Investigations of the scavenger receptor class a and complement receptor 3 two pattern recognition receptors

INVESTIGATIONS OF THE SCAVENGER RECEPTOR CLASS A AND COMPLEMENT RECEPTOR – TWO PATTERN RECOGNITION RECEPTORS BY GOH WEE KANG JASON B. Sc. (Hons), Murdoch University, Australia Msc (Med Genetics), University of Aberdeen, Scotland A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements I would like to especially thank my supervisor Associate Professor Lu Jinhua for giving me the opportunity to pursue a PhD in his laboratory and for the training in the course of study. I also thank Dr Alister Dodds (University of Oxford, UK) for his help in preparing the complement C3 fragments for the solid phase binding assay. Many thanks to Dr Alex Law (University of Oxford, UK) for the complement receptor vectors and antibodies, Dr Low Boon Chuan (Dept of Biological Sciences, NUS) for the RhoGTPase vectors, and Dr Gan Yunn Hwen (Dept of Biochemistry, NUS) for the GFP bacteria. Grateful thanks to Dr Chua Kaw Yan (NUS, Singapore) and her lab members for the generous loan of the flow cytometry machine. I would like to acknowledge past and present members of this laboratory for their help and companionship during my stay in the laboratory. In particular, I would like to thank Linda Wang for her friendship and support over the past six years. My appreciation also extends to staff of the DNA Sequencing Lab, NUMI for their help. I am grateful to the National University of Singapore for awarding me a research scholarship during the duration of my study. Last, but not least, I dedicated this dissertation in memory of my late father, Jack Goh, and to my mother, Ho Yeok Kuen. I love them very much and always will. My appreciation extends to my brother Jonathan and his family for their love and support. i Table of Contents Page Acknowledgements . i Table of contents ii Summary vii List of figures x List of Tables xiii Publications . xiv Abbreviations xv Chapter Introduction Page 1.1 1.2 1.3 1.4 1.4.1 1.4.1.1 1.4.1.2 1.5 1.6 1.6.1 1.6.1.1 1.6.2 1.6.2.1 1.6.2.2 1.6.2.3 1.6.2.3.1 1.6.2.3.2. 1.6.3 1.7 1.7.1 1.7.1.1 1.7.1.1.1 1.7.1.1.2 1.7.1.1.3 1.7.1.1.4 1.7.1.1.5 1.7.1.1.6 Innate Immunity Pathogen-associated molecular patterns (PAMP) . Pattern recognition receptors (PRR) . Sensing/signaling PRRs Toll-like receptors (TLR) TLR ligands and leucine-rich repeat (LRR) domain 10 Toll/Interleukin-1 receptor (TIR) domain and TLR-mediated signaling pathways 11 Endocytic/phagocytic PRRs . 13 Complement receptors and complements . 15 Complement system 15 Complement component C3 . 20 Complement receptor (CR3, CD11b/CD18, Mac-1,M2) 24 Ligand promiscuity of CR3 26 Inserted (I) domain in ligand recognition . 28 Integrin bi-directional signaling 31 Inside-out signaling pathways of integrins . 31 Outside-in signaling pathways of integrins 33 Complement receptor (CR4, CD11c/CD18, X2) 35 Scavenger receptors 37 Scavenger receptor class A (SR-A) 41 SR-A structure 42 Cytoplasmic domain of SR-A . 45 Transmembrane domain of SR-A . 46 Spacer domain of SR-A 46 -helical coiled-coil domain of SR-A 46 Collageneous domain of SR-A . 49 Cysteine-rich domain (SRCR) of SR-A 51 ii 1.7.1.2 1.7.1.3 1.7.1.3.1 1.7.1.3.2 1.7.1.3.3 1.7.1.3.4 1.7.1.3.5 1.8 Ligand binding properties of SR-A 53 Physiological roles of SR-A . 57 Modified lipoprotein endocytosis and atherosclerosis 57 Cell-cell and cell-extracellular matrix adhesion . 59 Antimicrobial host defence . 61 Apoptotic cell clearance 63 Bone remodeling or osteogenesis regulation . 