RESUMEN
The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of â¼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C-reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.
Asunto(s)
Proteína C-Reactiva/metabolismo , Activación de Complemento/fisiología , Proteínas del Sistema Complemento/metabolismo , Matriz Extracelular/metabolismo , Componente Amiloide P Sérico/metabolismo , Humanos , Ligandos , Unión Proteica , Proteínas RecombinantesRESUMEN
Hypersensitivity reactions to particulate drugs can partly be caused by complement activation and represent a major complication during intravenous application of nanomedicines. Several liposomal and micellar drugs and carriers, and therapeutic antibodies, were shown to activate complement and induce complement activation-related pseudoallergy (CARPA) in model animals. To explore the possible use of the natural complement inhibitor factor H (FH) against CARPA, we examined the effect of FH on complement activation induced by CARPAgenic drugs. Exogenous FH inhibited complement activation induced by the antifungal liposomal Amphotericin-B (AmBisome), the widely used solvent of anticancer drugs Cremophor EL, and the anticancer monoclonal antibody rituximab in vitro. An engineered form of FH (mini-FH) was more potent inhibitor of Ambisome-, Cremophor EL- and rituximab-induced complement activation than FH. The FH-related protein CFHR1 had no inhibitory effect. Our data suggest that FH or its derivatives may be considered in the pharmacological prevention of CARPA. FROM THE CLINICAL EDITOR: Although liposomes and micelles are already in use in the clinical setting as drug carriers, there remains the potential problem of hypersensitivity due to complement activation. In this article, the authors investigated the use of complement inhibitor factor H (FH) on complement activation and showed good efficacy. The results would therefore suggest the potential application of complement inhibitor in the future.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Factor H de Complemento/administración & dosificación , Hipersensibilidad a las Drogas/tratamiento farmacológico , Liposomas/efectos adversos , Activación de Complemento/inmunología , Factor H de Complemento/inmunología , Portadores de Fármacos/efectos adversos , Hipersensibilidad a las Drogas/inmunología , Hipersensibilidad a las Drogas/patología , Voluntarios Sanos , Humanos , Micelas , Nanomedicina , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Rituximab/efectos adversosRESUMEN
Complement is an essential humoral component of innate immunity; however, its inappropriate activation leads to pathology. Polymorphisms, mutations, and autoantibodies affecting factor H (FH), a major regulator of the alternative complement pathway, are associated with various diseases, including age-related macular degeneration, atypical hemolytic uremic syndrome, and C3 glomerulopathies. Restoring FH function could be a treatment option for such pathologies. In this article, we report on an engineered FH construct that directly combines the two major functional regions of FH: the N-terminal complement regulatory domains and the C-terminal surface-recognition domains. This minimal-size FH (mini-FH) binds C3b and has complement regulatory functions similar to those of the full-length protein. In addition, we demonstrate that mini-FH binds to the FH ligands C-reactive protein, pentraxin 3, and malondialdehyde epitopes. Mini-FH was functionally active when bound to the extracellular matrix and endothelial cells in vitro, and it inhibited C3 deposition on the cells. Furthermore, mini-FH efficiently inhibited complement-mediated lysis of host-like cells caused by a disease-associated FH mutation or by anti-FH autoantibodies. Therefore, mini-FH could potentially be used as a complement inhibitor targeting host surfaces, as well as to replace compromised FH in diseases associated with FH dysfunction.
