Complement Inhibitor C4b-binding Protein Research Articles

C4BP(β-)-mediated immunomodulation attenuates inflammation in DSS-induced murine colitis and in myeloid cells from IBD patients

The most recent and promising therapeutic strategies for inflammatory bowel disease (IBD) have engaged biologics targeting single effector components involved in major steps of the immune-inflammatory processes, such as tumor necrosis factor, interleukins or integrins. Nevertheless, these molecules have not yet met expectations regarding efficacy and safety, resulting in a significant percentage of refractory or relapsing patients. Thus, novel treatment options are urgently needed. The minor isoform of the complement inhibitor C4b-binding protein, C4BP(β-), has been shown to confer a robust anti-inflammatory and immunomodulatory phenotype over inflammatory myeloid cells. Here we show that C4BP(β-)-mediated immunomodulation can significantly attenuate the histopathological traits and preserve the intestinal epithelial integrity in dextran sulfate sodium (DSS)-induced murine colitis. C4BP(β-) downregulated inflammatory transcripts, notably those related to neutrophil activity, mitigated circulating inflammatory effector cytokines and chemokines such as CXCL13, key in generating ectopic lymphoid structures, and, overall, prevented inflammatory immune cell infiltration in the colon of colitic mice. PRP6-HO7, a recombinant curtailed analogue with only immunomodulatory activity, achieved a similar outcome as C4BP(β-), indicating that the therapeutic effect is not due to the complement inhibitory activity. Furthermore, both C4BP(β-) and PRP6-HO7 significantly reduced, with comparable efficacy, the intrinsic and TLR-induced inflammatory markers in myeloid cells from both ulcerative colitis and Crohn’s disease patients, regardless of their medication. Thus, the pleiotropic anti-inflammatory and immunomodulatory activity of PRP6-HO7, able to “reprogram” myeloid cells from the complex inflammatory bowel environment and to restore immune homeostasis, might constitute a promising therapeutic option for IBD.

Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite.

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.

Serum Complement Activation by C4BP-IgM Fusion Protein Can Restore Susceptibility to Antibiotics in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the etiological agent of gonorrhea, the second most common bacterial sexually transmitted infection worldwide. Reproductive sequelae of gonorrhea include infertility, ectopic pregnancy and chronic pelvic pain. Most antibiotics currently in clinical use have been rendered ineffective due to the rapid spread of antimicrobial resistance among gonococci. The developmental pipeline of new antibiotics is sparse and novel therapeutic approaches are urgently needed. Previously, we utilized the ability of N. gonorrhoeae to bind the complement inhibitor C4b-binding protein (C4BP) to evade killing by human complement to design a chimeric protein that linked the two N-terminal gonococcal binding domains of C4BP with the Fc domain of IgM. The resulting molecule, C4BP-IgM, enhanced complement-mediated killing of gonococci. Here we show that C4BP-IgM induced membrane perturbation through complement deposition and membrane attack complex pore insertion facilitates the access of antibiotics to their intracellular targets. Consequently, bacteria become more susceptible to killing by antibiotics. Remarkably, C4BP-IgM restored susceptibility to azithromycin of two azithromycin-resistant clinical gonococcal strains because of overexpression of the MtrC-MtrD-MtrE efflux pump. Our data show that complement activation can potentiate activity of antibiotics and suggest a role for C4BP-IgM as an adjuvant for antibiotic treatment of drug-resistant gonorrhea.

Safety and Immunogenicity of ChAd63/MVA Pfs25-IMX313 in a Phase I First-in-Human Trial

