Agustín Tortajada

Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation

Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C(3923ΔDG), which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H₂O) by spontaneous thioester hydrolysis, C(3923ΔDG) generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C(3b923ΔDG) and C3(H₂O)(923ΔDG) were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase-restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments.

C3 glomerulopathy–associated CFHR1 mutation alters FHR oligomerization and complement regulation

C3 glomerulopathies (C3G) are a group of severe renal diseases with distinct patterns of glomerular inflammation and C3 deposition caused by complement dysregulation. Here we report the identification of a familial C3G-associated genomic mutation in the gene complement factor H–related 1 (CFHR1), which encodes FHR1. The mutation resulted in the duplication of the N-terminal short consensus repeats (SCRs) that are conserved in FHR2 and FHR5. We determined that native FHR1, FHR2, and FHR5 circulate in plasma as homo- and hetero-oligomeric complexes, the formation of which is likely mediated by the conserved N-terminal domain. In mutant FHR1, duplication of the N-terminal domain resulted in the formation of unusually large multimeric FHR complexes that exhibited increased avidity for the FHR1 ligands C3b, iC3b, and C3dg and enhanced competition with complement factor H (FH) in surface plasmon resonance (SPR) studies and hemolytic assays. These data revealed that FHR1, FHR2, and FHR5 organize a combinatorial repertoire of oligomeric complexes and demonstrated that changes in FHR oligomerization influence the regulation of complement activation. In summary, our identification and characterization of a unique CFHR1 mutation provides insights into the biology of the FHRs and contributes to our understanding of the pathogenic mechanisms underlying C3G.

The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity

Mutations and polymorphisms in the gene encoding factor H (CFH) have been associated with atypical haemolytic uraemic syndrome, dense deposit disease and age-related macular degeneration. The disease-predisposing CFH variants show a differential association with pathology that has been very useful to unravel critical events in the pathogenesis of one or other disease. In contrast, the factor H (fH)-Ile(62) polymorphism confers strong protection to all three diseases. Using ELISA-based methods and surface plasmon resonance analyses, we show here that the protective fH-Ile(62) variant binds more efficiently to C3b than fH-Val(62) and competes better with factor B in proconvertase formation. Functional analyses demonstrate an increased cofactor activity for fH-Ile(62) in the factor I-mediated cleavage of fluid phase and surface-bound C3b; however, the two fH variants show no differences in decay accelerating activity. From these data, we conclude that the protective effect of the fH-Ile(62) variant is due to its better capacity to bind C3b, inhibit proconvertase formation and catalyze inactivation of fluid-phase and surface-bound C3b. This demonstration of the functional consequences of the fH-Ile(62) polymorphism provides relevant insights into the complement regulatory activities of fH that will be useful in disease prediction and future development of effective therapeutics for disorders caused by complement dysregulation.

Functional basis of protection against age-related macular degeneration conferred by a common polymorphism in complement factor B

Mutations and polymorphisms in complement genes have been linked with numerous rare and prevalent disorders, implicating dysregulation of complement in pathogenesis. The 3 common alleles of factor B (fB) encode Arg (fB32R), Gln (fB32Q), or Trp (fB32W) at position 32 in the Ba domain. The fB32Q allele is protective for age-related macular degeneration, the commonest cause of blindness in developed countries. Factor B variants were purified from plasma of homozygous individuals and were tested in hemolysis assays. The protective variant fB32Q had decreased activity compared with fB32R. Biacore comparison revealed markedly different proenzyme formation; fB32R bound C3b with 4-fold higher affinity, and formation of activated convertase was enhanced. Binding and functional differences were confirmed with recombinant fB32R and fB32Q; an intermediate affinity was revealed for fB32W. To confirm contribution of Ba to binding, affinity of Ba for C3b was determined. Ba-fB32R had 3-fold higher affinity compared with Ba-fB32Q. We demonstrate that the disease-protective effect of fB32Q is consequent on decreased potential to form convertase and amplify complement activation. Knowledge of the functional consequences of polymorphisms in complement activators and regulators will aid disease prediction and inform targeting of diagnostics and therapeutics.

Measurement of Factor H Variants in Plasma Using Variant-Specific Monoclonal Antibodies: Application to Assessing Risk of Age-Related Macular Degeneration