Asunto(s)
Complemento C3b/inmunología , Factor H de Complemento/genética , Factor H de Complemento/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Proteína C-Reactiva/metabolismo , Activación de Complemento , Complemento C3b/antagonistas & inhibidores , Complemento C3b/metabolismo , Vía Alternativa del Complemento , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Humanos , Malondialdehído/metabolismo , Unión Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Componente Amiloide P Sérico/metabolismoRESUMEN
Human complement factor H-related protein (CFHR) 4 belongs to the factor H family of plasma glycoproteins that are composed of short consensus repeat (SCR) domains. Although factor H is a well known inhibitor of the alternative complement pathway, the functions of the CFHR proteins are poorly understood. CFHR4 lacks SCRs homologous to the complement inhibitory domains of factor H and, accordingly, has no significant complement regulatory activities. We have previously shown that CFHR4 binds C-reactive protein via its most N-terminal SCR, which leads to classical complement pathway activation. CFHR4 binds C3b via its C terminus, but the significance of this interaction is unclear. Therefore, we set out to clarify the functional relevance of C3b binding by CFHR4. Here, we report a novel role for CFHR4 in the complement system. CFHR4 serves as a platform for the assembly of an alternative pathway C3 convertase by binding C3b. This is based on the sustained ability of CFHR4-bound C3b to bind factor B and properdin, leading to an active convertase that generates C3a and C3b from C3. The CFHR4-C3bBb convertase is less sensitive to the factor H-mediated decay compared with the C3bBb convertase. CFHR4 mutants containing exchanges of conserved residues within the C-terminal C3b-binding site showed significantly reduced C3b binding and alternative pathway complement activation. In conclusion, our results suggest that, in contrast to the complement inhibitor factor H, CFHR4 acts as an enhancer of opsonization by promoting complement activation.
Asunto(s)
Apolipoproteínas/metabolismo , Convertasas de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Vía Alternativa del Complemento/fisiología , Animales , Apolipoproteínas/genética , Sitios de Unión , Línea Celular , Convertasas de Complemento C3-C5/genética , Complemento C3b/genética , Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Humanos , Mutación , Properdina/genética , Properdina/metabolismo , SpodopteraRESUMEN
Human complement factor H-related protein 4 (CFHR4) is a plasma glycoprotein which appears in two isoforms. CFHR4 is a member of the factor H protein family, and shares structural similarity and sequence homology with the other CFHR proteins and with the complement regulator factor H. Given the structural and sequence similarity, we hypothesized that similar to factor H, CFHR4 binds to C-reactive protein (CRP). We have recombinantly expressed the two CFHR4 isoforms and analyzed their binding to both native and denatured, monomeric CRP. Here, we show that both CFHR4 isoforms bind in the presence of calcium to native pentameric CRP, but not to modified CRP. This is in contrast to factor H, which binds to modified CRP independent of calcium. Comparison of the two CFHR4 isoforms and a recombinant CFHR4 fragment for CRP binding indicates that the first domain of CFHR4 is relevant for this interaction. Interaction of the native proteins was demonstrated by co-precipitation of CFHR4 and CRP from serum of sepsis patients with elevated CRP levels. CFHR4 bound to necrotic cells and was localized in necrotic tumor tissue as demonstrated by immunohistological analyses. In addition, CFHR4 facilitated binding of native CRP to the surface of necrotic cells. Altogether these data identify CFHR4 as a novel ligand for native CRP, and suggest a role for CFHR4 in opsonization of necrotic cells.
Asunto(s)
Apolipoproteínas/metabolismo , Proteína C-Reactiva/metabolismo , Necrosis/metabolismo , Apolipoproteínas/química , Apolipoproteínas/aislamiento & purificación , Tampones (Química) , Calcio/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Neoplasias del Colon/patología , Humanos , Inmunoprecipitación , Concentración Osmolar , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.