BackgroundTransmission blocking vaccines targeting the sexual-stages of the malaria parasite could play a major role to achieve elimination and eradication of malaria. The Plasmodium falciparum Pfs25 protein (Pfs25) is the most clinically advanced candidate sexual-stage antigen. IMX313, a complement inhibitor C4b-binding protein that forms heptamers with the antigen fused to it, improve antibody responses. This is the first time that viral vectors have been used to induce antibodies in humans against an antigen that is expressed only in the mosquito vector.MethodsClinical trial looking at safety and immunogenicity of two recombinant viral vectored vaccines encoding Pfs25-IMX313 in healthy malaria-naive adults. Replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding Pfs25-IMX313, were delivered by the intramuscular route in a heterologous prime-boost regimen using an 8-week interval. Safety data and samples for immunogenicity assays were taken at various time-points.ResultsThe reactogenicity of the vaccines was similar to that seen in previous trials using the same viral vectors encoding other antigens. The vaccines were immunogenic and induced both antibody and T cell responses against Pfs25, but significant transmission reducing activity (TRA) was not observed in most volunteers by standard membrane feeding assay.ConclusionBoth vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. However, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine formulation.Trial Registration Clinicaltrials.gov NCT02532049.

Antibacterial Fusion Proteins Enhance Moraxella catarrhalis Killing.

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis. These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis, therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens.

C4BP-IgM protein as a therapeutic approach to treat Neisseria gonorrhoeae infections.

Gonorrhea is a sexually transmitted infection with 87 million new cases per year globally. Increasing antibiotic resistance has severely limited treatment options. A mechanism that Neisseria gonorrhoeae uses to evade complement attack is binding of the complement inhibitor C4b-binding protein (C4BP). We screened 107 porin B1a (PorB1a) and 83 PorB1b clinical isolates randomly selected from a Swedish strain collection over the last 10 years and noted that 96/107 (89.7%) PorB1a and 16/83 (19.3%) PorB1b bound C4BP; C4BP binding substantially correlated with the ability to evade complement-dependent killing (r = 0.78). We designed 2 chimeric proteins that fused C4BP domains to the backbone of IgG or IgM (C4BP-IgG; C4BP-IgM) with the aim of enhancing complement activation and killing of gonococci. Both proteins bound gonococci (KD C4BP-IgM = 2.4 nM; KD C4BP-IgG 980.7 nM), but only hexameric C4BP-IgM efficiently outcompeted heptameric C4BP from the bacterial surface, resulting in enhanced complement deposition and bacterial killing. Furthermore, C4BP-IgM substantially attenuated the duration and burden of colonization of 2 C4BP-binding gonococcal isolates but not a non-C4BP-binding strain in a mouse vaginal colonization model using human factor H/C4BP-transgenic mice. Our preclinical data present C4BP-IgM as an adjunct to conventional antimicrobials for the treatment of gonorrhea.

PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis.

Respiratory tract infections are one of the leading causes of mortality worldwide urging better understanding of interactions between pathogens causing these infections and the host. Here we report that an extracellular matrix component proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel antibacterial component of innate immunity. We detected the presence of PRELP in human bronchoalveolar lavage fluid and showed that PRELP can be found in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and the adventitia of blood vessels in lung tissue. PRELP specifically binds respiratory tract pathogens Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae, but not other bacterial pathogens tested. We focused our study on M. catarrhalis and found that PRELP binds the majority of clinical isolates of M. catarrhalis (n = 49) through interaction with the ubiquitous surface protein A2/A2H. M. catarrhalis usually resists complement-mediated serum killing by recruiting to its surface a complement inhibitor C4b-binding protein, which is also a ligand for PRELP. We found that PRELP competitively inhibits binding of C4b-binding protein to bacteria, which enhances membrane attack complex formation on M. catarrhalis and thus leads to increased serum sensitivity. Furthermore, PRELP enhances phagocytic killing of serum-opsonized M. catarrhalis by human neutrophils in vitro. Moreover, PRELP reduces Moraxella adherence to and invasion of human lung epithelial A549 cells. Taken together, PRELP enhances host innate immunity against M. catarrhalis through increasing complement-mediated attack, improving phagocytic killing activity of neutrophils, and preventing bacterial adherence to lung epithelial cells.

Complement in removal of the dead - balancing inflammation.

