CROSSTALK BETWEEN CRP AND FICOLINS REGULATES INNATE IMMUNITY 1

CROSSTALK BETWEEN CRP AND FICOLINS REGULATES INNATE IMMUNITY         ZHANG JING (B.Eng (Hons), Tianjin University, China)   A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCE AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to thank my supervisors, Prof. Ding and Prof. Ho, for the opportunity and the guidance that they have given to me not only for science but also for life. They have been great teachers and I have learnt so much from them. I would like to especially thank Prof. Ding for her painstaking and dedicated effort in imparting the many important skills that a researcher should possess. I would also like to thank Prof. Lu Jinhua, who is my thesis advisory member, for his suggestions and guidance to my research all through the four years. I would like to express my gratitude to my mentors, Dr. Ng Miang Lon, for sharing their years of experience and insights on research with me in my first year of phD study, most of which I can never learn from books. Special thanks also go to Dr. Jason Goh for help with the rFBG expression and purification.   I would also like to thank Prof. Steffen Thiel (University of Aarhus, Denmark)  for providing me ficolin proteins&clones that have been used in this thesis and giving me comments and suggestions about my experimental design and paper drafting; Prof. Teizo Fujita (Fukushima Medical University, Japan) for providing L-ficolin and H-ficolin cDNA plasmids; Prof. Anna Blom (Lund University, Sweden) for the C4BP protein. Dr. Cynthia Yingxin He for the help with the realtime imaging; Dr. Ganesh Anand for the help with hydrogen deuterium exchange; Dr. Yang Lifeng for the help with the computational modelling and Dr. Andrew Tan for useful discussion about experimental design. It has been a great pleasure to collaborate with Prof. Thiagarajan PS and Liu Bing to study the systematic function of C4BP. I would like to express my sincere thanks to the many former and present lab mates, Li Yue, Li Peng, Xiaowei, Nancy, Patricia, Belinda, Agnes, Diana, Sebastian, Siow Ting, Xiaolei, Sun Miao, Zehua, Jianing, Joanne, Guili, Cheryl, Imelda, Ruijuan, Yuan Quan, Peng Jun, Lifeng, Lin Bing, Shirly, Sia Lee, Soon Kok, Marianne, Sash, Porkodi, Cuicui, Rebecca, Zhiwei, Neha, Karthik, Fengting, Glenn, SunYe, Maz and Thuyen. I would like to express my gratefulness to many of them for always sharing their thoughts and lending their hands, ears and eyes to my work during my years of confusion and revelations here and to some of them for being such great lunchtime buddies! Special thanks also go to ficolin group members, who have been always inspiring me and giving me countless suggestions during my phD study. Many thanks also go to Michelle, Denise, and Xianhui, who have taught and helped me with my analysis of the mass spectrometry data; Tong Yan and Xiao Yong who helped me with confocal microscope ; Subha who came to my rescue when I got stuck with a problem in the lab. Most importantly, I would like to thank my family: Dad, Mom, and my hubby, Dong Bo and my parents-in law for all the joy, love and encouragement. I would especially like to thank my husband, Dong Bo for always being there for me, and for always trusting me. This thesis is dedicated to my Dad and Mom for all the years of love. Thank God for his continuous unfailing love. i TABLE OF CONTENTS Acknowledgements Table of Contents Summary List of Tables List of Figures List of Abbreviations List of Primers Flow Chart Page i ii vii ix x xiv xviii xxiii CHAPTER 1: INTRODUCTION ············································································1 1.1 Immune sentinels for bacteria recognition in human······································1 1.1.1 The innate immune system shapes the adaptive immune system································ 1.1.2 Pathogen-associated molecular patterns ····································································· LPS ···························································································································· GlcNAc ······················································································································ Phosphocholine ········································································································· 10 1.1.3 The innate immune system uses pattern recognition receptors ··································· 12 C-reactive protein ·········································································································18 Ficolins ·························································································································20 1.1.4 The ancient origin of PRR interactomes ····································································· 25 1.1.5 Non-self recognitions by human PRRs and the formation of PRR:PRR interactomes 28 1.2 Innate immune responses to combat the pathogens ········································30 1.2.1 Extracellular responses ······························································································· 32 Complement pathways ··································································································32 Other extracellular innate immune responses: Coagulation & prophenoloxidase activating system ······································································································ 36 1.2.2 Intracellular responses ································································································ 40 NF-kB activation ···········································································································40 Opsonization and phagocytosis ····················································································42 1.3 Infection-inflammation conditions and immune response ······························46 1.4 Aims and Rationale of experimental approaches ············································47 ii CHAPTER 2: MATERIALS AND METHODS ·····················································51 2.1 Materials ··············································································································51 2.1.1 Organisms ·················································································································· 51 2.1.2 Biochemicals and enzymes ························································································ 52 2.1.3 Medium and agar········································································································ 54 2.2 Purification of native serum type ficolins ·························································55 Native L-ficolin ············································································································ 56 Native H-ficolin ············································································································ 56 2.3 Expression and purification of recombinant M-ficolin and FBG ··················57 2.4 Analysis of the purified proteins········································································58 2.4.1 Bradford protein assay ······························································································· 58 2.4.2 SDS-PAGE and Western blotting immunodetection ··················································· 58 2.4.3 Silver staining ············································································································ 59 2.4.4 Mass spectrometry ····································································································· 60 2.5 Simulation of “normal” and “infection-inflammation conditions ·················60 2.6 In vitro bacterial killing assay ············································································61 2.7 Protein:protein interaction assays ·····································································62 2.7.1 ELISA ························································································································ 62 2.7.2 Surface Plasmon Resonance (SPR) ············································································ 64 2.7.3 Co-immunoprecipitation (Co-IP) ··············································································· 64 2.8 Cell culture and transfection··············································································65 2.8.1 Isolation of primary monocytes from buffy coat ························································ 65 2.8.2 Cell culture ················································································································· 65 2.8.3 Transfection by Lipofectamine 2000 ·········································································· 66 2.8.4 Transfection by Nucleofection ··················································································· 66 2.8.5 Transfection by DOTAP ····························································································· 67 2.9 Complement activity assay ·················································································67 2.9.1 C4 cleavage assay ······································································································ 67 2.9.2 Phagocytosis assay ····································································································· 68 2.9.3 C3 deposition assay by flow cytometry ······································································ 69 2.10 Effect of C4BP on the complement pathway under infectioninflammation condition····················································································70 iii 2.10.1 Manipulation of C4BP level in the serum································································· 70 2.10.2 Complement measurement by pull-down with GlcNAc- and PC-beads ··················· 71 2.11 Functional study of CRP:M-ficolin complex formation································71 2.11.1 PAMP stimulation ···································································································· 71 2.11.2 RNA extraction ········································································································ 72 2.11.3 Reverse Transcription PCR ······················································································ 73 2.11.4 Meaurement of cytokine production ········································································· 74 2.12 Localization of M-ficolin ··················································································74 2.12.1 Cell surface protein extraction ·················································································· 