Binding Of Complement Factor H Research Articles

A Mutation in Complement Factor H (W1183R) Enhances VWF Proteolysis By ADAMTS13 Under Shear

Introduction: Complement factor H (CFH) is the primary negative regulator of complement activation in the alternative pathway in both fluidic phase and on cell surface. Loss of function of CFH such as W1183R is associated with atypical hemolytic uremic syndrome (aHUS). Homozygous mutation W1206R in the murine CFH, corresponding to W1183R in human CFH, results in acute aHUS. Interestingly, in these mice, while plasma VWF antigen levels are significantly increased, plasma VWF multimer size is markedly reduced in ADAMTS13-depedent manner. However, the molecular mechanism underlying such a dramatic effect of the mutant CFH (W1206R) on VWF multimer distribution is not known. The goal of this study is to determine how wild type CFH and mutant CFH W1183R affects VWF proteolysis under shear, and the mechanism of action. Methods: Recombinant WT CFH, CFH W1183R, ADAMTS13, and VWF fragments were expressed in mammalian cells and purified to homogeneity. The effects of WT CFH or CFH W1183R on proteolytic cleavage of VWF73 peptide and full-length multimeric VWF were determined. Additionally, the binding and unfolding events were determined by micro scale thermophoresis (MST) and optical tweezer, respectively. Results : WT CFH showed no effect on the cleavage of peptide VWF73 substrate, but CFH W1183R enhanced the proteolytic cleavage of VWF73 by ADAMTS13 in a dose-dependent manner ( Fig. 1). Counterintuitively, WT CFH appeared to inhibit the proteolytic cleavage of multimeric VWF by ADAMTS13 under arterial shear, but CFH W1183R exhibited an opposite effect, modestly enhancing the proteolytic cleavage of multimeric VWF by ADAMTS13 under the same conditions. The MST binding assay demonstrated that both WT CFH and CFH W1183R bind to a short A1, a long AIM-A1, and A1-A2-A3 fragments with a dissociation constant ( K D) of 6.4 x 10 -7, 3.0 x 10 -9 M, and 3.3 x10 -9 mol/L, respectively. These results suggest that CFH may bind to the AIM, A1-A2 junction, and A2 domain. To further assess the impact of CFH binding to A2, we performed optical tweezer experiments. The results showed that both WT CFH (Lc: from 27.4 nm at 12 nM to 40 nm at 1.0 mM) and CFH W1183R (Lc: from 26 nm at 12 nM to 31.8 nm at 1.0 mM) resulted in significantly extended contour length (Lc) of VWF-A2 domain in a concentration-dependent manner ( Fig. 2), suggesting a dramatic conformational change in the A2 domain upon binding by WT CFH or CFH W1183R. Conclusions : Our results demonstrate that WT CFH at physiological concentrations may exhibit a modest inhibitory activity towards cleavage of VWF by ADAMTS13 under shear, but such an inhibitory activity disappears as the result of a point mutation in the C-terminal end of CFH (e.g., W1183R). Instead, the mutant CFH W1183R appears to enhance the VWF cleavage by ADAMTS13, likely through direct binding to the AIM, A1-A2 junction, and A2 domain of VWF, resulting in conformational changes in the central A2 domain.

A genome-wide association study identifies key modulators of complement factor H binding to malondialdehyde-epitopes

Genetic variants within complement factor H (CFH), a major alternative complement pathway regulator, are associated with the development of age-related macular degeneration (AMD) and other complementopathies. This is explained with the reduced binding of CFH or its splice variant factor H-like protein 1 (FHL-1) to self-ligands or altered self-ligands (e.g., malondialdehyde [MDA]-modified molecules) involved in homeostasis, thereby causing impaired complement regulation. Considering the critical role of CFH in inhibiting alternative pathway activation on MDA-modified surfaces, we performed an unbiased genome-wide search for genetic variants that modify the ability of plasma CFH to bind MDA in 1,830 individuals and characterized the mechanistic basis and the functional consequences of this. In a cohort of healthy individuals, we identified rs1061170 in CFH and the deletion of CFHR3 and CFHR1 as dominant genetic variants that modify CFH/FHL-1 binding to MDA. We further demonstrated that FHR1 and FHR3 compete with CFH for binding to MDA-epitopes and that FHR1 displays the highest affinity toward MDA-epitopes compared to CFH and FHR3. Moreover, FHR1 bound to MDA-rich areas on necrotic cells and prevented CFH from mediating its cofactor activity on MDA-modified surfaces, resulting in enhanced complement activation. These findings provide a mechanistic explanation as to why the deletion of CFHR3 and CFHR1 is protective in AMD and highlight the importance of genetic variants within the CFH/CFHR3/CFHR1 locus in the recognition of altered-self in tissue homeostasis.