Recognition and removal of apoptotic and necrotic cells must be efficient and highly controlled to avoid excessive inflammation and autoimmune responses to self. The complement system, a crucial part of innate immunity, plays an important role in this process. Thus, apoptotic and necrotic cells are recognized by complement initiators such as C1q, mannose binding lectin, ficolins, and properdin. This triggers complement activation and opsonization of cells with fragments of C3b, which enhances phagocytosis and thus ensures silent removal. Importantly, the process is tightly controlled by the binding of complement inhibitors C4b-binding protein and factor H, which attenuates late steps of complement activation and inflammation. Furthermore, factor H becomes actively internalized by apoptotic cells, where it catalyzes the cleavage of intracellular C3 to C3b. The intracellularly derived C3b additionally opsonizes the cell surface further supporting safe and fast clearance and thereby aids to prevent autoimmunity. Internalized factor H also binds nucleosomes and directs monocytes into production of anti-inflammatory cytokines upon phagocytosis of such complexes. Disturbances in the complement-mediated clearance of dying cells result in persistence of autoantigens and development of autoimmune diseases like systemic lupus erythematosus, and may also be involved in development of age-related macula degeneration.

The Complement Inhibitor Factor H Generates an Anti-Inflammatory and Tolerogenic State in Monocyte-Derived Dendritic Cells.

The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury, although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation, respectively, attenuating the activity of the C3/C5 convertases and, consequently, avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study, we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly, FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture, resembling immature MoDCs, lower expression of the maturation marker CD83 and the costimulatory molecules CD40, CD80, and CD86, decreased production of key proinflammatory Th1-cytokines (IL-12, TNF-α, IFN-γ, IL-6, and IL-8), and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover, FH-treated MoDCs show low Ag uptake and, when challenged with LPS, display reduced CCR7 expression and chemotactic migration, impaired CD4(+) T cell alloproliferation, inhibition of IFN-γ secretion by the allostimulated T cells, and, conversely, induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus, this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity, transplantation, and autoimmunity.

Yersinia pestis uses the Ail outer membrane protein to recruit vitronectin.

Yersinia pestis, the agent of plague, requires the Ail (attachment invasion locus) outer membrane protein to survive in the blood and tissues of its mammalian hosts. Ail is important for both attachment to host cells and for resistance to complement-dependent bacteriolysis. Previous studies have shown that Ail interacts with components of the extracellular matrix, including fibronectin, laminin and heparan sulfate proteoglycans, and with the complement inhibitor C4b-binding protein. Here, we demonstrate that Ail-expressing Y. pestis strains bind vitronectin - a host protein with functions in cell attachment, fibrinolysis and inhibition of the complement system. The Ail-dependent recruitment of vitronectin resulted in efficient cleavage of vitronectin by the outer membrane Pla (plasminogen activator protease). Escherichia coli DH5α expressing Y. pestis Ail bound vitronectin, but not heat-treated vitronectin. The ability of Ail to directly bind vitronectin was demonstrated by ELISA using purified refolded Ail in nanodiscs.

A Novel Interaction between Complement Inhibitor C4b-binding Protein and Plasminogen That Enhances Plasminogen Activation

The complement, coagulation, and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross-talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to Streptococcus pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the β-chain and protein S binds plasminogen much stronger than the main isoform containing the β-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the β-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, whereas the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation.

Binding of Complement Inhibitor C4b-binding Protein to a Highly Virulent Streptococcus pyogenes M1 Strain Is Mediated by Protein H and Enhances Adhesion to and Invasion of Endothelial Cells

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.

