The YeastCandida albicansBinds Complement Regulators Factor H and FHL-1
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ABSTRACT The human facultative pathogenic yeast Candida albicans causes mucocutaneous infections and is the major cause of opportunistic fungal infections in immunocompromised patients. C. albicans activates both the alternative and classical pathway of the complement system. The aim of this study was to assay whether C. albicans binds human complement regulators in order to control complement activation at its surface. We observed binding of two central complement regulators, factor H and FHL-1, from normal human serum to C. albicans by adsorption assays, immunostaining, and fluorescence-activated cell sorter (FACS) analyses. Specificity of acquisition was further confirmed in direct binding assays with purified proteins. The surface-attached regulators maintained their complement regulatory activities and mediated factor I-dependent cleavage of C3b. Adsorption assays with recombinant deletion mutant proteins were used to identify binding domains. Two binding sites were localized. One binding domain common to both factor H and FHL-1 is located in the N-terminal short consensus repeat domains (SCRs) 6 and 7, and the other one located in C-terminal SCRs 19 and 20 is unique to factor H. These data indicate that by surface acquisition of host complement regulators, the human pathogenic yeast C. albicans is able to regulate alternative complement activation at its surface and to inactivate toxic complement activation products.Keywords:
Complement control protein
Complement factor B
Virulence factor
As an important tool of the innate immune system, the complement system rapidly recognizes and clears invading microbes and host debris. Furthermore, it stimulates B- and T-cell development through cross talks with the adaptive immune system. Complement is activated through specific danger pattern recognition by the classical and lectin pathways, unspecific initiation and amplification by the alternative pathway, resulting in cell lysis by terminal pathway. The classical pathway, lectin and alternative pathways induce opsonization of C3b on targeted cells, which ultimately results in the clearance of targets. The complement system is tightly controlled to avoid unwanted damage of healthy host cells. Over-activation of complement due to mutations in complement proteins or regulators, or the presence of autoantibodies against the regulators, has been linked to diseases like atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD) and paroxysmal nocturnal hemoglobinuria (PNH). ‘Regulators of complement activity’ (RCA) proteins form a group of complement regulators. They down-regulate the activity of the central enzyme complexes of the complement system, i.e. C3 convertases, through two mechanisms: (1) they assist serine protease Factor I to induce degradation of C3b and C4b (cofactor activity) and (2) they accelerate the irreversible dissociation of C3 convertases, C3bBb and C4b2a (decay acceleration activity). Many viruses functionally mimic the host complement-regulator proteins to avoid complement attacks. Investigations on complement regulation elucidated the mechanisms of how regulators bind to C3b. The recent published structures of C3b in complex with factor H (FH), complement receptor 1 (CR1), membrane cofactor protein (MCP), smallpox inhibitor of complement enzymes (SPICE) and decay accelerating factor (DAF) imply that all the regulators utilize a common platform in C3b for binding. However, the molecular mechanisms of complement regulation, especially the activity control of C3 convertases, are still largely unexplored. In this thesis, we investigated the molecular basis of cofactor activity and decay accelerating activity in the alternative pathway, as well as the regulatory mechanisms in the classical pathway, through determining a series crystal structures and performing systematic biochemical and biophysical experiments.
Decay-accelerating factor
Complement control protein
Complement component 2
Paroxysmal nocturnal hemoglobinuria
Complement factor B
Lectin pathway
Properdin
Anaphylatoxin
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Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.
Complement factor B
Complement component 3
Complement component 2
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The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a-to our knowledge-new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite SbiratioC3ratioFactor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and beta(2)-glycoprotein I and interferes with innate immune recognition.
Complement factor B
Complement component 2
C3-convertase
Complement control protein
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Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs). Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I.
C3-convertase
Complement factor B
Complement control protein
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The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.
C3-convertase
iC3b
Complement factor B
Complement control protein
Cleavage (geology)
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Abstract Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.
Complement control protein
Complement factor B
Complement component 2
Poxviridae
Decay-accelerating factor
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Isolated complement components were used to study the regulation of the alternative complement pathway C3 convertase (EC 3.4.21.47), also called C3b,Bb, on M protein-carrying (M+) and M protein-lacking (M-) streptococci. Neither M- nor M+ streptococci directly affected the formation or dissociation of the surface-bound C3b,Bb or the inactivation of surface-bound C3b by factor I. However, the activity of the serum control protein of the alternative complement pathway, factor H, in controlling streptococcus-bound C3b and C3b,Bb was 6-8 times stronger on M+ organisms than on M- organisms. Furthermore, M+ streptococci of different serotypes and purified streptococcal M6 protein were shown to selectively bind factor H, the dissociation constants ranging from 4.5 X 10(-6) M to 6 X 10(-7) M. We conclude that the antiphagocytic activity of streptococcal M protein may be due to complement inhibition mediated by the binding of factor H. Binding of a regulatory protein appears to be a previously unrecognized route by which a pathogen is able to evade alternative pathway activation.
C3-convertase
Complement factor B
Complement control protein
Myeloma protein
Dissociation constant
Complement component 2
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C3-convertase
Complement factor B
Complement component 2
Properdin
Complement control protein
Decay-accelerating factor
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A typical haemolytic uraemic syndrome (aHUS) is a rare disease involving haemolytic anaemia, thrombocytopenia, and renal failure. There is growing evidence that the disease is associated with defective control of the alternative pathway of complement [1].
Genetic abnormalities in complement regulatory proteins, including complement factor H (CFH), membrane cofactor protein, and complement factor I, have been reported in 30%, 10%, and 5% of patients with aHUS, respectively [2–4]. CFH is a plasma protein produced by the liver that acts as a central regulator in the alternative pathway of complement activation. CFH acts as a cofactor for complement factor I in the inactivation of the central complement protein C3b to form iC3b. CFH also accelerates the decay of the C3 convertase C3bBb of the alternative pathway, and competes with factor B for binding to C3b [5].
iC3b
Complement factor B
Complement component 2
Decay-accelerating factor
Complement component 3
C3-convertase
Complement component 5
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