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The complement system is a proteolytic cascade triggered by pathogen and danger-associated molecular patterns, with resultant outcomes of inflammation, cellular activation, and opsonization of material for removal by phagocytosis. While first discovered as an activity in serum, it is now recognized that complement components play important roles at local and individual cell-intrinsic levels. In particular, apart from the extracellular serum activities of complement, it is now believed that complement also acts intracellularly, as part of a cellular signal transduction cascade that can stimulate cellular survival and activation, and individual immune cell phenotypes, via effects on cellular metabolism. This review will describe what is currently known about how complement functions in intracellular signal transduction, and outline the functional advantages of a compartmentalized and intracellular complement system., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)

The term "intracellular complement" has been introduced recently as an umbrella term to distinguish functions of complement proteins that take place intracellularly, rather than in the extracellular environment. However, this rather undefined term leaves some confusion as to the classification of what intracellular complement really is, and as to which intracellular compartment(s) it should refer to. In this review, we will describe the evidence for both canonical and non-canonical functions of intracellular complement proteins, as well as the current controversies and unanswered questions as to the nature of the intracellular complement. We also suggest new terms to facilitate the accurate description and discussion of specific forms of intracellular complement and call for future experiments that will be required to provide more definitive evidence and a better understanding of the mechanisms of intracellular complement activity., (© 2022 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.)

Platelet transfusion refractoriness is a serious clinical concern that complicates the management of thrombocytopenic patients. Previous studies have suggested a potential role for both complement and platelet activation based on in vitro analyses of platelet concentrates. In this study, the post-transfusion platelet response, as indicated by the corrected count increment at 1 and 24 h after prophylactic platelet transfusions, respectively, was correlated with the 1 h post-transfusion Δconcentration (1 h post-transfusion - pretransfusion) of complement and platelet activation biomarkers. The study was registered as a clinical trial at ClinicalTrials.gov (identifier: NCT02601131) and patients were recruited during inpatient care in the hematological department. Soluble terminal complement complexes, soluble P-selectin and soluble CD40 ligand were analyzed. Confirmed alloimmunized patients were excluded. Included subjects were either given platelet transfusions (n = 43) and categorized into four clinical study groups or included in a non-transfused control group (n = 10). In total, 54 transfusions were included. No transfusion-mediated complement activation was observed. The transfusions were associated with a significant increase in the concentration of soluble P-selectin ( p < .001), primarily corresponding to the passive infusion of soluble P-selectin-containing plasma residuals. The Δconcentration of soluble P-selectin was, however, not significantly correlated with the corrected count increments. Thus, significant correlations between biomarkers of complement and platelet activation and the post-transfusion platelet response could not be demonstrated in this study.

Components of the extracellular matrix (ECM), when exposed to body fluids may promote local complement activation and inflammation. Pathologic complement activation at the glomerular basement membrane and at the Bruch's membrane is implicated in renal and eye diseases, respectively. Binding of soluble complement inhibitors to the ECM, including factor H (FH), is important to prevent excessive complement activation. Since the FH-related (FHR) proteins FHR1 and FHR5 are also implicated in these diseases, our aim was to study whether these FHRs can also bind to ECM components and affect local FH activity and complement activation. Both FH and the FHRs showed variable binding to ECM components. We identified laminin, fibromodulin, osteoadherin and PRELP as ligands of FHR1 and FHR5, and found that FHR1 bound to these ECM components through its C-terminal complement control protein (CCP) domains 4-5, whereas FHR5 bound via its middle region, CCPs 3-7. Aggrecan, biglycan and decorin did not bind FH, FHR1 and FHR5. FHR5 also bound to immobilized C3b, a model of surface-deposited C3b, via CCPs 3-7. By contrast, soluble C3, C3(H 2 O), and the C3 fragments C3b, iC3b and C3d bound to CCPs 8-9 of FHR5. Properdin, which was previously described to bind via CCPs 1-2 to FHR5, did not bind in its physiologically occurring serum forms in our assays. FHR1 and FHR5 inhibited the binding of FH to the identified ECM proteins in a dose-dependent manner, which resulted in reduced FH cofactor activity. Moreover, both FHR1 and FHR5 enhanced alternative complement pathway activation on immobilized ECM proteins when exposed to human serum, resulting in the increased deposition of C3-fragments, factor B and C5b-9. Thus, our results identify novel ECM ligands of FH family proteins and indicate that FHR1 and FHR5 are competitive inhibitors of FH on ECM and, when bound to these ligands, they may enhance local complement activation and promote inflammation under pathological conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Papp, Papp, Uzonyi, Cserhalmi, Csincsi, Szabó, Bánlaki, Ermert, Prohászka, Erdei, Ferreira, Blom and Józsi.)