74 2.12.2 Immunofluoresence localization of M-ficolin by GFP labeling ································ 75 2.12.3 Immunofluoresence localization of M-ficolin by antibody staining ························· 75 2.12.4 Flow cytometric detection of M-ficolin ···································································· 75 2.13 Assay of NF-κB activity assay mediated by M-ficolin···································· 76 2.13.1 Dual luciferase assay ································································································ 76 2.13.2 Electrophoretic Mobility Shift Assay (EMSA) ························································· 77 2.13.3 Testing the specificity of NF-κB activation by inhibitors ········································· 77 2.14 Screening for interaction partner of M-ficolin ···············································78 2.14.1 Amplification and purification of human leukocyte double-stranded cDNA library 78 2.14.2 Cloning of bait genes into pGBKT7 ········································································· 78 2.14.3 Yeast-two hybrid screening assay ············································································· 79 2.15 In vivo protein interaction assay ······································································81 2.15.1 In situ proximity ligation assay (PLA) ····································································· 81 2.15.2 Co-localization assay of M-ficolin and GPCR43······················································ 82 2.15.3 Yeast three hybrid to confirm the formation of ternary complex between M-ficolin, CRP and GPCR43 ····································································································· 83 2.16 Characterization of binding sites between CRP and M-ficolin ····················84 2.16.1 Hydrogen deuterium exchange mass spectrometry (HDMS) ···································· 84 2.16.2 Computational prediction of binding sites between CRP and M-ficolin ··················· 87 Molecular Dynamics simulation ··················································································87 Zdock and Rdock ··········································································································88 2.16.3 Site-directed mutagenesis ························································································· 88 2.17 Statistical analysis ·····························································································89 iv CHAPTER 3: RESULTS ··························································································90 3.1 Characteristics of endogenous CRP levels in healthy and patient sera and purified CRP proteins ························································································90 3.2 Purity of the three purified ficolins ···································································93 3.2.1 Purity of the purified native L-, H- ficolins ································································ 93 3.2.2 Purify of the expressed and purified M-ficolin and FBG domains of Land Mficolins························································································································ 94 3.3 Definition of the “normal” and “infection-inflammation conditions” ···········95 3.4 CRP interacts with L- and M-ficolins but not H-ficolin ··································96 3.5 Characterization of CRP:L-ficolin ····································································99 3.5.1 The CRP binding region is delineated to FBG domain of the L-ficolin ······················ 99 3.5.2 CRP and L-ficolin interaction is pH- and calcium- sensitive ······································ 100 3.5.3 Infection-inflammation triggers CRP:L-ficolin interaction········································· 103 3.6 Functional significance of CRP:L-ficolin interaction ······································105 3.6.1 CRP:L-ficolin triggers two autonomous amplification pathways ······························· 105 3.6.2 CRP:L-ficolin upregulates opsonization and phagocytosis ········································· 106 3.6.3 CRP:L-ficolin interaction increases C3 deposition to enhance killing of P. aerogninosa ···················································································································109 3.6.4 CRP:L-ficolin crosstalk integrates the classical and lectin complement pathways to enhance antimicrobial activity ···················································································· 112 3.6.5 Synergism between CRP:L-ficolin under infection-inflammation condition enhances bacterial killing ··························································································· 114 3.7 The differential inhibitory effect of C4BP on the lectin pathway and classical pathway ································································································119 3.8 Characterization of CRP:M- ficolin ··································································123 3.8.1 Infection-inflammation condition enhanced CRP:M-ficolin binding affinity by 100-fold ······················································································································ 123 3.8.2 CRP interacts with M-ficolin via FBG domain ··························································· 124 3.8.3 CRP and M-ficolin interaction is pH- and calcium- sensitive ····································· 125 3.9 M-ficolin activates signal transduction in the immune cells ···························128 3.9.1 Secreted M-ficolin associates with the immune cell membrane ································· 128 3.9.2 M-ficolin mediates NF-κB activation ········································································· 131 3.9.3 M-ficolin is localized to the cell membrane by association with GPCR43 ················· 136 v 3.9.4 The M-ficolin association with GPCR43 is physiologically relevant ························· 141 3.10 CRP collaborates with M-ficolin to regulate IL-8 secretion ·························142 3.10.1 CRP, M-ficolin and GPCR43 form a ternary complex ·············································· 143 3.10.2 CRP regulates M-ficolin-mediated IL-8 secretion ···················································· 145 3.11 The molecular mechanisms underlying the pH- and calcium- sensitive formation of CRP:M-ficolin interaction ·························································147 3.11.1 Identification of binding interfaces between M-ficolin:CRP under infectioninflammation condition ··························································································· 148 3.11.2 Infection-inflammation condition of low pH and calcium expands M-ficolin structure and enhances its interaction with CRP ······················································ 154 3.11.3 Computational modeling of pH- and calcium- dependent CRP:M-ficolin interaction ················································································································ 160 3.11.4 Localization of the exact binding sites of CRP on M-ficolin ···································· 163 3.11.5 The biological implications of the pH- and calcium- sensitive interaction between CRP and M-ficolin ·································································································· 167 3.11.6 M-ficolin on monocytes attracts and induces monocyte clustering, regulated by CRP ························································································································· 171 3.12 CRP does not interact with H-ficolin ······························································174 CHAPTER 4: DISCUSSION ···················································································176 4.1 The functional divergence of L- and M-ficolins in evolution··························177 4.2 CRP:L-ficolin interaction connects the classical and lectin complement pathways and boost the antimicrobial activity ················································179 4.3 M-ficolin connects the extracellular surveillance to the intracellular signal transduction ·········································································································182 4.4 Molecular basis for anti- and pro-inflammatory roles of CRP through its interaction with M-ficolin ··················································································186 CHAPTER 5: GENERAL CONCLUSION AND FUTURE PERSPECTIVES ··189 5.1 Conclusion ···········································································································189 5.2 Future perspectives ·····························································································194 BIBLIOGRAPHY ·····································································································197 vi SUMMARY Early detection and efficient removal of virulent pathogens are fundamental to host survival. Although C-reactive protein (CRP) and ficolins have long been known to independently initiate the classical and lectin complement pathways respectively under physiological condition, how they function under pathophysiological condition remains poorly understood. This thesis reports that a defined local “infection-inflammation condition” induced a 100-fold increase in the interaction between CRP and L-ficolin. This leads to communication between the classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition have consistently proven the functional significance of the amplification pathways in boosting the complement-mediated antimicrobial activity. This was again supported by the effective killing of P. aeruginosa in the plasma under defined local infection-inflammation condition, where powerful antimicrobial activity is provoked by CRP:L-ficolin interactions. Therefore, we conclude that the local infection-inflammation condition triggers a strong CRP:L-ficolin interaction, eliciting autonomous complementamplification pathways which co-exist with and reinforce the classical and lectin pathways. Similar to L-ficolin, the M-ficolin was found to interact with CRP in a pH- and calcium-dependent manner. However, their biological consequences are divergent. We found that M-ficolin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, GPCR43, to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M- ficolin:GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis occurring at the local site of infection triggers a strong vii interaction between the CRP and the M-ficolin:GPCR43 complex. This ternary complex curtails IL-8 production, thus, preventing immune