65 Aims of study 66 Chapter Materials and Methods 2.1 2.2 2.2.1 2.2.1.1 2.2.1.2 2.2.1.3 2.2.2 2.2.2.1 2.2.2.2 2.2.2.3 2.2.2.4 2.2.2.5 2.2.2.6 2.2.2.7 2.2.2.8 2.2.2.9 2.2.2.10 2.2.2.11 2.2.2.12 2.2.2.13 2.2.2.14 2.2.2.15 2.2.3 2.2.4 2.2.4.1 2.2.4.2 2.2.4.3 2.2.4.4 2.2.4.5 2.2.4.6 2.2.4.7 2.2.4.8 2.3 2.3.1 2.3.2 2.3.2.1 2.3.2.2 2.3.3 Buffers and media . 68 Molecular biology . 68 RNA manipulation 68 Isolation of total RNA . 68 Quantitation of RNA . 68 Reverse transcription 69 Gene/plasmid DNA cloning 69 DNA primer synthesis 69 Polymerase chain reaction (PCR) . 70 Ethanol precipitation of DNA . 71 Restriction endonuclease digestion . 71 DNA agarose gel electrophoresis . 70 Isolation of DNA from agarose gels . 72 Quantitation of DNA 72 DNA ligation . 73 Preparation of competent cells 73 Transformation of competent cells . 74 Methods for the identification of positive clones . 74 Rapid isolation of plasmid DNA 75 Plasmid purification for transfection 75 Site-directed mutagenesis . 76 DNA sequencing . 77 Commerical expression vectors 78 Construction of expression vectors . 79 Expression vectors of wild-type/native receptors . 79 SR-AI collageneous domain mutant receptors . 80 SR-AI cysteine-rich (SRCR) domain mutant receptors 81 SR-AI cytoplasmic domain mutant receptors . 83 Construction of soluble SR-AI (psSR-AI-MH) expression vector . 84 Construction of soluble SRCR domain (SRCR) expression vector 85 Expression vectors of CR3 mutant receptors 86 Dominant negative expression vectors . 86 Cell biology . 87 Human embryonic kidney (HEK) cell-line culture . 87 Monocyte-derived dendritic cells (DCs) in vitro culture 88 Isolation of human peripheral blood monocytes . 88 Generation of DCs from blood monocytes . 89 Microbial and molecular stimuli used in the present studies 89 iii 2.3.3.1 2.3.3.2 2.3.4 2.3.5 2.3.6 2.3.7 2.3.8 2.3.9 2.3.10 2.3.11 2.4 2.4.1 2.4.2 2.4.3 2.4.3.1 2.4.3.2 2.4.4 2.4.5 2.4.6 2.4.7 2.4.8 2.4.9 2.4.10 2.5 Bacterial strains used as stimuli 89 Molecules/PAMPs 90 Pharmaceutical inhibitors . 91 Transient liposome-based cell transfection . 92 Dual luciferase assay 92 Treatment of transfected HEK 293T cells with various bacterial and molecular stimuli 94 Dendritic cell stimulation with various bacterial and molecular stimuli . 95 Enzyme-linked Immunosorbent Assay (ELISA) 96 Green fluorescent protein-E. coli DH5 binding assays 97 Confocal microscopy 98 Protein chemistry 99 Antibodies used in this study 99 Protein concentration determination . 100 Expression of recombinant proteins sSR-AI and sSRCR . 100 Calcium phosphate transfection 100 Purification of recombinant proteins 101 Preparation of C3 and its degradation fragments 101 Flow cytometry . 102 Cell surface biotinylation 102 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) . 103 Western blotting 104 Coomassie blue staining . 104 Solid Phase Protein Binding Assay 104 Statistical analysis . 105 Chapter Characterization of SR-AI as a receptor for the complement opsonin iC3b 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 Overview . 106 TLR4 but not SR-A is able to activate NF-B in response to LPS 108 E. coli DH5 induces SR-AI mediated NF-B activation . 110 LPS does not activate SR-AI-mediated NF-B signaling at high concentrations . 111 Neither smooth nor rough LPS activate SR-AI-mediated NF-B signaling 113 The SR-A ligands S. aureus and LTA does not induce SR-AImediated NF-B 114 The potential SR-A ligand B. subtilis induce SR-A mediated NF-B activation . 115 The potential SR-A ligand M. bovis fails to induce SR-AI mediated NF-B activation. . 115 DH5 induces IL-8 and MCP-1 production through SR-AI stimulation 116 The SR-A ligand fucoidan does not activate NF-B activation via SR-AI but inhibits that induced by DH5. . 117 DH5 only activates SR-AI signaling with fresh BCS 119 iv 3.12 3.13 3.14 3.14.1 3.14.2 3.14.3 3.15 3.15.1 3.15.2 3.16 3.17 3.18 3.19 DH5 activation of SR-AI requires serum opsonin activity. . 