Gram-negative bacteria from the genus Acinetobacter are responsible for life-threating hospital-related infections such as pneumonia, septicemia, and meningitis, especially in immunocompromised patients. Worryingly, Acinetobacter have become multi- and extensively drug resistant (MDR/XDR) over the last few decades. The complement system is the first line of defense against microbes, thus it is highly important to increase our understanding of evasion mechanisms used by Acinetobacter spp. Here, we studied clinical isolates of Acinetobacter spp. (n=50), aiming to characterize their recognition by the complement system. Most isolates tested survived 1 h incubation in 30% serum, and only 8 isolates had a lower survival rate, yet none of those isolates were fully killed. Intriguingly, four isolates survived in human whole blood containing all cell component. Their survival was, however, significantly reduced. Flow cytometry analyses revealed that most of the isolates were detected by human IgG and IgM. Interestingly, we could not detect any significant concentration of deposited C1q, despite observing C4b deposition that was abolished in C1q-deficient serum, indicating transient binding of C1q to bacteria. Moreover, several isolates were recognized by MBL, with C4b deposition abolished in MBL-deficient serum. C3b was deposited on most isolates, but this was not, however, seen with respect to C5b and formation of the membrane attack complex (MAC), indicating that many isolates could avoid complement-mediated lysis. India ink staining showed that isolates were capsulated, and capsule thickness varied significantly between isolates. Studies performed on a wild-type strain and capsule mutant strains, demonstrated that the production of a capsular polysaccharide is one mechanism that mediates resistance to complement-mediated bactericidal activity by preventing MAC deposition and lysis. Our data showed that most clinical Acinetobacter spp. isolates are highly serum resistant despite being efficiently recognized by the complement system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Magda, Bettoni, Laabei, Fairley, Russo, Riesbeck and Blom.)

The complement system is a well-characterized cascade of extracellular serum proteins that is activated by pathogens and unwanted waste material. Products of activated complement signal to the host cells via cell surface receptors, eliciting responses such as removal of the stimulus by phagocytosis. The complement system therefore functions as a warning system, resulting in removal of unwanted material. This review describes how extracellular activation of the complement system can also trigger autophagic responses within cells, up-regulating protective homeostatic autophagy in response to perceived stress, but also initiating targeted anti-microbial autophagy in order to kill intracellular cytoinvasive pathogens. In particular, we will focus on recent discoveries that indicate that complement may also have roles in detection and autophagy-mediated disposal of unwanted materials within the intracellular environment. We therefore summarize the current evidence for complement involvement in autophagy, both by transducing signals across the cell membrane, as well as roles within the cellular environment. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Rituximab is a pioneering anti-CD20 monoclonal antibody that became the first-line drug used in immunotherapy of B-cell malignancies over the last twenty years. Rituximab activates the complement system in vitro , but there is an ongoing debate on the exact role of this effector mechanism in therapeutic effect. Results of both  in vitro  and  in vivo  studies are model-dependent and preclude clear clinical conclusions. Additional confounding factors like complement inhibition by tumor cells, loss of target antigen and complement depletion due to excessively applied immunotherapeutics, intrapersonal variability in the concentration of main complement components and differences in tumor burden all suggest that a personalized approach is the best strategy for optimization of rituximab dosage and therapeutic schedule. Herein we critically review the existing knowledge in support of such concept and present original data on markers of complement activation, complement consumption, and rituximab accumulation in plasma of patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphomas (NHL). The increase of markers such as C4d and terminal complement complex (TCC) suggest the strongest complement activation after the first administration of rituximab, but not indicative of clinical outcome in patients receiving rituximab in combination with chemotherapy. Both ELISA and complement-dependent cytotoxicity (CDC) functional assay showed that a substantial number of patients accumulate rituximab to the extent that consecutive infusions do not improve the cytotoxic capacity of their sera. Our data suggest that individual assessment of CDC activity and rituximab concentration in plasma may support clinicians' decisions on further drug infusions, or instead prescribing a therapy with anti-CD20 antibodies like obinutuzumab that more efficiently activate effector mechanisms other than complement., (Copyright © 2020 Felberg, Taszner, Urban, Majeranowski, Jaskuła, Jurkiewicz, Stasiłojć, Blom, Zaucha and Okrój.)