Complement depletion and Coombs positivity in pneumococcal hemolytic uremic syndrome (pnHUS). Case series and plea to revisit an old pathogenetic concept

Hemolytic uremic syndrome is a rare complication of invasive pneumococcal infection (pnHUS). Its pathogenesis is poorly understood, and treatment remains controversial. The emerging role of complement in various forms of HUS warrants a new look at this “old” disease.We performed a retrospective analysis of clinical and laboratory features of three sequential cases of pnHUS since 2008 associated with pneumonia/pleural empyema, two due to Streptococcus pneumoniae serotype 19 A. Profound depletion of complement C3 (and less of C4) was observed in two patients. One patient was Coombs test positive. Her red blood cells (RBCs) strongly agglutinated with blood group compatible donor serum at 0 °C, but not at 37 °C. All three patients were treated with hemodialysis, concentrated RBCs, and platelets. Patient 2 received frozen plasma for hepatic failure with coagulation factor depletion. Intravenous immunoglobulin infusion, intended to neutralize pneumococcal neuraminidase in patient 3, was associated with rapid normalization of platelets and cessation of hemolysis. Two patients recovered without sequelae or disease recurrence. Patient 2 died within 2½ days of admission due to complicating Pseudomonas aeruginosa sepsis and multiorgan failure.Our observations suggest that pnHUS can be associated with dramatic, transient complement consumption early in the course of the disease, probably via the alternative pathway. A critical review of the literature and the reported cases argue against the postulated pathological role of preformed antibodies against the neuraminidase-exposed Thomsen-Friedenreich neoantigen (T antigen) in pnHUS. The improved understanding of complement regulation and bacterial strategies of complement evasion allows to propose a testable, new pathogenetic model of pnHUS. This model shifts emphasis from the action of natural anti-T antibodies toward impaired Complement Factor H (CFH) binding and function on desialylated membranes. Removal of neuraminic acid residues converts (protected) self to non-self surfaces that supports membrane attack complex (MAC) assembly. Complement activation is potentially exacerbated by decreased CFH availability following tight CFH binding to pneumococcal evasion proteins and/or by the presence of genetic variants of complement regulator proteins. Detailed clinical and experimental investigations are warranted to better understand the role of unregulated complement activation in pnHUS. Instead of avoidance of plasma, a new, integrated model is evolving, which may include short-term therapeutic complement blockade, particularly where genetic or functional APC dysregulation is suspected, in addition to bacterial elimination and, potentially, neuraminidase neutralization.

Complement factor H in AMD: Bridging genetic associations and pathobiology.

Age-Related Macular Degeneration (AMD) is a complex multifactorial disease characterized in its early stages by lipoprotein accumulations in Bruch's Membrane (BrM), seen on fundoscopic exam as drusen, and in its late forms by neovascularization ("wet") or geographic atrophy of the Retinal Pigmented Epithelial (RPE) cell layer ("dry"). Genetic studies have strongly supported a relationship between the alternative complement cascade, in particular the common H402 variant in Complement Factor H (CFH) and development of AMD. However, the functional significance of the CFH Y402H polymorphism remains elusive. In this article, we critically review the literature surrounding the functional significance of this polymorphism. Furthermore, based on our group's studies we propose a model in which CFH H402 affects CFH binding to heparan sulfate proteoglycans leading to accelerated lipoprotein accumulation in BrM and drusen progression. We also review the literature on the role of other complement components in AMD pathobiologies, including C3a, C5a and the membrane attack complex (MAC), and on transgenic mouse models developed to interrogate invivo the effects of the CFH Y402H polymorphism.

Staphylococcus aureus SdrE captures complement factor H's C-terminus via a novel ‘close, dock, lock and latch' mechanism for complement evasion

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine–aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE–CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH1206–1226), which binds SdrE N2 and N3 domains (SdrEN2N3) with high affinity, and determined the crystal structures of apo-SdrEN2N3 and the SdrEN2N3–CFH1206–1226 complex. Comparison of the structure of the CFH–SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrEN2N3 adopts a ‘close’ state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel ‘close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a ‘clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion.