over-activation. Our finding implies a possible mechanism which the host employs to expand its repertoire of immune functioncum-regulation tactics by promiscuous protein-protein networking. To understand the detailed mechanism of low pH- and low calcium- triggered CRP:M-ficolins interaction, we delineated the precise binding interface between M-ficolin and CRP. We found that the flexible C-terminus of the fibrinogen-like (FBG) domain of M-ficolin undergoes dramatic conformational change under acidosis and hypocalcaemia, which exposes unique motifs to augment its interaction with CRP. In silico analyses indicate that in contrast to normal condition, under infection-inflammation condition, the relocation of CRP binding site to the conserved pathogen sugar-ligand binding pocket and a simultaneous 100-fold increase in the affinity of M-ficolin:CRP complex might diverge the M-ficolin from pathogen recognition. This conformational change possibly helps to restore homeostasis. Therefore, infection-induced microenvironment perturbations act as a molecular switch to transform the FBG domain conformation and regulate its function. Overall, we have demonstrated that both the L- and M- ficolins interact with CRP to send the extracellular environmental cues into the cell to elicit intracellular immune response. This explains the intrinsic necessity to have two different ficolin isoforms, the L- and Mficolins, each with high homology in our human body. Our findings provide new molecular insights into the host immune response to infection under infectioninflammation conditions. Our precise delineation of the interaction interface between CRP and M-ficolin will be useful for the future development of immunomodulators. (492 words) viii LIST OF TABLES Table No. Title CHAPTER 1.1 Examples of major pattern recognition receptors and their ligands. 1.2 The tissue distribution, ligand spectrum and functions of different ficolin isoforms in different species. CHAPTER 3.1 The potential interaction partners of M-ficolin screened from human leukocyte cDNA library. 3.2 Summary of H/2H exchange data for FBG. 3.3 Summary of H/2H exchange data for CRP. 3.4 Parameters for computational simulation and docking analysis. Page 17 24 138 150 151 162 ix   Figure 3.50: pH-sensitive and calcium-sensitive regions are highlighted on the primary sequence of FBG (M-ficolin115 – M-ficolin326). The amino acids composed of domain A (purple letter), B (grey letter) and P (blue letter) were highlighted. The black boxes are the pH-sensitive regions. The blue underline shows the calcium-sensitive regions under pH 7.4 and the red underline shows the calcium-sensitive regions under pH 6.5. The green boxes indicate the conserved calcium binding sites. The disulfide bond (Cys270 – Cys283) that stabilizes the calcium binding site is indicated in orange. The grey shaded region is the secretory peptide. FBG domain starts from Pro115 and ends at Ala326 3.11.3 Computational modeling of pH- and calcium- dependent CRP:M-ficolin interaction To further confirm the proposed pH and calcium regulating mechanism of CRP:rM-FBG, computational molecular dynamics (MD) simulation was applied based on the crystal structure of M-ficolin FBG (PDB: 2JHM) to demonstrate the conformational change of whole rM-FBG molecule. This was aimed at understanding how the FBG plays a dominant role in controlling its interaction with CRP under pathophysiological condition. The rM-FBG structures were simulated for 15 ns at a constant temperature of 300 K until 160 the root mean square deviation (RMSD) of the simulated structures of rM-FBG under pH 7.4 and 6.5 reached equilibration. Structures in the last ns after equilibration were extracted and used in our experiments (Figure 3.51). By analyzing the simulated rM-FBG structures we found that the solvent accessible surface area for rM-FBG structure under pH 6.5 is larger than that of rM-FBG structure under pH 7.4 (Table 3.4). This is consistent with our HDMS results and corroborates that low pH induces an extended structure of rM-FBG which might expose its binding sites to CRP leading to the high affinity of CRP:rM-FBG under infection-inflammation condition. Figure 3.51: Simulated structures of FBG under infection-inflammation and normal conditions. The green color ball indicates the location of the bound calcium and the surrounding area is the calcium binding site. To confirm the detailed binding pattern between CRP and rM-FBG, the simulated rM-FBG structures under normal and infection-inflammation conditions were used to dock on the CRP molecule. As the slight conformational change of CRP does not contribute significantly, to CRP:M-ficolin interaction, crystal structure of CRP (PDB code 1B09) was used for computational docking analysis. The random docking results were further optimized in accordance with the HDMS results (Figure 3.52, upper panel). We found that consistent with the binding sites on CRP and rM-FBG under pH 6.5 and 161 7.4 identified by HDMS, the predicted binding interface was listed below (Figure 3.52, lower panel). The entire molecule docking analysis further confirmed that the rM-FBG binding sites on CRP were less variant, whereas due to the flexible conformational change of the C-terminal domain of rM-FBG, more CRP binding sites translocated to the C-terminal region of rM-FBG under pathophysiological condition, thus explaining