Annexin A2 and A5 serve as new ligands for C1q on apoptotic cells

C1q is the initiator of the classical complement pathway and opsonizes apoptotic cells to facilitate phagocytosis. Deficiency of C1q is the strongest known risk factor for development of systemic lupus erythematosus (SLE), which appears to be related to ensuing impaired clearance of apoptotic material. The objective of the current study was to investigate new ligands for C1q on the surface of apoptotic cells. We revealed that the two phospholipid-binding proteins annexin A2 and A5 are, beside DNA, significant C1q ligands. We furthermore, demonstrated that C1q binds directly to histones exposed on the surface of dying cells but we did not detect significant interaction with phosphatidylserine. The complement inhibitors C4b-binding protein and factor H also interact with dying cells, most likely to decrease complement activation beyond the level of C3 to allow noninflammatory clearance. Despite the fact that C4b-binding protein, factor H, and C1q share some ligands on dying cells, we showed that these three proteins did not compete with one another for binding to apoptotic cells. We additionally demonstrated that the way in which apoptosis is induced influenced both the degree of apoptosis and the binding of C1q. The knowledge, that annexin A2 and A5 act as ligands for C1q on apoptotic cells, sheds new light on the pathophysiology of autoimmune diseases.

Complement inhibitor C4b-binding protein is involved in protein AA amyloidosis

Complement inhibitor C4b-binding protein is involved in protein AA amyloidosis

Annexin A2 and A5 Serve as New Ligands for C1q on Apoptotic Cells

C1q is the initiator of the classical complement pathway and opsonizes apoptotic cells to facilitate phagocytosis. Deficiency of C1q is the strongest known risk factor for development of systemic lupus erythematosus (SLE), which appears to be related to ensuing impaired clearance of apoptotic material. The objective of the current study was to investigate new ligands for C1q on the surface of apoptotic cells. We revealed that the two phospholipid-binding proteins annexin A2 and A5 are, beside DNA, significant C1q ligands. We furthermore, demonstrated that C1q binds directly to histones exposed on the surface of dying cells but we did not detect significant interaction with phosphatidylserine. The complement inhibitors C4b-binding protein and factor H also interact with dying cells, most likely to decrease complement activation beyond the level of C3 to allow noninflammatory clearance. Despite the fact that C4b-binding protein, factor H, and C1q share some ligands on dying cells, we showed that these three proteins did not compete with one another for binding to apoptotic cells. We additionally demonstrated that the way in which apoptosis is induced influenced both the degree of apoptosis and the binding of C1q. The knowledge, that annexin A2 and A5 act as ligands for C1q on apoptotic cells, sheds new light on the pathophysiology of autoimmune diseases.

PRELP Protein Inhibits the Formation of the Complement Membrane Attack Complex

PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA), the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b-binding protein, which was suggested to locally down-regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3-convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins.

Islet Amyloid Polypeptide Triggers Limited Complement Activation and Binds Complement Inhibitor C4b-binding Protein, Which Enhances Fibril Formation

Islet amyloid polypeptide (IAPP) is synthesized in pancreatic β-cells and co-secreted with insulin. Aggregation and formation of IAPP-amyloid play a critical role in β-cell death in type 2 diabetic patients. Because Aβ-fibrils in Alzheimer disease activate the complement system, we have here investigated specific interactions between IAPP and complement factors. IAPP fibrils triggered limited activation of complement in vitro, involving both the classical and the alternative pathways. Direct binding assays confirmed that IAPP fibrils interact with globular head domains of complement initiator C1q. Furthermore, IAPP also bound complement inhibitors factor H and C4b-binding protein (C4BP). Recombinant C4BP mutants were used to show that complement control protein (CCP) domains 8 and 2 of the α-chain were responsible for the strong, hydrophobic binding of C4BP to IAPP. Immunostaining of pancreatic sections from type 2 diabetic patients revealed the presence of complement factors in the islets and varying degree of co-localization between IAPP fibrils and C1q, C3d, as well as C4BP and factor H but not membrane attack complex. Furthermore, C4BP enhanced formation of IAPP fibrils in vitro. We conclude that C4BP binds to IAPP thereby limiting complement activation and may be enhancing formation of IAPP fibrils from cytotoxic oligomers.