Complement Factor H Inhibits CD47-Mediated Resolution of Inflammation

Variants of the CFH gene, encoding complement factor H (CFH), show strong association with age-related macular degeneration (AMD), a major cause of blindness. Here, we used murine models of AMD to examine the contribution of CFH to disease etiology. Cfh deletion protected the mice from the pathogenic subretinal accumulation of mononuclear phagocytes (MP) that characterize AMD and showed accelerated resolution of inflammation. MP persistence arose secondary to binding of CFH to CD11b, which obstructed the homeostatic elimination of MPs from the subretinal space mediated by thrombospsondin-1 (TSP-1) activationof CD47. The AMD-associated CFH(H402) variantmarkedly increased this inhibitory effect on microglial cells, supporting a causal link to disease etiology. This mechanism is not restricted to the eye, as similar results were observed in a model of acute sterile peritonitis. Pharmacological activation of CD47 accelerated resolution of both subretinal and peritoneal inflammation, with implications for the treatment of chronic inflammatory disease.

Complement Factor H Antibodies from Lung Cancer Patients Induce Complement-Dependent Lysis of Tumor Cells, Suggesting a Novel Immunotherapeutic Strategy.

Characterization of the humoral immune response in selected patients with cancer who uniformly do well may lead to the development of novel therapeutic strategies. We have previously shown an association between patients with early-stage nonmetastatic lung cancer and autoantibodies to complement factor H (CFH). CFH protects normal and tumor cells from destruction by the alternative complement pathway by inactivating C3b, a protein that is essential for formation of a lytic complex on the cell surface. Here, we show that CFH autoantibodies in lung cancer patients recognize a conformationally distinct form of CFH in vitro, are IgG3 subclass, and epitope map to a crucial functional domain of CFH known to interact with C3b. Purified CFH autoantibodies inhibited binding of CFH to A549 lung tumor cells, increased C3b deposition, and caused complement-dependent tumor cell lysis. This work demonstrates that CFH autoantibodies isolated from patients with lung cancer can kill tumor cells in vitro, suggesting that they may perform this function in vivo as well. Development of specific antibodies to the conformationally distinct epitope of CFH may lead to a useful biologic therapy for lung cancer.

Molecular Interactions between Complement Factor H and Its Heparin and Heparan Sulfate Ligands.

Complement factor H (CFH) is the major regulator of the central complement protein C3b in the alternative pathway of complement activation. A molecular view of the CFH interaction with native heparan sulfate (HS) is central for understanding the mechanism of how surface-bound CFH interacts with C3b bound to host cell surfaces. HS is composed of sulfated heparin-like S-regions that alternate with desulfated NA-regions. Solution structural studies of heparin (equivalent to the S-regions) and desulfated HS (the NA-regions) by scattering and ultracentrifugation showed that each structure was mostly extended and partially bent, but with greater bending and flexibility in the NA-regions compared to the S-regions. Their solution structures have been deposited in the Protein Data Bank. The largest HS oligosaccharides showed more bent and flexible structures than those for heparin. A folded-back domain structure for the solution structure of the 20 domains in CFH was determined likewise. CFH binds to the S-regions but less so to the NA-regions of HS. The bivalent interaction of CFH–heparin was observed by ultracentrifugation, and binding studies of CFH fragments with heparin-coated sensor chips. In common with other CFH interactions with its physiological and pathophysiological ligands, the CFH–heparin and CFH–C3b interactions have moderate micromolar dissociation constants KD, meaning that these complexes do not fully form in vivo. The combination of the solution structures and binding studies indicated a two-site interaction model of CFH with heparin at cell surfaces. By this, the bivalent binding of CFH to a cell surface is co-operative. Defective interactions at either of the two independent CFH–heparin sites reduce the CFH interaction with surface-bound C3b and lead to immune disorders.

Human complement factor H is a novel diagnostic marker for lung adenocarcinoma

Human complement factor H (CFH), a central complement control protein, is a member of the regulators of complement activation family. Recent studies suggested that CFH may play a key role in the resistance of complement-mediated lysis in various cancer cells. In this study, we investigated the role of CFH in human lung cancer. Expression of CFH was analyzed in lung cancer cell lines by RT-PCR, Western blotting and immunofluorescence. In primary lung tumors, the protein expression of CFH was evaluated by immunohistochemistry (IHC) on tissue microarray (TMA). Binding of CFH to lung cancer cells was detected by flow cytometry. mRNA expression of CFH was detected in 6 out of 10 non-small cell lung cancer (NSCLC) cell lines, but in none of the small cell lung cancer (SCLC) cell lines. In line with Western blotting, immunofluorescence analysis demonstrated CFH protein expression in 3 NSCLC cell lines, and the immunoreaction was mainly associated with cell cytoplasm and membrane. In primary lung tumors, 54 out of 101 samples exhibited high expression of CFH and high expression was significantly correlated with lung adenocarcinoma (p=0.009). Also, in adenocarcinoma of the lung, Kaplan-Meier survival analysis showed a tendency that CFH-positive tumors had worse prognosis in comparison to CFH-negative tumors (p=0.082). Additionally, shorter survival time of patients with adenocarcinoma (<20 months) was associated with higher staining of CFH (p=0.033). Our data showed that non-small cell lung cancer cells expressed and secreted CFH. CFH might be a novel diagnostic marker for human lung adenocarcinoma.