Monoclonal antibodies ofatumumab (anti-CD20) and alemtuzumab (anti-CD52) which are approved for usage in patients with chronic lymphocytic leukemia (CLL), efficiently activate the classical complement pathway. However complement is an exhaustible component and high doses of its activators may deplete complement-dependent cytotoxicity (CDC) potential, thus reducing the effect of repeated mAb dosing. Widely used method to measure CDC activity of patients' serum is hemolytic assay (CH50) on sheep erythrocytes. Despite its simplicity, such CH50 assay may not reflect pivotal interactions between patient serum and human complement inhibitors on the surface of target cells. We propose calcein release assay performed on tumor cells similar to those targeted by therapeutic antibodies as an alternative method. We analyzed serum samples collected from 12 patients participating in the clinical study, receiving s.c. 30 mg alemtuzumab three times per week combined with i.v. ofatumumab at an initial dose of 300 mg in week 3 further escalated to 2000 mg every other week. All serum samples were measured by hemolytic assay on sheep erythrocytes as well as using calcein release assay on CD20-positive Raji cells. Our data show that results obtained from both assays are related to each other at the level of the whole group (n = 96 samples, Spearman r = 0.504, p < .001) but may substantially differ when analyzing individual patients. Furthermore, by using CDC assay on Raji cells, we found that in the presented clinical study CDC serum potential was not significantly affected when measured before consecutive administrations in most of the patients., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The complement system resembles a double-edged sword since its activation can either benefit or harm the host. Thus, regulation of this system is of utmost importance and performed by several circulating and membrane-bound complement inhibitors. The pool of well-established regulators has recently been enriched with proteins that either share structural homology to known complement inhibitors such as Sushi domain-containing (SUSD) protein family and Human CUB and Sushi multiple domains (CSMD) families or extracellular matrix (ECM) macromolecules that interact with and modulate complement activity. In this review, we summarize the current knowledge about newly discovered complement inhibitors and discuss their implications in complement regulation, as well as in processes beyond complement regulation such cancer development. Understanding the behavior of these proteins will introduce new mechanisms of complement regulation and may provide new avenues in the development of novel therapies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Complement activation is associated with common rheumatic diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic vasculitis. Evidence linking complement activation to these diseases includes the presence of complement deposition in affected tissues, decreased levels of complement proteins and high levels of complement activation fragments in the blood and/or synovial fluid of patients with these diseases, as well as data from experimental models. Eculizumab, a monoclonal antibody that inhibits the complement component C5, is now approved for the treatment of rare conditions involving complement hyperactivation, and the success of this therapy has renewed interest in understanding the utility of complement inhibition in rheumatological practice, particularly for SLE. For example, inhibiting C5 is a potential means of reducing glomerular inflammation in lupus nephritis or treating thrombotic microangiopathy in SLE. The complement system is one of multiple mediators of tissue injury in complex diseases such as SLE, and identifying the disease context in which complement activation has a predominant role is a challenge. An added difficulty in RA is identifying a role for therapeutic complement inhibition within the diverse treatment modalities already available. In this Review, evidence for the therapeutic potential of complement manipulation in rheumatology practice is evaluated.

The complement system is an arm of innate immunity that aids in the removal of pathogens and dying cells. Due to its harmful, pro-inflammatory potential, complement is controlled by several soluble and membrane-bound inhibitors. This family of complement regulators has been recently extended by the discovery of several new members, and it is becoming apparent that these proteins harbour additional functions. In this review, the current state of knowledge of the physiological functions of four complement regulators will be described: cartilage oligomeric matrix protein (COMP), CUB and sushi multiple domains 1 (CSMD1), sushi domain-containing protein 4 (SUSD4) and CD59. Complement activation is involved in both the development of and defence against cancer. COMP expression is pro-oncogenic, whereas CSMD1 and SUSD4 act as tumour suppressors. These effects may be related in part to the complex influence of complement on cancer but also depend on unrelated functions such as the protection of cells from endoplasmic reticulum stress conveyed by intracellular COMP. CD59 is the main inhibitor of the membrane attack complex, and its deficiency leads to complement attack on erythrocytes and severe haemolytic anaemia, which is now amenable to treatment with an inhibitor of C5 cleavage. Unexpectedly, the intracellular pool of CD59 is crucial for insulin secretion from pancreatic β-cells. This finding is one of several relating to the intracellular functions of complement proteins, which until recently were only considered to be present in the extracellular space. Understanding the alternative functions of complement inhibitors may unravel unexpected links between complement and other physiological systems, but is also important for better design of therapeutic complement inhibition., (© 2017 The Association for the Publication of the Journal of Internal Medicine.)

Aim: Complement-mediated atypical haemolytic uraemic syndrome (aHUS) is a rare disease with high mortality and morbidity if left untreated. The diagnostic work-up is complicated and the manifestations overlap with other conditions. Therefore, we hypothesize that complement-mediated aHUS is an under diagnosed disease., Methods: A cohort of 768 referrals referred to the Coagulation Unit in Malmo, Sweden, for analysis of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), 2007-2012, were retrospectively reviewed. Subjects were included on the basis of presence of haemolytic anaemia, thrombocytopaenia, renal failure and ADAMTS13 > 0.05. They were excluded if tested positive for Escherichia coli. Included subjects were categorized as "suspected HUS" with and without potential causes and triggers. Levels of C3 and C4, presence of complement factor H (CFH)-specific antibodies and associated deficiency in complement factor H related protein 1 (CFHR1) were analyzed on frozen samples., Results: In total, 134/316 (42%) unique subjects fulfilled inclusion criteria; 103 were categorized as "suspected HUS associated with potential causes/triggers" and 31 subjects categorized as "suspected HUS" without such association. One case of complement-mediated aHUS had been confirmed during the treatment period. Laboratory analyses performed showed that in total 78 cases had findings consistent with complement-mediated aHUS: 24 cases indicated presence of CFH-specific antibodies whereof five cases had isolated low C3 titres and six cases had deficiency of CFHR1. Additionally 54 cases indicated isolated alternative pathway consumption., Conclusion: The results suggest that the presence of complement-mediated aHUS was under diagnosed in this cohort calling for improvement of diagnostic availability., (© 2016 Asian Pacific Society of Nephrology.)