Functional Recruitment of Human Complement Inhibitor C4b-Binding Protein to Outer Membrane Protein Rck of Salmonella

Resistance to complement mediated killing, or serum resistance, is a common trait of pathogenic bacteria. Rck is a 17 kDa outer membrane protein encoded on the virulence plasmid of Salmonella enterica serovars Typhimurium and Enteritidis. When expressed in either E. coli or S. enterica Typhimurium, Rck confers LPS-independent serum resistance as well as the ability to bind to and invade mammalian cells. Having recently shown that Rck binds the inhibitor of the alternative pathway of complement, factor H (fH), we hypothesized that Rck can also bind the inhibitor of the classical and lectin pathways, C4b-binding protein (C4BP). Using flow cytometry and direct binding assays, we demonstrate that E. coli expressing Rck binds C4BP from heat-inactivated serum and by using the purified protein. No binding was detected in the absence of Rck expression. C4BP bound to Rck is functional, as we observed factor I-mediated cleavage of C4b in cofactor assays. In competition assays, binding of radiolabeled C4BP to Rck was reduced by increasing concentrations of unlabeled protein. No effect was observed by increasing heparin or salt concentrations, suggesting mainly non-ionic interactions. Reduced binding of C4BP mutants lacking complement control protein domains (CCPs) 7 or 8 was observed compared to wt C4BP, suggesting that these CCPs are involved in Rck binding. While these findings are restricted to Rck expression in E. coli, these data suggest that C4BP binding may be an additional mechanism of Rck-mediated complement resistance.

NC4 Domain of Cartilage-specific Collagen IX Inhibits Complement Directly Due to Attenuation of Membrane Attack Formation and Indirectly through Binding and Enhancing Activity of Complement Inhibitors C4B-binding Protein and Factor H

Collagen IX containing the N-terminal noncollagenous domain 4 (NC4) is unique to cartilage and a member of the family of fibril-associated collagens with both collagenous and noncollagenous domains. Collagen IX is located at the surface of fibrils formed by collagen II and a minor proportion of collagen XI, playing roles in tissue stability and integrity. The NC4 domain projects out from the fibril surface and provides sites for interaction with other matrix components such as cartilage oligomeric matrix protein, matrilins, fibromodulin, and osteoadherin. Fragmentation of collagen IX and loss of the NC4 domain are early events in cartilage degradation in joint diseases that precedes major damage of collagen II fibrils. Our results demonstrate that NC4 can function as a novel inhibitor of the complement system able to bind C4, C3, and C9 and to directly inhibit C9 polymerization and assembly of the lytic membrane attack complex. NC4 also binds the complement inhibitors C4b-binding protein and factor H and enhances their cofactor activity in degradation of activated complement components C4b and C3b. NC4 interactions with fibromodulin and osteoadherin inhibited binding to C1q and complement activation by these proteins. Taken together, our results suggest that collagen IX and its interactions with matrix components are important parts of a machinery that protects the cartilage from complement activation and chronic inflammation seen in diseases like rheumatoid arthritis.

Islet amyloid polypeptide triggers limited complement activation and binds complement inhibitor C4b-binding protein, which enhances fibril formation

Islet amyloid polypeptide (IAPP) is synthesized in pancreatic β-cells and co-secreted with insulin. Aggregation and formation of IAPP-amyloid play a critical role in β-cell death in type 2 diabetic patients. Because Aβ-fibrils in Alzheimer disease activate the complement system, we have here investigated specific interactions between IAPP and complement factors. IAPP fibrils triggered limited activation of complement in vitro, involving both the classical and the alternative pathways. Direct binding assays confirmed that IAPP fibrils interact with globular head domains of complement initiator C1q. Furthermore, IAPP also bound complement inhibitors factor H and C4b-binding protein (C4BP). Recombinant C4BP mutants were used to show that complement control protein (CCP) domains 8 and 2 of the α-chain were responsible for the strong, hydrophobic binding of C4BP to IAPP. Immunostaining of pancreatic sections from type 2 diabetic patients revealed the presence of complement factors in the islets and varying degree of co-localization between IAPP fibrils and C1q, C3d, as well as C4BP and factor H but not membrane attack complex. Furthermore, C4BP enhanced formation of IAPP fibrils in vitro. We conclude that C4BP binds to IAPP thereby limiting complement activation and may be enhancing formation of IAPP fibrils from cytotoxic oligomers.