization of 2OST in complex with 3’- phosphoadenosine 5’-phosphate (PAP). The substrate recognition mechanism of 2OST was ii examined by way of extensive structurally guided mutational analysis. Several residues were identified, including Arg-189, Tyr-94, and His-106, that are responsible for dictating the substrate specificity of 2OST. Despite success with the crystallization of 2OST, the crystallization of 6OST has been an ongoing process. A promising expression construct for crystallization purposes has been identified for 6OST-1. Using a homology model of 6OST- 3 with structurally known 3OST-3, several residues involved in PAPS and substrate binding were proposed. iii To my mother, Faye S. Barfield, whose everlasting love and support helped guide me throughout my educational endeavors. To my fiancé, Joshua R. Horne, whose continuous love, support, and optimism made completion of this dissertation possible. iv ACKNOWLEDGEMENTS First and foremost, I would like to offer my sincerest gratitude to my advisor, Dr. Jian Liu, for his guidance throughout my graduate career. His encouragement, optimism, and support helped offer me the strength and perseverance to strive through completion of these studies over the past five years. Dr. Liu is an upstanding mentor who takes pride in the success of his students and I will always be grateful to him for helping me become a better scientist. Next, I would like to acknowledge Dr. Lars Pedersen for assistance with the structural analysis of 2OST. I would also like to thank Dr. Pedersen for serving as a member of my dissertation committee, providing me with continuous counseling throughout completion of the crystallization studies for 2OST, 6OST, and TPST. I must thank Andrea Moon in Dr. Lars Pedersen’s laboratory for teaching me how to manipulate the structural analysis software, PyMol, and Dr. Raj Gosavi for providing me with a library of TPST enzymes. To Dr. Jerry Turnbull, I would like to thank him for providing me with an amazing opportunity to study abroad at the University of Liverpool. I send my warmest appreciation to my other dissertation committee members, Dr. Mike Jarstfer, Dr. Scott Singleton, and Dr. Qisheng Zhang, for providing me with honest and thought-provoking suggestions regarding my research study. Finally, I am so grateful for the former and current labmates of the Liu laboratory. I express thanks to Dr. Ding Xu for teaching me the expression, purification and mutational v analysis of the heparan sulfate sulfotransferases. I would also like to thank former labmates, Dr. Ronald Copeland, Dr. Miao Chen, Dr. Tanya Burch, and Lan Yu, as well as current labmates Dr. Renpeng Liu, Liz Pempe, Dr. Yongmei Xu, Dr. Kai Li, Justin Roberts, Ryan Bullis, Dr. Xianxuan Zhou, Truong Pham, and Dr. Juzheng Sheng for their assistance and expertise during my time in the lab. I would like to send my sincerest gratitude to Courtney Jones, Dr. Renpeng Liu, Liz Pempe, and Sherket Peterson for proofreading and providing me with helpful suggestions to improve my dissertation. Special thanks to the “Liu ladies”, Sherket Peterson and Courtney Jones, who continuously offered their advice and support both in and outside the laboratory. Without the help and support of everyone involved, completion of this dissertation would not have been possible. vi TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... XIV LIST OF FIGURES ......................................................................................................... XV LIST OF ABBREVIATIONS ...................................................................................... XVIII INTRODUCTION............................................................................................................. 1 HEPARAN SULFATE PROTEOGLYCANS .............................................................................. 1 Chemical Structure of Heparan Sulfate and Heparin ................................................. 2 BIOSYNTHESIS OF HEPARIN AND HEPARAN SULFATE ....................................................... 5 Chain Initiation............................................................................................................ 5 Chain Polymerization .................................................................................................. 9 Chain Modification .................................................................................................... 12 CURRENT METHODOLOGIES FOR STRUCTURAL ANALYSIS OF HEPARAN SULFATE ........ 36 Chemical Degradation Using Nitrous Acid............................................................... 37 Enzymatic Degradation Using Heparin Lyases ........................................................ 39 PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL FUNCTIONS OF HEPARAN SULFATE ......... 40 Anticoagulation ......................................................................................................... 41 Cell Proliferation ....................................................................................................... 44 Inflammation .............................................................................................................. 46 Viral Infection ............................................................................................................ 48 ix Tumor Progression .................................................................................................... 52 STATEMENT OF PROBLEM ...................................................................................... 55 MATERIALS AND METHODS ................................................................................... 65 PREPARATION OF COMPETENT CELLS ............................................................................ 65 CHEMICAL TRANSFORMATION OF COMPETENT CELLS ................................................... 57 EXPRESSION OF HEPARAN SULFATE SULFOTRANSFERASES IN ORIGAMI B/ORIGAMI BCHAP CELL LINES .......................................................................... 57 PROTEIN PURIFICATION COUPLED TO THE FPLC SYSTEM .............................................. 58 Nickel Sepharose Fast Flow Affinity Chromatography for His6-Tagged Proteins ... 58 Amylose Affinity Chromatography for MBP Fusion Proteins ................................... 58 Glutathione Sepharose 4 Fast Flow Affinity Chromatography for GST fusion proteins ...................................................................................................................... 59 Hi-Load Superdex 75/200 Gel Filtration Chromatography ...................................... 59 PROTEIN PROPERTY ANALYSIS USING GEL FILTRATION CHROMATOGRAPHY USING HPLC ............................................................................................................................. 60 SITE-DIRECTED MUTATIONAL ANALYSIS ...................................................................... 61 Design of Primers and Polymerase Chain Reaction ................................................. 61 Small Scale Expression and Purification of Mutant Enzymes ................................... 62 Sulfotransferase Activity Analysis of Mutant Enzymes.............................................. 63 LARGE SCALE PREPARATION AND PURIFICATION OF K5 POLYSACCHARIDE .................. 64 MODIFICATION OF HEPARAN SULFATE POLYSACCHARIDES USING PAPS REGENERATION SYSTEM ................................................................................................ 66 DISACCHARIDE ANALYSIS OF HEPARAN SULFATE POLYSACCHARIDES .......................... 66 Enzymatic Degradation Using Heparin Lyases ........................................................ 66 x Chemical Degradation
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