Type 2 Diabetes (T2D) is a disease of increasing importance and represents a growing burden on global healthcare and human health. In T2D, loss of effectiveness of insulin signaling in peripheral tissues cannot be compensated for by adequate insulin secretion, leading to hyperglycemia and resultant complications. In recent years, inflammation has been identified as a central component of T2D, both in inducing peripheral insulin resistance as well as in the pancreatic islet, where it contributes to loss of insulin secretion and death of insulin-secreting beta cells. In this review we will focus on non-traditional roles of complement proteins which have been identified in T2D-associated inflammation, beta cell secretory function, and in maintaining homeostasis of the pancreatic islet. Improved understanding of both traditional and novel roles of complement proteins in T2D may lead to new therapeutic approaches for this global disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Joint diseases are often characterized by inflammatory processes that result in pathological changes in joint tissues, including cartilage degradation and release of components into the synovial fluid. The complement system plays a central role in promoting the inflammation. Because several cartilage proteins are known to interact with complement, causing either activation or inhibition of the system, we aimed to investigate these interactions comprehensively. Bovine cartilage explants were cultured with IL-1α to induce cartilage degradation, followed by incubation with human serum. Label-free selected reaction monitoring mass spectrometry was used to specifically quantify complement proteins interacting with the cartilage explant. In parallel, the time-dependent degradation of cartilage was detected using mass spectrometry analysis (liquid chromatography-tandem mass spectrometry). Complement proteins resulting from activation of the classical, alternative, and terminal pathways were detected on IL-1α-stimulated cartilage at time points when clear alterations in extracellular matrix composition had occurred. Increased levels of the complement activation product C4d, as detected by ELISA in serum after incubation with IL-1α-stimulated cartilage, confirmed the selected reaction monitoring results indicating complement activation. Further, typical activated (cleaved) C3 fragments were detected by Western blotting in extracts of IL-1α-stimulated cartilage. No complement activation was triggered by cartilage cultured in the absence of IL-1α. Components released from IL-1α-stimulated cartilage during culture had an inhibitory effect on complement activation. These were released after a longer incubation period with IL-1α and may represent a feedback reaction to cartilage-triggered complement activation observed after a shorter incubation period., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Autoantibodies termed C3-nephritic factor (C3NeF), which stabilize convertases of the alternative complement pathway, often stimulate autoinflammatory diseases. However, knowledge about analogous autoantibodies acting on the classical pathway (C4NeF) is limited to a few reports, which indicate association with kidney dysfunction, systemic lupus erythematous, and infections. C4NeF may appear independently from C3NeF, but the lack of a routine diagnostic method predisposes C4NeF for being an underestimated player in autoinflammatory episodes. We tested the activity of classical convertases directly in serum/plasma to screen samples from 13 patients with C3 glomerulopathies and identified one patient showing significantly prolonged half-life of these enzymes. Observed effect was reproduced by immunoglobulins purified from patient's plasma and additionally confirmed on classical convertase built from purified components. Isolated immunoglobulins protected classical convertases from both spontaneous and inhibitor-driven decay but not from C4b proteolysis. The patient had a decreased serum level of C3, elevated sC5b-9, and normal concentrations of factor B and C4. Neither C3NeF nor other autoantibodies directed against alternative pathway proteins (factor H, factor B, factor I, C3, and properdin) were found. Genetic analysis showed no mutations in C3, CFB, CFH, CFI, MCP, THBD, and DGKE genes. Renal biopsy revealed a membranoproliferative pattern with intense C3 deposits. Our results underline the importance of C4NeF as an independent pathogenic factor and a need for the implementation of routine examination of classical convertase activity. Proposed method may enable robust inspection of such atypical cases.

Helicobacter pylori is an important human pathogen and a common cause of peptic ulcers and gastric cancer. Despite H. pylori provoking strong innate and adaptive immune responses, the bacterium is able to successfully establish long-term infections. Vitronectin (Vn), a component of both the extracellular matrix and plasma, is involved in many physiological processes, including regulation of the complement system. The aim of this study was to define a receptor in H. pylori that binds Vn and determine the significance of the interaction for virulence. Surprisingly, by using proteomics, we found that the hydrogen peroxide-neutralizing enzyme catalase KatA is a major Vn-binding protein. Deletion of the katA gene in three different strains resulted in impaired binding of Vn. Recombinant KatA was generated and shown to bind with high affinity to a region between heparin-binding domain 2 and 3 of Vn that differs from previously characterised bacterial binding sites on the molecule. In terms of function, KatA protected H. pylori from complement-mediated killing in a Vn-dependent manner. Taken together, the virulence factor KatA is a Vn-binding protein that moonlights on the surface of H. pylori to promote bacterial evasion of host innate immunity.

Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The complement system plays a major role in the autoimmune disease, systemic lupus erythematosus (SLE). However, the role of complement in SLE is complex since it may both prevent and exacerbate the disease. In this review, we explore the latest findings in complement-focused research in SLE. C1q deficiency is the strongest genetic risk factor for SLE, although such deficiency is very rare. Various recently discovered genetic associations include mutations in the complement receptors 2 and 3 as well as complement inhibitors, the latter related to earlier onset of nephritis. Further, autoantibodies are a distinct feature of SLE that are produced as the result of an adaptive immune response and how complement can affect that response is also being reviewed. SLE generates numerous disease manifestations involving contributions from complement such as glomerulonephritis and the increased risk of thrombosis. Furthermore, since most of the complement system is present in plasma, complement is very accessible and may be suitable as biomarker for diagnosis or monitoring of disease activity. This review highlights the many roles of complement for SLE pathogenesis and how research has progressed during recent years., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Type 2 diabetes is triggered by reduced insulin production, caused by genetic and environmental factors such as inflammation originating from the innate immune system. Complement proteins are a component of innate immunity and kill non-self cells by perforating the plasma membrane, a reaction prevented by CD59. Human pancreatic islets express CD59 at very high levels. CD59 is primarily known as a plasma membrane protein in membrane rafts, but most CD59 protein in pancreatic β cells is intracellular. Removing extracellular CD59 disrupts membrane rafts and moderately stimulates insulin secretion, whereas silencing intracellular CD59 markedly suppresses regulated secretion by exocytosis, as demonstrated by TIRF imaging. CD59 interacts with the exocytotic proteins VAMP2 and Syntaxin-1. CD59 expression is reduced by glucose and in rodent diabetes models but upregulated in human diabetic islets, potentially reflecting compensatory reactions. This unconventional action of CD59 broadens the established view of innate immunity in type 2 diabetes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system., (© 2013 S. Karger AG, Basel.)

Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP) domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint.

Rheumatoid arthritis (RA) is a highly disabling disease affecting all structures of the joint. Understanding the pathology behind the development of RA is essential for developing targeted therapeutic strategies as well as for developing novel markers to predict disease onset. Several molecules normally hidden within the cartilage tissue are exposed to complement components in the synovial fluid upon cartilage breakdown. Some of these have been shown to activate complement and toll-like receptors, which may enhance an already existing inflammatory response, thereby worsening the course of disease. Other cartilage-resident molecules have in contrast shown to possess complement-inhibitory properties. Knowledge about mechanisms behind pathological complement activation in the joints will hopefully lead to methods which allow us to distinguish patients with pathological complement activation from those where other inflammatory pathways are predominant. This will help to elucidate which patients will benefit from complement inhibitory therapies, which are thought to aid a specific subset of patients or patients at a certain stage of disease. Future challenges are to target the complement inhibition specifically to the joints to minimize systemic complement blockade., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Inter alpha inhibitor (IαI) is an abundant serum protein consisting of three polypeptides: two heavy chains (HC1 and HC2) and bikunin, a broad-specificity Kunitz-type proteinase inhibitor. The complex is covalently held together by chondroitin sulfate but during inflammation IαI may interact with TNF-stimulated gene 6 protein (TSG-6), which supports transesterification of heavy chains to hyaluronan. Recently, IαI was shown to inhibit mouse complement in vivo and to protect from complement-mediated lung injury but the mechanism of such activity was not elucidated. Using human serum depleted from IαI, we found that IαI is not an essential human complement inhibitor as was reported for mice and that such serum has unaltered hemolytic activity. However, purified human IαI inhibited classical, lectin and alternative complement pathways in vitro when added in excess to human serum. The inhibitory activity was dependent on heavy chains but not bikunin and detected at the level of initiating molecules (MBL, properdin) in the lectin/alternative pathways or C4b in the classical pathway. Furthermore, IαI affected formation and assembly of the C1 complex and prevented assembly of the classical pathway C3-convertase. Presence and putative interactions with TSG-6 did not affect the ability of IαI to inhibit complement thus implicating IαI as a potentially important complement inhibitor once enriched onto hyaluronan moieties in the course of local inflammatory processes. In support of this, we found a correlation between IαI/HC-containing proteins and hemolytic activity of synovial fluid from patients suffering from rheumatoid arthritis.

The complement system is key to innate immunity and its activation is necessary for the clearance of bacteria and apoptotic cells. However, insufficient or excessive complement activation will lead to immune-related diseases. It is so far unknown how the complement activity is up- or down- regulated and what the associated pathophysiological mechanisms are. To quantitatively understand the modulatory mechanisms of the complement system, we built a computational model involving the enhancement and suppression mechanisms that regulate complement activity. Our model consists of a large system of Ordinary Differential Equations (ODEs) accompanied by a dynamic Bayesian network as a probabilistic approximation of the ODE dynamics. Applying Bayesian inference techniques, this approximation was used to perform parameter estimation and sensitivity analysis. Our combined computational and experimental study showed that the antimicrobial response is sensitive to changes in pH and calcium levels, which determines the strength of the crosstalk between CRP and L-ficolin. Our study also revealed differential regulatory effects of C4BP. While C4BP delays but does not decrease the classical complement activation, it attenuates but does not significantly delay the lectin pathway activation. We also found that the major inhibitory role of C4BP is to facilitate the decay of C3 convertase. In summary, the present work elucidates the regulatory mechanisms of the complement system and demonstrates how the bio-pathway machinery maintains the balance between activation and inhibition. The insights we have gained could contribute to the development of therapies targeting the complement system.