119 DH5 induces CR-3-mediated NF-B activation in presence of fresh BCS . 121 DH5 stimulation of SR-AI requires bacteria opsonization with human complement C3. 123 DH5 induces SR-AI-mediated NF-B activation in the presence of human serum . 123 SR-AI is opsonized with C3 complement in fresh human serum . 125 SR-AI is activated by DH5bound human complement C3 127 Involvement of the collageneous domain of SR-AI in the signaling response to opsonized DH5. . 129 SR-AI response to opsonized DH5 does not involve the postulated ligand binding region of receptor – the lysine cluster at the Cterminal end of the collageneous domain . 129 Other basic residues in the proximal region of the collageneous domain are also not required for SR-AI signaling induced by opsonized DH5. 131 The SRCR domain of SR-AI is required for its recognition of opsonized DH5 . 133 Purified SR-AI and SRCR bind serum-opsonized E. coli DH5 . 138 Purified SR-AI binds iC3b but not C3 or C3b 139 Conclusion 142 Chapter Characterization of the cytoplasmic domain of SR-AI in DH5a-induced intracellular signaling of receptor 4.1 4.2 4.3 4.4 4.5 4.6 4.7 Overview . 143 The cytoplasmic tail of SR-A is not involved in DH5 induced NFB activation . 146 DH5 induced, SR-AI mediated NF-B activation is not dependent of the adaptor molecule MyD88 . 149 SR-A mediated NF-B activation following DH5 stimulation is dependent on phosphatidylinositol-3-kinase (PI3K) 150 SR-A mediated NF-B activation in response to DH5 involves the Rho GTPase Cdc42 . 152 SR-A mediated NF-B activation following DH5 stimulation is reduced by the membrane cholesterol sequestering compound methylcyclodextrin (MCDextrin) but not affected by the actin polymerization inhibitor cytochalasin D . 154 Conclusion 156 v Chapter Opsonization of bacteria with complement C3 induces Racmediated NF-B activation and inhibits dendritic cell production of interleukin-12 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 Overview . 157 Serum-opsonized E. coli DH5stimulates CR3 signaling of NF-B activation . 159 Serum-opsonized DH5 does not elicit NF-B activation through CR4. 161 The cytoplasmic tails of both subunits of CR3 are not involved in DH5 induced NF-B activation 163 CR3 is activated by opsonic C3 deposited on DH5 . 164 Rac is required for DH5-elicited CR3 signaling of NF-B activation . 168 Cytochalasin D inhibits CR3 signaling of NF-B activation and IL-8 production in CR3-239T cells . 170 Opsonic C3 is a negative regulator of DH5-induced IL-12 production. 172 IFN- abrogates C3-mediated inhibition of IL-12 production 175 Inhibition of Rac enhances DH5 induction of IL-12. . 176 Cytochalasin D inhibits rather than enhances DH5-induced IL-12 production from DCs. . 177 DTxB enhances IL-12 production by DH5C3+ 178 Rac inhibition does not enhance IL-12 induction without opsonic C3 Co-stimulation. . 179 DCs ingestion of DH5 is not significantly affected by Rac inhibition and C3 deficiency . 180 Conclusion. . 182 Chapter Discussion 6.1 6.2 6.3 6.4 6.5 6.5.1 6.5.2 6.6 6.7 SR-AI is a novel complement C3-binding receptor 183 The role of SR-AI as an opsonic PRR in the recognition of complement-opsonized Gram-positive and Gram-negative bacteria species . 186 The involvement of the SRCR domain of SR-AI in the recognition of C3-opsonized bacteria . 188 Elucidation of SR-A-mediated signaling pathways 191 The functional role the cytoplasmic domain of SR-A and CR3 in receptor signaling – Possible involvement of a signaling coreceptor(s) . 195 Recognized co-receptors of SR-A 196 Recognized co-receptors of CR3 198 The suppression of IL-12 by opsonic C3 receptors 200 Summary and future studies . 