A RECOMBINANT SYSTEM TO MODEL PROTEOGLYCAN AGGREGATE INTERACTIONS AND AGGRECAN DEGRADATION by HAZUKI ELEANOR MIWA Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisors: Dr. Thomas A. Gerken and Dr. Thomas M. Hering Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY January 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2005 by Hazuki Eleanor Miwa All rights reserved Dedications I would like to dedicate this thesis to my parents, Johji and Satsuki Bernice Miwa and my daughter, Miko Nina Miwa. Table of Contents Title page Committee Sign-off Sheet Copyright page CWRU Waiver Dedication page Table of Contents vi List of Tables xv List of Figures xvi Acknowledgements xxi List of Abbreviations xxiii Abstract xxix Chapter 1. Introduction and Background 1.1 General introduction 1.1.1 Cartilage and osteoarthritis 1 1.2 Aggregate interactions 1.2.1 Structure and function of proteoglycan aggregates in the cartilage extracellular matrix 4 1.2.2 Functions of the link protein family members 6 1.2.3 Divalent cation binding properties of link protein 8 1.2.4 Structural organization of aggrecan 9 - vi - 1.2.5 Structure and biosynthesis of KS and CS on aggrecan 12 1.2.6 Structural characteristics of link protein and the homologous G1 domain of aggrecan and other lecticans 14 1.2.7 HA binding site of PTR domain 19 1.3 Aggrecan degradation 1.3.1 Aggrecan catabolism by members of ADAMTS family 21 1.3.2 Neo-epitope antibodies 24 1.3.3 Regulation of ADAMTS4 activity 28 1.3.4 Aggrecan structure and substrate specificity of ADAMTS4 31 1.4 Focus of thesis work 36 Part I. Characterization of Recombinant Link Protein and Recombinant Aggrecan Chapter 2. Expression, Purification, and Refolding of a Pair of Recombinant Proteoglycan Tandem Repeat Domains of Link Protein from Escherichia coli Summary 38 2.1 Introduction 39 2.2 Results and Discussion 2.2.1 Cloning of link protein fragments from bovine and human link protein 41 2.2.2 Expression and purification of MBP/full-length and truncated recombinant bovine and human link protein fusion proteins in E. coli 45 2.2.3 Factor Xa digestion of MBP-bovine PTR1+2 fusion protein 49 2.2.4 Factor Xa digestion of MBP-human link proteins 54 2.2.5 Enterokinase digestion of MBP/E-bPTR1+2 56 - vii - 2.2.6 Refolding of monomeric PTR1+2 domains 58 2.2.7 Zinc (II) binding of various link protein constructs 62 2.2.8 Alternative approaches for expressing recombinant PTR1+2 domains 65 2.3. Conclusions 75 2.4. Experimental Procedures 2.4.1 Materials 76 2.4.2 Construction of E. coli link protein expression vectors 77 2.4.3 Expression and purification of MBP/full-length and truncated recombinant bovine and human link protein fusion proteins in E. coli 80 2.4.4. Factor Xa digestion of MBP fusion link proteins 82 2.4.5 SDS-PAGE and Western blot analysis 82 2.4.6 Enterokinase digestion of MBP fusion link protein 83 2.4.7 Sephacryl S-300 chromatography and Refolding of MBP/bPTR1+2 84 2.4.8 Zinc binding analysis of MBP/rhLP fusion proteins expressed in E. coli 85 2.4.9 Enzyme linked immunosorbent assay (ELISA) 86 2.4.10 Construction of bovine PTR1+2 into the pBAD/Thio-TOPO vector 86 2.4.11 Pilot expression of thioredoxin fusion PTR1+2 domains in E. coli 87 2.4.12 Construction of bovine PTR1+2 into the pPICZα vector 88 2.4.13 Pilot expression of PTR1+2 domains in Pichia pastoris 89 2.4.14 Large-scale expression and purification of the PTR1+2 domains in Pichia pastoris 90 2.4.15 N-terminal amino acid sequencing 91 - viii - Acknowledgements 92 Chapter 3. Biochemical and Functional Characterization of Full-length Recombinant Aggrecan and Cartilage Link Protein Expressed in Mammalian Cells Summary 93 3.1 Introduction 94 3.2 Results 3.2.1 Construction of recombinant aggrecan expression vectors 95 3.2.2 Mammalian expression of recombinant aggrecan 97 3.2.3 Hydrodynamic sizes of recombinant aggrecan monomer and attached glycosaminoglycans 106 3.2.4 Expression of recombinant link protein 108 3.2.5 Zinc binding of human recombinant link protein 115 3.2.6 Recombinant proteoglycan ternary aggregate formation 116 3.3 Discussion 121 3.4 Experimental Procedures 3.4.1 Materials 128 3.4.2 Purification of aggrecan and link protein from bovine cartilage and chondrocyte cultures 129 3.4.3 Construction of full-size aggrecan expression vectors 132 3.4.4 Construction of link protein expression vectors 134 3.4.5 Cell culture 135 3.4.6 Expression and purification of recombinant aggrecan in mammalian cells 136 - ix - 3.4.7 Composite agarose