ROLE OF MYELOPEROXIDASE MEDIATED OXIDATIVE MODIFICATION AND APOLIPOPROTEIN COMPOSITION IN HIGH DENSITY LIPOPROTEIN FUNCTION by ARUNDHATI UNDURTI Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Stanley L. Hazen Department of Microbiology and Molecular Biology Cell Biology Program CASE WESTERN RESERVE UNIVERSITY August, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________Arundhati Undurti candidate for the ______________________degreePhD *. Alan Levine (signed)_______________________________________________ (chair of the committee) Stanley Hazen ________________________________________________ Jonathan Smith ________________________________________________ Menachem Shoham ________________________________________________ Mark Chance ________________________________________________ ________________________________________________ (date) _______________________06-30-2010 *We also certify that written approval has been obtained for any proprietary material contained therein. For Amma and Nana TABLE OF CONTENTS List of Figures 3 List of Tables 7 Abbreviations 8 Acknowledgements 11 Abstract 13 Chapter I: Introduction Pathogenesis of Atherosclerosis Endothelial Dysfunction 17 Fatty Streak Formation 17 Advanced Lesion Formation 18 Thrombotic Complications 18 Role of Lipoproteins in Atherosclerosis Lipoprotein Classification and Metabolism 19 High Density Lipoprotein 20 Reverse Cholesterol Transport 21 Scavenger receptor B1 22 Spherical HDL 23 Non cholesterol efflux activities of HDL 24 Lipoprotein Oxidation in Atherogenesis Myeloperoxidase 25 Mechanism of MPO mediated atherosclerosis development 26 Chapter II: Modification of High Density Lipoprotein by Myeloperoxidase Generates a Pro-inflammatory Particle Abstract 35 Introduction 37 Materials and Methods 40 Results 47 Discussion 58 1 Chapter III: The Apolipoprotein Composition of High Density Lipoprotein Influences Cholesterol Efflux and Non cholesterol Efflux Activities of the Lipoprotein Abstract 75 Introduction 76 Materials and Methods 79 Results 85 Discussion 90 Chapter IV: Generation of Two Fusion Proteins of the Extracellular Domain of Scavenger Receptor B1 to Identify Structure-Function Relationships between High Density Lipoprotein and Scavenger Receptor B1 Introduction 104 Materials and Methods 107 Results 118 Discussion 121 Chapter V: Discussion and Future Directions 153 References 164 2 LIST OF FIGURES Chapter I Figure I-1: The Structure of an artery 28 Figure I-2: Rupture of the fibrous cap and thrombotic complications 29 Figure I-3: Reverse cholesterol transport 30 Figure I-4: Formation of spherical HDL 31 Figure I-5: VCAM-1 expression is controlled by the transcription factor NF-ț% 32 Figure I-6: HDL signaling pathway 33 Chapter II - Figure II-1: Oxidation of HDL by the MPO/H2O2/Cl system has functional consequences for classic atheroprotective activities of HDL 61 Figure II-2: HDL protects HUVEC and BAEC from multiple apoptogenic triggers while MPO-oxidized HDL fails to do so 62 Figure II-3: Exposure of HDL to the MPO oxidant system inhibits the anti- apoptotic activity of the particle as monitored by loss of capacity to both inhibit caspase-3 activity and induce eNOS activity 64 Figure II-4: HDL oxidized by physiologically relevant levels MPO-generated oxidants inhibits the anti-inflammatory activity of the particle in HUVEC and promotes VCAM-1 protein expression in BAEC independent of TNF-Į 65 Figure II-5: MPO-oxidized HDL induces bovine aortic endothelial cell NF-ț% DFWLYDWLRQ,..DFWLYDWLRQDQGSKRVSKRU\ODWLRQRI,ț%Į 67 Figure II-6: MPO-oxidized HDL fails to bind to the physiologic HDL receptor, 3 scavenger receptor B1 (SR-B1) and gains binding to an alternate receptor on endothelial cells 69 Figure II-7: The scavenger receptors CD36 and SR-A1 do not recognize HDL modified by the MPO/H2O2/Cl- system 71 Figure II-8: ApoA1 tyrosine, tryptophan and methionine residues do not appear to be involved in endothelial activation by MPO-oxidized HDL 72 Chapter III Figure III-1: Spherical HDL A1 and spherical HDL A1/A2 are similar in diameter 94 Figure III-2: Spherical HDL containing both apoA1 and apoA2 is less anti- apoptotic than spherical HDL containing only apoA1 95 Figure III-3: Spherical HDL containing both apoA1 and apoA2 is less efficient at inhibiting TNF-ĮLQGXFHGVXUIDFH9&$0-1 protein expression than spherical HDL containing only apoA1 97 Figure III-4: Spherical HDL containing both apoA1 and apoA2 is more pro- inflammatory than spherical HDL containing only apoA1 upon MPO mediated oxidation 98 Figure III-5: MPO-oxidized sHDL A1 and MPO-oxidized sHDL A1/A2 induces bovine aortic endothelial cell NF-ț%DFWLYDWLRQDQG,..DFWLYDWLRQ 99 Figure III-6: Spherical HDL containing apoA1 