205 References . 207 Appendix 254 vi Summary One of the fundamental aspects of innate immunity is the ability of immune cells, particularly antigen-presenting cells (APCs) such as macrophages and dendritic cells(DCs), to detect and respond to potential microbial pathogens or even endogeneous molecules that have become altered and pose a threat to the system. These cells employ an array of pattern recognition receptors (PRRs) that can recognize these pathogens or altered endogeneous molecules either directly by binding to pathogen associated molecular patterns (PAMPs) found on the microbial cell surfaces or indirectly via opsonins such as complement components and immunoglobulins which have deposited on the surfaces of cellular or molecular targets. In this study, we focused on two major PRRs found on APCs, the opsonic complement receptor (CR3) and non-opsonic scavenger receptor class A (SR-A). We will attempt to investigate certain aspects of their ligand binding properties and delineate downstream signaling pathways upon ligand engagement. SR-A is a non-opsonic PRR important for the clearance of infectious and endogenous molecular and cellular debris. Although ligand binding properties of SR-A have been extensively studied in relation to modified low density lipoproteins (LDL), the mechanisms governing its broad ligand specificity and interaction with other ligands, particularly PAMPs, is not completely elucidated. In addition, its signaling properties remain poorly understood. In this study, we express SR-A isoform (SR-AI) on human embryonic kidney (HEK) 293T cells, which lack most PRRs including SR-A, and report that E. coli DH5stimulation of these transfected cells mediated NF-B activation and chemokine production i.e. interleukin (IL-8) and monocyte chemoattractant protein (MCP-1). Opsonization with complement C3 is required for vii E. coli DH5to stimulate SR-AI signaling as it was abolished by heat inactivation of sera, C3 depletion of the sera or anti-C3 antibodies. Selected point mutations in the scavenger receptor cysteine rich (SRCR) but not the collageneous domain abolish SRAI signaling response to the opsonized E. coli DH5. Purified SR-AI binds to iC3b but not to C3 or C3b which suggests SR-AI as a complement receptor for opsonic iC3b. In contrast to DH5, SR-AI cannot mediate NF-B activation in response to the SR-A ligands LPS and fucoidan. We have also established that the cytoplasmic tail of SR-AI is not required for DH5-induced NF-B activation, suggesting the possibility of one or more co-receptors involved in SR-AI signaling. The identity of the co-receptor is presently unknown but does not include Toll-like receptors (TLRs). The co-receptor appears to co-localize with SR-AI on lipid rafts of HEK293T cells and its signaling involves phosphatidylinositol-3-kinase (PI3K) and RhoGTPase cdc42. Complement C3 opsonizes microorganisms for enhanced phagocytosis via mainly CR3 and the related complement receptor (CR4). In addition, cross-linking of CR3 inhibits IL-12 production although the signaling mechanism(s) concerned is not known. In this study, we investigate CR3 and CR4 signaling after expression of these C3 receptors on HEK 293T cells. DH5 opsonized with normal serum (DH5C3+) activated CR3, but not CR4, culminating in NF-B activation and IL-8 production. CR3 activation was not elicited when DH5 was opsonized with C3-deficient sera (DH5C3-) or pre-incubated in normal serum with anti-C3 antibodies. CR3-mediated NF-B activation in response to DH5C3+ was inhibited by dominant negative Rac (N17Rac). DH5C3+ and DH5C3- were both ingested by dendritic cells (DCs), but DH5C3- induced 1.8 folds more IL-12 from DCs than DH5C3+. The Rac inhibitor NSC23766 and the PI3K inhibitor LY294002 both enhanced DH5C3+, but not viii DH5C3-, induction of IL-12 from DCs suggesting inhibition of IL-12 production by opsonic C3 involves Rac and PI3K. 