Objective: Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage, where it catalyzes collagen fibrillogenesis. Elevated amounts of COMP are found in serum during increased turnover of cartilage associated with active joint disease, such as rheumatoid arthritis (RA) and osteoarthritis (OA). This study was undertaken to investigate the ability of COMP to regulate complement, a capacity that has previously been shown for some other cartilage proteins., Methods: Regulation of complement by COMP was studied using functional in vitro assays. Inter-actions between complement proteins and COMP were investigated by direct binding assay and electron microscopy. Circulating COMP and COMP-C3b complexes in serum and synovial fluid from RA and OA patients and healthy controls were measured with a novel enzyme-linked immunosorbent assay., Results: We found in vivo evidence of complement activation by released COMP in the general circulation of patients with RA, but not patients with OA. COMP induced activation and deposition of C3b and C9 specifically via the alternative pathway of complement, which was attributable to direct interaction between COMP and properdin. Furthermore, COMP inhibited the classical and the lectin complement pathways due to direct interaction with the stalk region of C1q and mannose-binding lectin, respectively., Conclusion: COMP is the first extracellular matrix protein for which an active role in inflammation has been demonstrated in vivo. It can activate one complement pathway at the same time as it has the potential to inhibit another. The net outcome of these interactions is most likely determined by the type of released COMP fragments, which may be disease specific., (Copyright © 2010 by the American College of Rheumatology.)

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K(D)) for vitronectin binding to UspA2 was 2.3 x 10(-8) M, and the N-terminal region encompassing residues UspA2 30-170 bound vitronectin with a K(D) of 7.9 x 10(-8) M. Electron microscopy verified that the active binding domain (UspA2(30-177)) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2(30-177) bound to the C-terminal region of vitronectin residues 312-396. Finally, when human serum was pre-incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N-terminal domain of UspA2 and the C-terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis-dependent serum resistance.

The complement system can be specifically targeted to tumor cells due to molecular changes on their surfaces that are recognized by complement directly or via naturally occurring antibodies. However, tumor cells often overexpress membrane-bound complement inhibitors protecting them from complement attack. We have previously shown that non-small cell lung cancer (NSCLC) cells, additionally to membrane-bound inhibitors, produce substantial amounts of soluble regulators such as factor I (FI) and factor H (FH). Since low oxygen concentration is associated with rapidly growing solid tumors, we studied how NSCLC cells protect themselves from complement attack under hypoxic conditions. Unexpectedly, mRNA levels and secretion of both FI and FH were significantly decreased already after 24 h exposure to hypoxia while cell viability measured by XTT assay and annexin V/7-AAD staining was affected only marginally. Furthermore, we observed decrease of mRNA level and loss of membrane-bound complement inhibitor CD46 and increased deposition of early (C3b) and terminal (C9) complement components on hypoxic NSCLC cells. All three complement pathways (classical, lectin and alternative) were employed to deposit C3b on cell surface. Taken together, our results imply that under hypoxic conditions NSCLC give up some of their available defense mechanisms and become more prone to complement attack.

This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.

Infestation of skin by the parasitic itch mite Sarcoptes scabiei afflicts 300 million people worldwide and there is a need for novel and efficient therapies. We have previously identified a multigene family of serine proteases comprising multiple catalytically inactive members (scabies mite-inactivated protease paralogs (SMIPPs)), which are secreted into the gut of S. scabiei. SMIPPs are located in the mite gut and in feces excreted into the upper epidermis. Scabies mites feed on epidermal protein, including host plasma; consequently, they are exposed to host defense mechanisms both internally and externally. We found that two recombinantly expressed SMIPPs inhibited all three pathways of the human complement system. Both SMIPPs exerted their inhibitory action due to binding of three molecules involved in the three different mechanisms which initiate complement: C1q, mannose-binding lectin, and properdin. Both SMIPPs bound to the stalk domains of C1q, possibly displacing or inhibiting C1r/C1s, which are associated with the same domain. Furthermore, we found that binding of both SMIPPs to properdin resulted in prevention of assembly of the alternative pathway convertases. However, the SMIPPs were not able to dissociate already formed convertases. Immunohistochemical staining demonstrated the presence of C1q in the gut of scabies mites in skin burrows. We propose that SMIPPs minimize complement-mediated gut damage and thus create a favorable environment for the scabies mites.

Complement is part of the innate immune defence and not only recognizes microbes but also unwanted host molecules to enhance phagocytosis and clearance. This process of opsonisation must be tightly regulated to prevent immunopathology. Endogenous ligands such as dying cells, extracellular matrix proteins, pentraxins, amyloid deposits, prions and DNA, all bind the complement activator C1q, but also interact with complement inhibitors C4b-binding protein and factor H. This contrasts to the interaction between C1q and immune complexes, in which case no inhibitors bind, resulting in full complement activation. Disturbances to the complement regulation on endogenous ligands can lead to diseases such as age-related macular degeneration, neurological and rheumatic disorders. A thorough understanding of these processes might be crucial to developing new therapeutic strategies.