RESUMEN
Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated cell lysis due to deficiency of GPI-anchored complement regulators. Blockage of the lytic pathway by eculizumab is the only available therapy for PNH patients and shows remarkable benefits, but regularly yields PNH erythrocytes opsonized with fragments of complement protein C3, rendering such erythrocytes prone to extravascular hemolysis. This effect is associated with insufficient responsiveness seen in a subgroup of PNH patients. Novel C3-opsonin targeted complement inhibitors act earlier in the cascade, at the level of activated C3 and are engineered from parts of the natural complement regulator Factor H (FH) or complement receptor 2 (CR2). This inhibitor class comprises three variants of "miniFH" and the clinically developed "FH-CR2" fusion-protein (TT30). We show that the approach of FH-CR2 to target C3-opsonins was more efficient in preventing complement activation induced by foreign surfaces, whereas the miniFH variants were substantially more active in controlling complement on PNH erythrocytes. Subtle differences were noted in the ability of each version of miniFH to protect human PNH cells. Importantly, miniFH and FH-CR2 interfered only minimally with complement-mediated serum killing of bacteria when compared to untargeted inhibition of all complement pathways by eculizumab. Thus, the molecular design of each C3-opsonin targeted complement inhibitor determines its potency in respect to the nature of the activator/surface providing potential functionality in PNH.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Complemento C3/genética , Inactivadores del Complemento/farmacología , Eritrocitos/efectos de los fármacos , Proteínas Opsoninas/genética , Animales , Anticuerpos Monoclonales Humanizados/biosíntesis , Anticuerpos Monoclonales Humanizados/inmunología , Células Cultivadas , Activación de Complemento/efectos de los fármacos , Factor H de Complemento/genética , Factor H de Complemento/inmunología , Inactivadores del Complemento/inmunología , Inactivadores del Complemento/metabolismo , Vía Alternativa del Complemento , Eritrocitos/inmunología , Eritrocitos/patología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Expresión Génica , Hemoglobinuria Paroxística/tratamiento farmacológico , Hemoglobinuria Paroxística/genética , Hemoglobinuria Paroxística/metabolismo , Hemoglobinuria Paroxística/patología , Hemólisis/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Ingeniería de Proteínas , Conejos , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Complement is an essential part of innate immunity as it participates in host defense against infections, disposal of cellular debris and apoptotic cells, inflammatory processes and modulation of adaptive immune responses. Several soluble and membrane-bound regulators protect the host from the potentially deleterious effects of uncontrolled and misdirected complement activation. Factor H is a major soluble regulator of the alternative complement pathway, but it can also bind to host cells and tissues, protecting them from complement attack. Interactions of factor H with various endogenous ligands, such as pentraxins, extracellular matrix proteins and DNA are important in limiting local complement-mediated inflammation. Impaired regulatory as well as ligand and cell recognition functions of factor H, caused by mutations or autoantibodies, are associated with the kidney diseases: atypical hemolytic uremic syndrome and dense deposit disease and the eye disorder: age-related macular degeneration. In addition, factor H binds to receptors on host cells and is involved in adhesion, phagocytosis and modulation of cell activation. In this review we discuss current concepts on the physiological and pathophysiological roles of factor H in light of new data and recent developments in our understanding of the versatile roles of factor H as an inhibitor of complement activation and inflammation, as well as a mediator of cellular interactions. A detailed knowledge of the functions of factor H in health and disease is expected to unravel novel therapeutic intervention possibilities and to facilitate the development or improvement of therapies.
RESUMEN
C-reactive protein (CRP) is a pattern recognition molecule that binds several microbial and host ligands. Ligand-bound CRP activates the complement system via the classical pathway. Previously, we identified human complement factor H-related protein 4 (CFHR4), a member of the factor H protein family, as a CRP binding protein. Here, we investigated the molecular basis and the functional relevance of the interaction of CFHR4 with native CRP. Using recombinantly expressed CFHR4 fragments, the CRP binding site was localized to the first short consensus repeat (SCR) domain of CFHR4. Peptide arrays identified residues 35-41 of CFHR4 to be involved in CRP binding. Substitutions of the positively charged amino acids of this motif resulted in strongly reduced CRP binding. Sequence comparisons revealed that such a motif is not present in the related SCR6 domain of factor H, or in the homologous domains of the four other CFHR proteins. Homology modelling based on SCR6 of factor H showed that the CRP binding site is surface exposed on SCR1 of CFHR4. CFHR4-bound CRP was able to activate complement, determined by C3 fragment deposition. Recombinant CFHR4 proteins with mutations in the identified binding site showed reduced CRP binding, which in turn resulted in reduced complement activation. In summary, these data reveal the molecular basis of the specific interaction of CFHR4 with native CRP and suggest a role for CFHR4 in enhancing opsonization via CRP binding.