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The factor H protein family. Immunopharmacology 42, 53-60. 253 APPENDIX: MEDIA and SOLUTIONS Molecular biology LB-broth (Luria-Bertani medium) Bacto-tryptone 1.0% Yeast extract 0.5% NaCl 0.5% Adjust pH to 7.5 with M NaOH prior to autoclaving. Media was sterilized by autoclaving at 15 LB/in2 for 15 min. Thermolabile antibiotics were filter-sterilized through 0.2 m filters and added to the autoclaved LBbroth. Final concentrations for ampicillin was 100 g/ml. LB agar plates LB-broth was prepared as above with 15 g/L of Bacto-agar added. Media was sterilized by autoclaving. 50 x TAE buffer (for DNA gel electrophoresis) Tris base Acetic acid EDTA pH 7.8 2M 1M 0.1 M 10 x DNA loading buffer Ficoll400 EDTA(pH 8.0) 0.1 M Bromophenol blue 20% (w/v) 0.25% (w/v) Ethidium bromide The stock solution was made at 10 mg/ml and stored in a light-tight bottle. Final working concentration was 0.5 μg/ml. 10X TNE buffer Tris base Sodium chloride EDTA pH 7.4 100mM 2M 10mM Cell Biology x Phosphate buffered saline (PBS) KH2PO4 Na2HPO4 NaCl KCl 1.76 mM 10.4 mM 137 mM 2.7 mM FACS Wash buffer x PBS BCS Sodium azide 2% (v/v) 0.05% (w/v) 254 Protein Chemistry Cell lysis buffer Tris-HCl, pH 7.5-8.0 NaCl EDTA EGTA KCl Triton-X 100 Leupeptin Protease Aprotinin inhibitors PMSF 50 mM 300 mM 2mM 2mM 20mM 1% (v/v) 100 g/ml 100 g/ml mM PMSF = phenylmethylsulphonyl fluoride 2x HBS buffer NaCl KCl Na2HPO4.2H2O KCl D-glucose (anhydrous) HEPES 280 mM 10 mM 1.5 mM 2.7 mM 12mM 50mM Wash buffer Tris-HCl, pH 8.0 20 mM NaCl 500 mM Imidazole 10 mM Elution Buffer Tris-HCl, pH 8.0 20 mM NaCl 500 mM Imidazole 250 mM SDS-PAGE gel electrophoresis and Western Blotting Stacking gel preparation (for mini SDS-PAGE gel) dH2O 0.5 M Tris-HCL, pH 6.8 10% (w/v) SDS 30% Acrylamide/Bisacrylamide solution 29:1 (3.3%C) 10% APS TEMED 3.05 ml 1.25 ml 50 l 0.65 ml 25 l l Separating gel preparation (for mini SDS-PAGE gel)-12.5% gel dH2O 1.5 M Tris-HCL, pH 8.8 10% (w/v) SDS 30% Acrylamide/Bisacrylamide solution 29:1 (3.3%C) 10% APS TEMED 3.17 ml 2.5 ml 100 l 4.16 ml 50 l l 255 10 x SDS-PAGE electrophoresis buffer Tris base Glycine SDS Adjust the pH to 8.3. 250 mM 2.5 M 1% (w/v) x Reducing sample loading buffer Tris-HCL, pH 6.8 Glycerol SDS Bromophenol Blue Dithiothreitol (DTT) 250 mM 50% (v/v) 10% (w/v) 1% (w/v) 0.5 M 10 x Western blot transfer buffer Tris base Glycine 250 mM 1.92 M TBS-T buffer Tris-HCL, pH 7.5 NaCl Tween-20 50 mM 150 mM 0.1% (v/v) Blocking Buffer Non-fat milk 5% (prepared in TBS-T buffer) 256 [...]... three activation pathways of complement: the Classical, Mannose-Binding Lectin, and Alternative Pathways The three pathways converge at the point of cleavage of C3 The classical pathway is initiated by the binding of the C1 complex to antibodies bound to the bacterial cell surface C1s first cleaves C4 and then cleaves C2, leading to the formation of a C4b 2a enzyme complex (classical pathway C3 convertase)... convertase) The mannosebinding lectin pathway is initiated by binding of the complex of MBL and MASP 1and MASP2 to arrays of mannose groups on the bacterial cell surface MASP2 acts as a protease like C1s, and facilitate classical pathway C3 convertase formation The alternative pathway is initiated by the covalent binding of a small amount of C3b to hydroxyl groups on cell-surface carbohydrates and proteins and. .. alternative pathway Although each pathway has a unique combination of initiating proteins, all three converge in the activation of the