only or spherical HDL containing both apoA1 and apoA2 are equally efficient at promoting cholesterol efflux from macrophages 101 Figure III-7: ApoA2 transgenic mice show less reverse cholesterol transport compared to C57Bl/6J mice 102 Chapter IV Figure IV-1: Extracellular domain of SR-B1 125 Figure IV-2: Primer sequences for the directional cloning of extracellular domain of SR-B1 126 4 Figure IV-3a: Vector map of p-ENTR/d-TOPO 127 Figure IV-3b: Directional TOPO cloning of extracellular domain of SR-B1 128 Figure IV-4: SR-B1 extracellular domain is successfully cloned into p-ENTR/ d-TOPO vector 129 Figure IV-5a: The mammalian expression vector pSeCTag2C 130 Figure IV-5b: The multiple cloning site of the mammalian vector pSeCTag2C 131 Figure IV-6a: Restriction digest of pSecTag2C with Not I fails to cut the vector 132 Figure IV-6b: Primers for introducing a Not I site into pSecTag2C 133 Figure IV-6c: Restriction digest of pSecTag2C after introduction of a Not I site 133 Figure IV-7: The extracellular domain of SR-B1 is successfully ligated into the pSecTag2C vector 134 Figure IV-8: Western blot of whole cell extract and media of 293T cells transfected with pSecTag2C vector containing SR-B1 extracellular domain 135 Figure IV-9a: Coomassie gel of nickel column purification of SR-B1 fusion protein from the media of 293T cells 137 Figure IV-9b: Western blot analysis of purified SR-B1 fusion protein 138 Figure IV-10: SR-B1 fusion protein can bind to HDL 140 Figure IV-11a: The pMAL-c4X vector map 141 Figure IV-11b: .Forward and reverse primer sequences for introducing a multiple cloning site with an N-terminal Tev cleavage site into the pMAL-c4X vector 141 Figure IV-12: SR-B1 extracellular domain is successfully ligated into pMAL-c4x vector 143 Figure IV-13: SR-B1 fusion protein is produced in E.coli 144 Figure IV-14: Purification scheme for MBP-SR-B1-6x His fusion protein from E.coli 145 Figure IV-15a: Coomassie gel of purification of MBP-SR-B1-6x His fusion 5 protein with amylose beads 146 Figure IV-15b: Western blot analysis of MBP-SR-B1-6x His fusion protein purified with amylose beads 147 Figure IV-16: Coomassie gel of MBP-SR-B1-6x His purified on a nickel column 149 6 LIST OF TABLES Table IV-1: Purification table for SR-B1 fusion protein purified from media of 293T cells on a nickel column 139 Table IV-2: Purification table of MBP-SR-B1-6x His fusion protein purified with amylose beads 148 Table IV-3: Purification table of MBP-SR-B1-6x His fusion protein purified on nickel column after prior purification with amylose beads 150 Table IV-4: Analysis of binding between MBP-SR-B1-6x His fusion protein and HDL (apoA1) 151 7 ABBREVIATIONS ABCA1—ATP binding cassette transporter A1 ABCG1—ATP binding cassette transporter G1 AEBSF—4-(2-Aminoethyl) benzenesulfonyl fluoride ApoA1—apolipoprotein A1 ApoA2—apolipoprotein A2 ApoE—apolipoprotein E ATP—adenosine triphosphate BAEC—bovine aortic endothelial cells 8-Br-cAMP—8-bromo-cyclic adenosine monophosphate Cl-Tyr—chloro-tyrosine CVD—cardiovascular disease DKO—double knock-out DPM—disintegrations per minute DTPA—diethylene triamine pentaacetic acid DTT—dithiothreitol ELISA—enzyme linked immunosorbent assay EMSA—electrophoretic mobility shift assay eNOS—endothelial nitric oxide synthase Fc—fragment, crystallizable FPLC—fast performance liquid chromatography GST—glutathione-S-transferase H-D exchange MS—hydrogen-deuterium exchange mass spectrometry HCl—hydrogen chloride HEK 293—human embryonic kidney cells HDL—high density lipoprotein HPLC—high performance liquid chromatography HRP—horse radish peroxidase HUVEC—human umbilical vein endothelial cells IDL—intermediate density lipoprotein 8 IPTG— LVRSURS\Oȕ-D-1-thiogalactopyranoside KCl—potassium chloride IKK—,ț%NLQDVH KO—knock out LCAT—lecithin cholesterol acyl transferase LDL—low density lipoprotein MAPK—mitogen activated protein kinase MBP—maltose binding protein MBP-SR-B1-6x His—fusion protein of extracellular domain of scavenger receptor B1 with N-terminal MBP tag and C-terminal 6x histidine tag MgCl2—magnesium chloride MPM—mouse peritoneal macrophages MPO—myeloperoxidase - MPO/H2O2/Cl — myeloperoxidase/hydrogen peroxide/chloride oxidation system NaCl—sodium chloride NF-ț%—nuclear factor- ț% NO—nitric oxide oxHDL—oxidized high density lipoprotein oxLDL—oxidized low density lipoprotein PBS—phosphate buffered saline PCR—polymerase chain reaction PMSF-- phenylmethylsulfonyl fluoride POPC—1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine RCT—reverse cholesterol transport rHDL—reconstituted high density lipoprotein sHDL A1—spherical HDL containing only apoA1 sHDL A1/A2—spherical HDL containing both apoA1 and apoA2 SANS—small angle neutron scattering