Complement factor H and age-related macular degeneration: the role of glycosaminoglycan recognition in disease pathology

AMD (age-related macular degeneration) is the major cause of blindness in the western world, associated with the formation of extracellular deposits called drusen in the macula, i.e. the central region of the retina. These drusen contain cellular debris and proteins, including components of the complement system such as the regulator CFH (complement factor H); dysregulation of complement is thought to play a major role in the development of AMD. CFH acts through its capacity to recognize polyanionic structures [e.g. sulfated GAGs (glycosaminoglycans)] found on host tissues, and thereby inactivates any C3b that becomes deposited. Importantly, a common polymorphism in CFH (Y402H) has been strongly associated with an increased risk of AMD. This polymorphism, which causes a tyrosine to histidine coding change, has been shown to alter the binding of CFH to sulfated GAGs, as well as to other ligands including C-reactive protein, necrotic cells and bacterial coat proteins. Of these, the change in the GAG-recognition properties of CFH is likely to be of most significance to AMD. Recent research has revealed that the disease-associated 402H allotype interacts less well (compared with 402Y) with binding sites within the macula (e.g. Bruch's membrane), where the GAGs heparan sulfate and dermatan sulfate play a major role in mediating the interaction with CFH. Reduced binding of the 402H allotype could result in impaired regulation of complement leading to chronic local inflammation that may contribute to the accumulation of drusen and thus the initiation, development and progression of AMD.

Impaired Binding of the Age-related Macular Degeneration-associated Complement Factor H 402H Allotype to Bruch's Membrane in Human Retina

Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruch's membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMD-associated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruch's membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruch's membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.

Genome-wide association study identifies variants in the CFH region associated with host susceptibility to meningococcal disease

Meningococcal disease is an infection caused by Neisseria meningitidis. Genetic factors contribute to host susceptibility and progression to disease, but the genes responsible for disease development are largely unknown. We report here a genome-wide association study for host susceptibility to meningococcal disease using 475 individuals with meningococcal disease (cases) and 4,703 population controls from the UK. We performed, in Western European and South European cohorts (consisting of 968 cases and 1,376 controls), two replication studies for the most significant SNPs. A cluster of complement factor SNPs replicated independently in both cohorts, including SNPs within complement factor H (CFH) (rs1065489 (p.936D<E), P = 2.2 x 10(-11)) and in CFH-related protein 3 (CFHR3)(rs426736, P = 4.6 x 10(-13)). N. meningitidis is known to evade complement-mediated killing by the binding of host CFH to the meningococcal factor H-binding protein (fHbp). Our study suggests that host genetic variation in these regulators of complement activation plays a role in determining the occurrence of invasive disease versus asymptomatic colonization by this pathogen.

The AMD‐associated complement factor H (CFH) polymorphism Y402H results in decreased CFH localisation to Bruch’s membrane

Abstract Purpose CFH down‐regulates the alternative pathway of the complement system by binding to polyanionic structures on host cells/tissues and inactivating surface associated C3b. Recently, the Y402H polymorphism in CFH has been shown to be a major risk factor for AMD. Here we investigated the functional consequences of the Y402H polymorphism by testing the hypothesis that the resultant amino acid substitution alters CFH binding to macular tissue Methods The 402H and 402Y forms of full‐length CFH and recombinant CFH fragments (composed of CCP6‐8) were labelled with different fluorophores (402Y with AlexFluor‐488 and 402H with AlexaFluor‐594). These were simultaneously incubated with frozen sections of human macular tissue from donor eyes and the relative binding of the two forms was investigated. In some experiments the tissue sections were digested with glycosidic enzymes prior to incubation with the fluorescently‐labelled proteins. Results Whilst the 402H and 402Y variants showed similar levels of binding to the RPE, there was a marked reduction in binding of the 402H form to Bruch’s membrane. The binding of both forms to Bruch’s membrane was dependent upon interactions with heparan sulfates, and to a lesser extent dermatan sulfates. Conclusion Complement mediated damage is important in the pathogenesis of AMD and the relative failure of the 402H form of CFH to localise to Bruch’s membrane may result in over activation of the complement system at the retinal pigment epithelium/Bruch's membrane interface.