the enhanced interaction under such conditions. The lower docking energy under pH 6.5 than pH 7.4 indicates that CRP:M-ficolin forms a more stable complex under mild acidic condition (Table 3.4). The larger buried area during the complex formation under infection-inflammation condition compared to normal condition further explained the enhanced binding under infection-inflammation condition (Table 3.4). Overall, we showed by MD simulation and docking analysis of whole rM-FBG molecule that under different conditions of the interaction between CRP:rM-FBG, the infection-inflammation condition triggers conformational change in rM-FBG, leading to the enhanced interaction with CRP. Table 3.4. Parameters for computational simulation and docking analysis Condition rM-FBG SAS (Å2) E_elec2 (kcal/mol) E_sol (kcal/mol) E_RDOCK (kcal/mol) ZDOCK score Buried area (Å2) Infection-inflamma tion condition Normal condition 10961.2 -29.46 -5.4 -31.92 16.02 985.8 10502.2 -32.57 5.2 -24.11 11.04 859.28 SAS is the area of solvent accessible surface during the complex formation. E_Elec2 is Electrostatic energy after second minimization with ionic residues in charged state E_Sol is Desolvation energy based on the Atomic Contact Energy (ACE) E_R Dock Final RDOCK energy: E_RDock=E_sol+beta*E_elec2 (beta=0.9) ZDock Score is the shape complementarity score including electrostatics and desolvation energy terms calculated by the ZDOCK program. Higher score is better. 162 Figure 3.52: Computational docking model of rM-FBG (red); CRP (blue) at normal and infection-inflammation conditions, optimized according to HDMS results. The CRP structure is from the crystal structure (PDB: 1B09). The surface view and ribbon view of the docking models are shown in the figure. The predicted CRP-binding sites on rM-FBG and rM-FBG-binding sites on CRP under physiological and pathophysiological conditions are tabulated below. 3.11.4 Localization of the exact binding sites of CRP on M-ficolin. As CRP was shown to bind to an extra region of M-ficolin which is exposed under pathophysiological condition and enhancing their binding affinity by 100 times, it was 163 imperative for us to identify the exact critical pH- and calcium- sensitive binding site(s) of CRP on the M-ficolin molecule. According to our HDMS results, we hypothesized that the pH and the calcium-sensitive CRP binding sites are located in the flexible C-terminal binding region of rM-FBG (284-326). It is reported that the Ca2+ ion is bound to a loop, which is stabilized by Cys270 and Cys283 disulphide bond, in the P domain. This binding is coordinated by the side chain carboxylate oxygen atoms of Asp262, Asp264, and the main chain carbonyl oxygen atoms of Ser266 and Ser268 as well as two water molecules in monomer A (Tanio et al. 2007). As pH and Ca2+ ions dramatically affect interaction between M-ficolin and CRP, the contact points may be potentially close to these amino acids. To locate the potential critical sites, we performed a sequence alignment of H-, Land M- ficolins (Figure 3.53A). Since both L- and M- ficolins but not H-ficolin interacted with CRP, we reasoned that amino acids that are conserved in L- and M-ficolins but not in H-ficolin (highlighted) are likely to connote functional significance. This sequence analysis revealed the importance of primary amino acids Val240-Ala326 near the calcium binding loop (Garlatti et al. 2007). We identified five amino acids as the potential contact points: Lys259, Phe274, Gln275, His284 and Leu293 which are proximal to the Ca2+ binding loop and the pH sensitive binding region. Our computational prediction suggested that mutations in these sites may only cause mild conformational change around the calcium binding loop without disrupting the overall structure of the protein (Figure 3.53B). In order to test which of the five amino acids is critical for the pH and calcium 164 sensitive interaction of CRP:rM-FBG, we cloned the wildtype rM-FBG to the mammalian expression vector. By site-directed mutagenesis, five mutated rM-FBG gene fragments (L259A, F274A, Q275A, H284A and L293A) in the same expression vector were constructed. Wildtype and mutated proteins were expressed (Figure 3.53C) and purified and their binding to CRP was tested under different pH and calcium conditions. ELISA showed that the H284A mutation caused a dramatic loss-of-binding to CRP compared to the wildtype proteins at pH 6.5 but not at pH 7.4 (Figure 3.54), indicating that His284 is the critical determinant of M-ficolin:CRP interaction under mild acidosis. No significant difference was observed for the other mutated proteins and wildtype protein. This is consistent with a previous report that His284 regulates the pH-sensitive ligand binding property of rM-FBG (Tanio et al. 2009). The replenishment of calcium to physiological level of 2.5 mM did not abolish the binding activity at the H284 site, thus authenticating our finding that calcium has a minimal inhibitory effect at pH 6.5. Taken together, the above findings suggest that pathophysiological microenvironmental perturbation caused conformational changes to M-ficolin, which regulates its association with CRP via a critical amino acid His284. As many other ligands of M-ficolin such as GlcNAc, GalNAc or sialic acid also dock to the same region of M-ficolin (Garlatti et al. 2007), it is conceivable that binding of CRP to the conformationally competent M-ficolin might promote/inhibit the PAMP/sugar/pathogen recognition by M-ficolin, which may interfere with pathogen recognition of M-ficolin. 