Rhesus rhadinovirus (RRV) is currently the closest known, fully sequenced homolog of human Kaposi sarcoma-associated herpesvirus. Both these viruses encode complement inhibitors as follows: Kaposi sarcoma-associated herpesvirus-complement control protein (KCP) and RRV-complement control protein (RCP). Previously we characterized in detail the functional properties of KCP as a complement inhibitor. Here, we performed comparative analyses for two variants of RCP protein, encoded by RRV strains H26-95 and 17577. Both RCP variants and KCP inhibited human and rhesus complement when tested in hemolytic assays measuring all steps of activation via the classical and the alternative pathway. RCP variants from both RRV strains supported C3b and C4b degradation by factor I and decay acceleration of the classical C3 convertase, similar to KCP. Additionally, the 17577 RCP variant accelerated decay of the alternative C3 convertase, which was not seen for KCP. In contrast to KCP, RCP showed no affinity to heparin and is the first described complement inhibitor in which the binding site for C3b/C4b does not interact with heparin. Molecular modeling shows a structural disruption in the region of RCP that corresponds to the KCP-heparin-binding site. This makes RRV a superior model for future in vivo investigations of complement evasion, as RCP does not play a supportive role in viral attachment as KCP does.

Pathogenic microbes acquire human complement inhibitors to circumvent the innate immune system. In this study, we identify two novel host-pathogen interactions, factor H (FH) and factor H-like protein 1 (FHL-1), the inhibitors of the alternative pathway that binds to Hib. A collection of clinical Haemophilus influenzae isolates was tested and the majority of encapsulated and unencapsulated bound FH. The isolate Hib 541 with a particularly high FH-binding was selected for detailed analysis. An increased survival in normal human serum was observed with Hib 541 as compared with the low FH-binding Hib 568. Interestingly, two binding domains were identified within FH; one binding site common to both FH and FHL-1 was located in the N-terminal short consensus repeat domains 6-7, whereas the other, specific for FH, was located in the C-terminal short consensus repeat domains 18-20. Importantly, both FH and FHL-1, when bound to the surface of Hib 541, retained cofactor activity as determined by analysis of C3b degradation. Two H. influenzae outer membrane proteins of approximately 32 and 40 kDa were detected with radiolabeled FH in Far Western blot. Taken together, in addition to interactions with the classical, lectin, and terminal pathways, H. influenzae interferes with the alternative complement activation pathway by binding FH and FHL-1, and thereby reducing the complement-mediated bactericidal activity resulting in an increased survival. In contrast to incubation with active complement, H. influenzae had a reduced survival in FH-depleted human serum, thus demonstrating that FH mediates a protective role at the bacterial surface.

Escherichia coli K1 and Group B Streptococcus (GBS) are the most common bacteria that cause meningitis during the neonatal period. Complement, the first line of defence in the host, acts on these bacteria to opsonize with various components of complement for subsequent presentation to phagocytes. To counteract these opsonization effects, E. coli and GBS bind to the complement regulators C4 binding protein and Factor H, respectively. Nonetheless, the deposition of complement components on these two bacteria from neonatal serum and their effect on the host cell interaction is unclear. Here we demonstrated that the deposition of complement proteins from adult serum prevented the invasion of E. coli into human brain microvascular endothelial cells, whereas the invasion of GBS was enhanced. In contrast, treatment with cord serum had no effect on the invasion of both these bacteria. We also examined the effect of the deposited complement proteins on phagocytosis using THP-1 cells and THP-1 cells differentiated into macrophages. Escherichia coli treated with adult serum neither attached nor entered these cells, whereas GBS was phagocytosed and survived efficiently. We further demonstrate that the inhibitory effect of complement proteins is the result of the bound complement inhibitors C4b-binding protein, in the case of E. coli, and Factor H, in the case of GBS. Taken together, these results suggest that E. coli and GBS utilize contrasting mechanisms of complement-mediated interactions with their target cells for successful establishment of disease.

Apoptosis, followed by rapid phagocytic clearance, is the primary mechanism by which organisms dispose of unwanted cells. The intracellular and extracellular composition of an apoptotic cell changes to decrease immunogenicity and enhance its uptake. By changing their extracellular composition, apoptotic cells acquire the capacity to bind complement initiation molecules such as C1q and MBL. Binding of these molecules can lead to complement activation. Membrane bound complement inhibitors are down-regulated during apoptosis, which would leave the cell less protected against complement activation; however, recent data show that fluid-phase complement inhibitors may compensate for this loss of regulation. Importantly, binding of complement is a process that mainly takes place during the late stages of apoptosis. Most cells will be cleared before that stage under steady state conditions, but during overwhelming apoptosis or impaired phagocytosis, apoptotic cells may remain in tissues for a longer time and acquire complement proteins. Based on the data from deficiencies of early complement components and the development of systemic lupus erythematosus with accumulation of dead cells, it is clear that, under certain conditions, apoptotic cells persist, becoming necrotic and overloading the scavenging capacities of the complement system. Although the complement system is also involved in inducing apoptosis in target cells, this review will focus on the role of complement in the clearance of apoptotic cells.