complement component C3 and a common lytic pathway involving the formation of the membrane attack complex (MAC) on the target cell surface The various complement pathways are schematically represented in Figure 1 .3 15 Figure 1 .3 Various pathways of complement activation The. .. like 3 lipoarabinomannan (LAM) and lipomannan (LM), and proteoglycans such as arabinogalactan A B C Figure 1.1 Bacterial cell surface PAMPs (A) Schematic representations of the general structure of the cell envelope of Gram-positive bacteria, Gram-negative bacteria, and Mycobacterium (B) Schematic representation of the chemical structure of lipopolysaccharide (LPS) The core is covalently bound to the. .. recruited to the cytoplasmic tail of activated TLR and associates via the TIR domain Subsequently, IRAKs and TRAF 6 are recruited and activated in a signaling complex The phosphorylation of TAK1 by IRAKs activates the former kinase which, in turn phosphorylate IKK complexes IKK then phosphorylates IB leading to ubiquitinylation and proteasome-mediated degradation of the latter, and the release of activated...List of Figures Page 1.1 Bacterial cell surface PAMPs 4 1.2 The MyD88-dependent signaling pathway 13 1 .3 Various pathways of complement activation .16 1.4 Ribbon diagrams of human component C3 and C3b 22 1.5 Activation and degradation of complement C3 23 1.6 Ribbon drawing of the extracellular segment of crystallized integrin v 3 illustrating various domains of the  and. .. (MKK4) and 6 (MKK6), which, in turn, can activate p38 and c-jun NH2-terminal kinase (JNK) MAPK signaling pathways (Johnson and Lapadat, 2002) These MAPKs can then activate the 12 transcription factor, activation protein 1 (AP-1) (Shaulian and Karin, 2002), which promotes the transcription of various pro-inflammatory genes PAMP Toll Like Receptor Cell membrane IRAK 1 IRAK 2 IRAK 4 TRAF6 TAB1 P TAB2 TAK1... target surfaces near the site of complement activation The surface-bound C3b can then recruit factor B and, following factor D mediated cleavage, form the alternative pathway C3 convertase C3bBb This convertase can recruit and cleave further C3 molecules, generating more C3 convertase in a self-sustaining cycle of C3 activation This positive amplification loop is the key 18 defining feature of the alternative... the alternative complement pathway (Muller-Eberhard and Gotze, 1972) C3bBb can also proceed to, like the classical pathway C3 convertase C4bC 2a, interact with a C3b molecule to form the C5 convertase, C3bBb3b (Daha et al., 1976; Medicus et al., 1976) and culminate in the formation of MAC The fact that the alternative pathway is initiated by a fluid phase convertase could potentially result in the indiscriminant... groups Activated C1s also splits C2 into C 2a and C2b The larger enzymatically active C 2a fragment associates with surface-bound C4b to form the classical pathway C3 convertase, C4b 2a (Muller-Eberhard et al., 1967), which can enzymatically generate large quantities of C 3a and C3b from native C3 Reminiscent of C4b, C3b can effectively bind target surfaces via its thiolester group In addition to cleaving C3, . complement receptor 3 (CR3) and non-opsonic scavenger receptor class A (SR -A) . We will attempt to investigate certain aspects of their ligand binding properties and delineate downstream signaling pathways. 1 13 3. 6 The SR -A ligands S. aureus and LTA does not induce SR-AI- mediated NF-B 114 3. 7 The potential SR -A ligand B. subtilis induce SR -A mediated NF-B activation 115 3. 8 The potential. INVESTIGATIONS OF THE SCAVENGER RECEPTOR CLASS A AND COMPLEMENT RECEPTOR 3 – TWO PATTERN RECOGNITION RECEPTORS BY GOH WEE KANG JASON B. Sc. (Hons), Murdoch University, Australia