polyacrylamide gel analysis and chemiluminescent Western blot analysis of aggrecan 137 3.4.8 3.0% and 3.5 % SDS-PAGE gel analysis of full-sized recombinant aggrecan 138 3.4.9 Aggrecan monomer size determination by Sepharose CL-2B chromatography139 3.4.10 GAG size determination by Sepharose CL-6B chromatography 139 3.4.11 Expression and purification of recombinant bovine link protein 140 3.4.12 PNGase F digestion of recombinant link protein 141 3.4.13 Biotin labeled HA binding of link protein 142 3.4.14 Trypsin digestion of proteoglycan aggregates 142 3.4.15 Zinc (II) binding analysis of human recombinant link protein 143 3.4.16 Analysis of proteoglycan aggregate formation 144 Acknowledgements 145 Part II. Aggrecan Degradation Chapter 4. Characterization of the Substrate Specificity of ADAMTS4 against Aggrecan Core Protein Summary 146 4.1 Introduction 149 4.2 Results 4.2.1 Aggrecan structure and aggrecan catabolites 151 4.2.2 Characterization of ADAMTS4-p68 154 4.2.3 Substrate specificity of p68 and p40 156 - x - 4.2.4 KS and CS affect substrate specificity of ADAMTS4 159 4.2.5 N-linked oligosaccharides inhibit the cleavage within the IGD by ADAMTS4 165 4.2.6 Characterization of FLAG-tagged full-length aggrecan 169 4.2.7 Representative digestion of wild-type FLAG-rbAgg expressed in COS-7 cells with ADAMTS4 173 4.2.8 Anti-NITEGE reactive fragments have intact FLAG epitope and are differentially glycosylated 175 4.2.9 KS is not required for ADAMTS4 cleavage within the IGD of FLAG-rbAgg 177 4.2.10 Substrate specificity of ADAMTS4 on chondroitin sulfate-free FLAG-aggrecan. 179 4.2.11 Construction of mutagenized full-length bovine aggrecan expression vectors. 185 4.2.12 Expression of full-length FLAG-rbAgg mutant aggrecans and their susceptibility to ADAMTS4-p40 191 4.2.13 Substrate specificity of ADAMTS4-p68 on mutant aggrecans lacking potentially glycosylated residues 197 4.2.14 The role of the extended structure N-terminal to the ADAMTS4 cleavage site within the IGD. 200 4.2.15 Representative digestion of wild-type FLAG-rbAgg expressed in COS-7 cells with MMP13 205 4.2.16 MMP13 digestion of mutant aggrecans 206 - xi - 4.2.17 Keratan sulfate synthesis in COS-7, CHO-K1, and RCS cells 210 4.2.18 Co-expression of sulfotransferase and FLAG-rbAgg construct 214 4.2.19 Susceptibility of KS-FLAG-rbAgg to ADAMTS4 216 4.3 Discussion 220 4.3.1 Effects of chondroitin sulfate and keratan sulfate on substrate specificity of ADAMTS4 220 4.3.2 Substrate specificity of ADAMTS4-p68 222 4.3.3 Substrate specificity of ADAMTS4-p40 224 4.3.4 Effects of Keratan sulfate substitution on cleavage within the IGD 225 4.3.5 Effects of non-GAG oligosaccharides on aggrecan cleavage by ADAMTS4 227 4.3.6 S377 is important for substrate recognition by ADAMTS4 228 4.3.7 The potential role of the T352IQTVT357 sequence on ADAMTS4 cleavage of aggrecan 229 4.3.8 Sulfation of CS by KS sulfotransferases? 232 4.3.9 The susceptibility of KS-FLAG-rbAgg to ADAMTS4 234 4.3.10 MMP13 cleavage sites within the aggrecan core protein 236 4.3.11 Conclusions 237 4.4 Experimental Procedures 4.4.1 Materials 239 4.4.2 Site-directed mutagenesis 240 4.4.3 ADAMTS4 digestion of de-glycosylated cartilage-derived steer aggrecan 241 4.4.4 Removal of N-linked oligosaccharides from cartilage-derived aggrecan 241 - xii - 4.4.5 Secondary structure prediction 242 4.4.6 ADAMTS4 digestion of recombinant aggrecan (FLAG-rbAgg) 242 4.4.7 MMP13 digestion of recombinant aggrecan (FLAG-rbAgg) 243 4.4.8 3.0 % SDS-PAGE of full-size FLAG-rbAgg 244 4.4.9 Western blot analysis 244 4.4.10 Semi-quantification of enzymatically cleaved products 245 4.4.11 Immunocytochemistry 245 4.4.12 Expression of keratan sulfate in COS-7, CHO, and RCS cells 246 4.4.13 Co-expression of FLAG-rbAgg with sulfo- and glycosyl- transferases 247 Acknowledgements 248 Chapter 5. Summary and Future Studies 5.1 General summary 249 5.2 Future studies -Proteoglycan aggregate interactions- 5.2.1 Refolding PTR1+2 domains from E. coli 252 5.2.2 Functional characterization of the cartilage link protein-HA interaction 253 5.2.3 Effect of glycosylation on HA binding of the G1 domain of aggrecan and link protein 257 -Aggrecan degradation- 5.2.4 The presence of chondroitin sulfate and keratan sulfate on aggrecan core protein affects the substrate specificity of ADAMTS4 isoforms 259 5.2.5 Characterization of glycosylation in the IGD of recombinant aggrecan expressed - xiii - in COS-7 cells 260 5.2.6 The role of Ser 377 in ADAMTS4 recognition 261 5.2.7.