Complement activation contributes to a pathological process in a number of autoimmune and inflammatory diseases, including rheumatoid arthritis (RA). In this review we summarize current knowledge of complement contribution to RA, based on clinical observations in patients and in vivo animal models, as well as on experiments in vitro aiming at elucidation of underlying molecular mechanisms. There is strong evidence that both the classical and the alternative pathways of complement are pathologically activated during RA as well as in animal models for RA. The classical pathway can be initiated by several triggers present in the inflamed joint such as deposited autoantibodies, dying cells, and exposed cartilage proteins such as fibromodulin. B cells producing autoantibodies, which in turn form immune complexes, contribute to RA pathogenesis partly via activation of complement. It appears that anaphylatoxin C5a is the main product of complement activation responsible for tissue damage in RA although deposition of membrane attack complex as well as opsonization with fragments of C3b are also important. Success of complement inhibition in the experimental models described so far encourages novel therapeutic approaches to the treatment of human RA.

Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.

Kaposi's sarcoma-associated human herpesvirus (KSHV) is thought to cause Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Previously, we reported that the KSHV complement control protein (KCP) encoded within the viral genome is a potent regulator of the complement system; it acts both as a cofactor for factor I and accelerates decay of the C3 convertases (Spiller, O. B., Blackbourn, D. J., Mark, L., Proctor, D. G., and Blom, A. M. (2003) J. Biol. Chem. 278, 9283-9289). KCP is a homologue to human complement regulators, being comprised of four complement control protein (CCP) domains. In this, the first study to identify the functional sites of a viral homologue at the amino acid level, we created a three-dimensional homology-based model followed by site-directed mutagenesis to locate complement regulatory sites. Classical pathway regulation, both through decay acceleration and factor I cleavage of C4b, required a cluster of positively charged amino acids in CCP1 stretching into CCP2 (Arg-20, Arg-33, Arg-35, Lys-64, Lys-65, and Lys-88) as well as positively (Lys-131, Lys-133, and His-135) and negatively (Glu-99, Glu-152, and Asp-155) charged areas at opposing faces of the border region between CCPs 2 and 3. The regulation of the alternative pathway (via factor I-mediated C3b cleavage) was found to both overlap with classical pathway regulatory sites (Lys-64, Lys-65, Lys-88 and Lys-131, Lys-133, His-135) as well as require unique, more C-terminal residues in CCPs 3 and 4 (His-158, His-171, and His-213) and CCP 4 (Phe-195, Phe-207, and Leu-209). We show here that KCP has evolved to maintain the spatial structure of its functional sites, especially the positively charged patches, compared with host complement regulators.

The complement system is an important part of innate immunity providing immediate protection against pathogens without a need for previous exposure. Its importance is clearly shown by the fact that patients lacking complement components suffer from fulminant and recurring infections. Complement is an explosive cascade, and in order to control it there are inhibitors present on every human cell and also circulating in blood. However, many infectious agents have developed strategies to prevent clearance and destruction by complement. Some pathogens simply hijack the host's complement inhibitors, while others are able to produce their own homologues of human inhibitors. Knowledge of these mechanisms on a molecular level may aid development of vaccines and novel therapeutic strategies that would be more specific than the use of antibiotics that, apart from causing resistance problems, also affect the normal flora, the outcome of which could be devastating. In this study the structural requirements and functional consequences of interactions between the major soluble inhibitor of complement C4b-binding protein and Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes, Escherichia coli K1, Moraxella catarrhalis and Candida albicans are described. Furthermore, a novel inhibitor produced by Kaposi's sarcoma-associated herpesvirus is identified and characterized in detail: KCP. It is shown that KCP inhibits classical C3-convertase and presents activated complement factors C4b and C3b for destruction by a serine proteinase, factor I. Using molecular modelling and site-directed mutagenesis, it was possible to localize sites on the surface of KCP required for complement inhibition and it is concluded that KCP uses molecular mechanisms identical to human inhibitors.

C4b-binding protein (C4BP) is a potent inhibitor of the classical pathway of complement. Structural analysis of our 3D model of the C4BP alpha-chain suggested that a cluster of positively charged amino acids at the interface between complement control protein (CCP) modules 1 and 2 could be involved in ligand binding. Nine C4BP mutants, where the positively charged amino acids were changed to glutamines, were expressed. We found that all of these displayed lower apparent affinity for C4b and that the site was also a specific heparin-binding site. The mutants demonstrated decreased ability to serve as factor I cofactors in a degradation of C4b. Also, their capacity to prevent the assembly of C3-convertase and to accelerate its decay were decreased. In conclusion, we found a binding site on C4BP that is functionally important for the regulation of C3-convertase. Many strains of Streptococcus pyogenes bind C4BP, via surface M proteins, which plays an important role in pathogenesis. Using the set of C4BP mutants we identified a key recognition surface for M proteins which overlaps with the C4b-binding site. The analysis of all mutants and inhibition with monoclonal antibodies indicated that the binding sites for C4b and M proteins are only overlapping but not identical. Furthermore, we found that the molecular mechanisms involved in these two interactions differ, since the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b-binding. We suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional non-covalent forces cause tight binding of C4BP to streptococcal M proteins.