165 Figure 3.53: Location of the possible critical binding sites on M-ficolin that might regulate its pH and calcium sensitive interaction with CRP. (A) The sequence comparison of the C-terminii of L-, H- and M-ficolins (upper panel). The amino acids common in L- and M-ficolins are in yellow and the calcium binding sites are in blue. Site-directed mutations are indicated by black triangles. The lower panel shows schematic illustrations of the ficolins with site-directed mutations (black squares). (B) The predicted structures of M-ficolin after mutating FBG residues: K259A, F274A, Q275A, L293A and H284A, using Discovery Studio 2.1 (Accelry Inc., San Diego, CA). The colored regions in the proteins indicate the predicted structural variations for different mutants. These regions are mainly located at the P domain of M-ficolin. (C) Purification of wildtype FBG, and K259A, F274A, Q275A, L293A and H284A mutants. E1-E6 indicates sequential elutions from the purification columns. 166 Figure 3.54: ELISA to test the binding affinity between the FBG mutants and CRP. One μg binding protein directly immobilized onto replicate wells was the positive control. The relative binding was calculated as a percentage against the positive control. 3.11.5 The biological implications of the pH- and calcium- sensitive interaction between CRP and M-ficolin. During local infections such as trauma-induced infection (Baranov et al. 2007) and intra-abdominal infection (Simmen et al. 1994), the level of CRP, an acute phase protein, dramatically increases and microenvironmental perturbation such as acidosis and hypocalcaemia prevails at the late stage of infection when excessive leukocyte recruitment (van Zwieten et al. 1981; Issekutz et al. 1982) or overwhelming immune activation (Zhang et al. 2009) occurs, thus creating a need to restore homeostasis. Ficolin respresents an important group of C-type lectin, with widespread occurrence of homologues from lower species such as horseshoe crab to human. Being a critical group of PRR, ficolins were found to recognize a wide range of microbes (Zhu et al. 2006), playing critical role in the frontline host defense. M-ficolin, a PRR located on the surface of the monocytes, has been proposed to be a phagocytic receptor upon recognizing the 167 bacteria (Teh et al. 2000). Therefore, the association of CRP to M-ficolin might regulate the host defense functions of M-ficolin. It was documented that at physiological pH, rM-FBG is an active conformer for PAMP/sugar recognition whereas under acidic condition, rM-FBG becomes a non-active form and does not recognize PAMP/sugar. Here we found that mildly low pH- and calcium- induced conformational change in M-ficolin markedly increased its interaction with CRP. Therefore, at physiological condition, CRP associates weakly with rM-FBG to the sites away from the C-terminal region, which might expose the GlcNAc-binding site of rM-FBG to bacterial PAMPs, thus promoting the pathogen recognition of monocytes. At such condition, rM-FBG forms an active form for PAMP/sugar recognition but a non-active form for CRP binding. In contrast, under acidosis, the flexible C-terminus in the FBG domain of M-ficolin exposes more binding sites for CRP, which completely blocked the sugar ligand (GlcNAc)-binding sites of rM-FBG (Figure 3.55A, upper panel). This might inhibit the pathogen recognition and the subsequent directional movement of monocytes towards the bacteria (Figure 3.55A, lower panel). This speculation was supported by the fact that CRP dose-dependently inhibited the binding of M-ficolin to immobilized GlcNAc-BSA on the ELISA plate (Figure 3.55B). Therefore, under acidic condition, rM-FBG forms an active conformer for CRP binding but is non-active towards PAMP/sugar recognition. Undoubtedly, the association of CRP to rM-FBG will further strengthen the transit from active to non-active form for pathogen recognition when the microenvironmental perturbation occurs thus warning the host of an overactive immune 168 response. These results explains the dual opposing role of CRP in regulating the IL-8 secretion and might imply a refined regulatory mechanism where the flexible structure of M-ficolin changes its conformation upon environmental perturbation during infection to accommodate CRP at a different site. This structure-activity relationship fine tunes the M-ficolin for effective pathogen recognition and controls the homeostatic recruitment of monocytes to prevent immune over-activation. It has been found that in the P domain, the region containing the calcium binding site and its corresponding stabilizing disulfide bond region are highly conserved (Endo et al. 2004). By aligning ficolin isoforms from different species, we also found that the identified pH sensitive residue His284 is conserved in 12 different species (Figure 3.56). The conservation of our identified pH and calcium sensitive CRP binding site on rM-FBG (His284) implies the necessity of CRP:M-ficolin interaction in vivo. 169 A B Figure 3.55: CRP inhibited the pathogen recognition of M-ficolin. (A) The computationally predicted binding interface of CRP(blue):FBG(red) overlaps with GlcNAc binding site (yellow) on FBG under infection-inflammation condition, whereas under normal condition, the GlcNAc binding site on FBG was exposed (upper panel). Therefore, we hypothesized that crosstalk between CRP and M-ficolin might block the pathogen recognition site of FBG under infection-inflammation condition but not under normal condition (lower panel). (B) Increasing amounts of CRP inhibit the binding of FBG to GlcNAc-BSA immobilized on the ELISA plate. FBG and CRP were preincubated at room temperature for h before adding to the ELISA wells, which were precoated with GlcNAc-BSA. FBG was identified using anti-His fusion antibody. *: p[...]