Functional analyses indicate a pathogenic role of factor H autoantibodies in atypical haemolytic uraemic syndrome

Atypical haemolytic uraemic syndrome (aHUS) is associated with defective complement regulation. Recently, an autoimmune aHUS form has been described that is associated with complement factor H (CFH) autoantibodies. The aim of this study was to address the pathologic relevance of CFH autoantibodies in aHUS. CFH autoantibodies were identified and antibody levels were analysed in three aHUS patients during the disease course by the ELISA method. Epitope mapping was performed using recombinant factor H fragments and domain-mapped monoclonal antibodies. The effect of the antibodies on cell-protective activity of CFH was measured by haemolytic assays. CFH:autoantibody complexes were analysed by ELISA. All three autoantibodies bound to the C-terminal domain of CFH, which is essential for CFH binding to cell surfaces. In patient 1, plasma exchanges and immune adsorption temporarily reduced the autoantibody titre and led to temporary clinical improvement. In patient 2, plasma exchanges and long-term immunosuppression strongly reduced the CFH autoantibody level, and induced a stable remission of aHUS. Patient 3 had lower autoantibody levels that decreased during the follow-up and is in good clinical condition. The patients' plasma samples caused enhanced lysis of sheep erythrocytes, and the degree of lysis correlated with the CFH autoantibody titre and the amount of CFH:autoantibody complexes. An addition of purified CFH to aHUS plasma or removal of IgG inhibited the haemolytic activity. These results support a direct role of the autoantibodies in aHUS pathology by inhibiting the regulatory function of CFH at cell surfaces and suggest that reduction of the autoantibody titre is beneficial for the patients.

Efficient destruction of human immunodeficiency virus in human serum by inhibiting the protective action of complement factor H and decay accelerating factor (DAF, CD55).

Activation of the human complement system leads to complement deposition on human immunodeficiency virus (HIV) and HIV-infected cells without causing efficient complement-mediated lysis. Even in the presence of HIV-specific antibodies, only a few particles are destroyed, demonstrating that HIV is intrinsically resistant to human complement. Here we report that, in addition to decay accelerating factor (DAF) being partially responsible, human complement factor H (CFH), a humoral negative regulator of complement activation, is most critical for this resistance. In the presence of HIV-specific antibodies, sera devoid of CFH (total genetic deficiency or normal human serum depleted of CFH by affinity chromatography) lysed free virus and HIV-infected but not uninfected cells. In the presence of CFH, lysis of HIV was only obtained when binding of CFH to gp41 was inhibited by a monoclonal antibody against a main CFH-binding site in gp41. Since CFH is an abundant protein in serum, and high local concentration of CFH can be obtained at the surface of HIV as the result of specific interactions of CFH with the HIV envelope, it is proposed that the resistance of HIV and HIV-infected cells against complement-mediated lysis in vivo is dependent on DAF and CFH and can be overcome by suppressing this protection. Neutralization of HIV may be achieved by antibodies against DAF and, more importantly, antibodies against CFH-binding sites on HIV envelope proteins.

HIV glycoprotein 41 and complement factor H interact with each other and share functional as well as antigenic homology.

We have shown that complement factor H (CFH) interacts with HIV-1 at the level of the sequence Env 105-119, contained in the C1 domain of gp120. CFH interaction with HIV was evident only after dissociation of the Env complex induced by exposure to sCD4. We hypothesized that CFH could act as a gp41 analog in the interaction with Env 105-119. A panel of partially overlapping, synthetic peptides reproducing the extracellular portion of gp41 was therefore used to compete the binding of CFH to Env 105-119. Three sets of peptides that competed this interaction were identified. These peptides defined a region of functional homology between the gp41 molecule and CFH (Env 580-600), and two regions of interaction (Env 620-640 and Env 650-670). In addition to this, a monoclonal antibody directed against peptide Env 580-600 and a polyclonal mouse antiserum raised against recombinant gp41 were shown to recognize CFH in Western blots and ELISA, respectively, also defining a region of antigenic homology between gp41 and CFH. These data provide evidence for interaction and molecular mimicry between an HIV structural protein and a negative regulator of the complement pathway. We show here that CFH can interact with both HIV Env proteins, suggesting a possible and efficient mechanism of downregulation of the complement cascade at the surface of infected cells.