... the interaction between CRP and L-/H-/M- ficolins 3.6 Binding of CRP to rL-FBG 3.7 Biochemical characterization of the interaction between CRP and rL-FBG 4 6 8 9 12 14 20 21 27 32 35 39 44 45 79 80 82 84 85 91 92 94 95 98 99 10 2 x 3.8 3.9 3 .10 3 .11 3 .12 3 .13 3 .14 3 .15 3 .16 3 .17 3 .18 3 .19 3.20 3. 21 3.22 3.23 3.24 3.25 3.26 3.27 3.28 3.29 3.30 3. 31 3.32 Infection-inflammation triggers CRP and L-ficolin... collectin and ficolins 1. 7 Molecular structure of human pentameric calcium-binding CRP 1. 8 Structural and sequence analysis of ficolins 1. 9 Summary of the PRR:PRR interactomes in horseshoe crab 1. 10 Cascade of innate immune response to combat the invading pathogens 1. 11 The three distinct pathways of complement activation 1. 12 Induction of prophenoloxidase (proPO) cascade in invertebrate immunity 1. 13 Schematic... condition 11 1 Co-existence of the amplification pathways and the classical and lectin pathways 11 3 Real-time observation of the bacterial killing effect of serum under normal (pH 7.4, 2.5 mM calcium) or infection-inflammation condition (pH 6.5, 2 mM calcium) 11 5 The synergistic action of CRP and L-ficolin enhanced antimicrobial activity of complement 11 8 Binding of CRP and L-/ H- ficolins to P aeruginosa 11 9... complex in serum 10 4 Downstream functional analysis of amplification pathways 1 and 2 by C4 cleavage assay 10 6 The phagocytosis assay of the opsonized beads generated in amplification pathways 1 and 2 under infection-inflammation condition (magnification: 400×) 10 8 The connection of the classical pathway and lectin pathway as a consequence of CRP: ficolin cross talk 10 9 Crosstalk between CRP and ficolins generated... GPCR43 :CRP and CRP: M-ficolin in PBMCs at pH 7.4 and pH 6.5 14 4 The effects of CRP- depleted or CRP- containing FBS on GlcNAc -induced IL-8 secretion at pH 7.4 and 6.5 14 6 The effect of CRP on regulating the expression of IL-8 in wildtype 14 6 or M-ficolin- U937 cells HDMS kinetic curves for deuterium incorporation of individual peptide fragments for rM-FBG 15 2 The CRP binding interface on FBG 15 2 HDMS... slower than the innate immunity, although it is commonly believed that stronger attacks against the invading microorganisms are mounted during the activation of adaptive immunity Over all, the innate immunity and the adaptive immunity integrated into an intact sophisticated host immune system where innate immunity serves as a rapid and efficient frontline defense strategy 1. 1 .1 The innate immune system... deposited C4BP and C3 on the GlcNAc beads or PC beads 12 2 under infection-inflammation condition or normal condition CRP: M-ficolin interaction was significantly enhanced under local infection-inflammation conditions 12 3 Dose-dependent binding of rM-FBG to CRP was shown by ELISA 12 4 SPR analysis of and the binding of rM-FBG to CRP 12 5 CRP and M-ficolin interaction is pH and calcium sensitive 12 7 A model... the monocytes 13 1 Dose-dependent binding of M-ficolin to the membrane extract of PBMCs immobilized on the plates 13 1 M-ficolin mediates IL-8 secretion upon PAMP/sugar stimulation 13 4 IL8 expression in wildtype and M-ficolin- U937 cells when stimulated with 10 ng/ml PGN, LTA, LA, ReLPS and LPS 13 4 RANTES secretion by monocytes stimulated with 10 0 mM GlcNAc, 4 ng/ml ReLPS, LPS and Lipid A 13 5 Effect of... with Xba1 restriction site at the end of the primer Reverse primer for cloning H-ficolin into pGBKT7 with EcoR1 restriction site at the end of the primer 11 ECoHFCNBR CATGCCATGGCTCAC TATCGAAGCATCATC CG 12 ECoCRPAF GGAATTCATGGAGAA GCTGTTGTG Forward primer for cloning CRP into pGADT7with EcoR1 restriction site at the end of the primer 13 BamCRPAR CGGGATCCTTATCAG GGCCACAGCT Reverse primer for cloning CRP into... structure of FBG (PDB:2JHM) 15 8 pH-sensitive and calcium-sensitive regions are highlighted on the 16 0 primary sequence of rM-FBG (M-ficolin 115 –M-ficolin326) Simulated structures of FBG under physiological and pathophysiological conditions 16 1 Computational docking model of rM-FBG (red); CRP (blue) at physiological and pathophysiological conditions, optimized according to HDMS results 16 3 Location of the possible . calcium). 11 5 3 .15 The synergistic action of CRP and L-ficolin enhanced antimicrobial activity of complement. 11 8 3 .16 Binding of CRP and L-/ H- ficolins to P. aeruginosa. 11 9 3 .17 The deposited. 3 .12 Crosstalk between CRP and ficolins generated more C3 deposition on the P. aeruginosa under the infection-inflammation condition. 11 1 3 .13 Co-existence of the amplification pathways and. conditions. 12 3 3 .19 Dose-dependent binding of rM-FBG to CRP was shown by ELISA. 12 4 3.20 SPR analysis of and the binding of rM-FBG to CRP. 12 5 3. 21 CRP and M-ficolin interaction is pH and calcium