PURPOSE The Y402H polymorphism in the complement regulator factor H (fH) is strongly associated with age-related macular degeneration (AMD) across diverse populations. Persons homozygous for histidine at this position have up to 12-fold greater risk for AMD than those homozygous for tyrosine. Knowledge of fH-Y402H status is, therefore, valuable in predicting risk and focusing preventive measures in the elderly. This knowledge requires genetic analysis, which is unavailable in most laboratories and which provides no information about the levels of fH protein, a putative linked determinant of disease risk. METHODS The authors describe novel monoclonal antibodies that distinguish the two fH allelic variants in plasma. ELISA with these antibodies not only reliably identifies the fH-Y402H status, confirmed by genotyping, but also quantifies the concentration of total fH and the fH-Y402 and fH-H402 variants. RESULTS In young adult control subjects, mean fH concentration was 233 mg/L. In elderly control subjects, mean fH concentration was 269 mg/L, whereas in a matching AMD cohort, mean fH concentration was 288 mg/L. Total fH concentration was similar in each subgroup of young and elderly control subjects; however, in the AMD group, fH concentration was significantly higher in the heterozygous subgroup. Measurement of the two variants in this subgroup showed that both were elevated to a similar degree. CONCLUSIONS The novel monoclonal antibody MBI-7 was used to develop a robust assay for measurement of fH and the variants in plasma. The simplicity of the assay means that it may be used by any clinical laboratory to identify polymorphic status and to quantify plasma levels in persons at risk for AMD.

Complement dysregulation and disease: From genes and proteins to diagnostics and drugs

During the last decade, numerous studies have associated genetic variations in complement components and regulators with a number of chronic and infectious diseases. The functional characterization of these complement protein variants, in addition to recent structural advances in understanding of the assembly, activation and regulation of the AP C3 convertase, have provided important insights into the pathogenic mechanisms involved in some of these complement related disorders. This knowledge has identified potential targets for complement inhibitory therapies which are demonstrating efficacy and generating considerable expectation in changing the natural history of these diseases. Comprehensive understanding of the genetic and non-genetic risk factors contributing to these disorders will also result in targeting of the right patient groups in a stratified medicine approach through better diagnostics and individually tailored treatments, thereby improving management of patients.

3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase

Generation of the alternative pathway C3-convertase, the central amplification enzyme of the complement cascade, initiates by the binding of factor B (fB) to C3b to form the proconvertase, C3bB. C3bB is subsequently cleaved by factor D (fD) at a single site in fB, producing Ba and Bb fragments. Ba dissociates from the complex, while Bb remains bound to C3b, forming the active alternative pathway convertase, C3bBb. Using single-particle electron microscopy we have determined the 3-dimensional structures of the C3bB and the C3bBb complexes at ≈27Å resolution. The C3bB structure shows that fB undergoes a dramatic conformational change upon binding to C3b. However, the C3b-bound fB structure was easily interpreted after independently fitting the atomic structures of the isolated Bb and Ba fragments. Interestingly, the divalent cation-binding site in the von Willebrand type A domain in Bb faces the C345C domain of C3b, whereas the serine-protease domain of Bb points outwards. The structure also shows that the Ba fragment interacts with C3b separately from Bb at the level of the α′NT and CUB domains. Within this conformation, the long and flexible linker between Bb and Ba is likely exposed and accessible for cleavage by fD to form the active convertase, C3bBb. The architecture of the C3bB and C3bBb complexes reveals that C3b could promote cleavage and activation of fB by actively displacing the Ba domain from the von Willebrand type A domain in free fB. These structures provide a structural basis to understand fundamental aspects of the activation and regulation of the alternative pathway C3-convertase.

Genetics of atypical hemolytic uremic syndrome (aHUS)

Hemolytic uremic syndrome (HUS) is a rare, life-threatening disease characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. The atypical form of HUS (aHUS), representing 5 to 10% of cases, lacks the association with infection by Shiga toxin producing Escherichia coli strains that characterizes the commonest clinical presentation of HUS. In the majority of aHUS cases, the disease results from the complement-mediated damage to the microvascular endothelium because of inherited defects in complement genes or autoantibodies against complement regulatory proteins. Incomplete penetrance of aHUS in carriers of mutations is common to all aHUS-associated complement genes and it is now established that the overall genetic predisposition to aHUS of an individual results from the combination of different inherited factors. Moreover, the patients genotype influences the clinical evolution, the response to plasma therapies, and the recurrence after transplantation. Here, we describe the genetic component of aHUS, the lessons that we have learned from the functional characterization of the aHUS-associated mutations, and the benefits of a comprehensive genetic analysis of the patients.

Factor H-related proteins determine complement-activating surfaces.

Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

Variant-specific quantification of factor H in plasma identifies null alleles associated with atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with complement alternative pathway defects in over half the cases. Point mutations that affect complement surface regulation are common in factor H (CFH); however, sometimes individuals have null mutations in heterozygosis. The latter are difficult to identify, although a consistently low plasma factor H (fH) concentration is suggestive; definitive proof requires demonstration that the mutant sequence is not expressed in vitro. Here, novel reagents and assays that distinguish and individually quantify the common factor H-Y402H polymorphic variants were used to identify alleles of the CFH gene, resulting in low or null expression of full-length fH and also normal or increased expression of the alternative splice product factor H-like-1 (FHL-1). Our assay identified three Y402H heterozygotes with low or absent fH-H402 but normal or increased FHL-1-H402 levels in a cohort of affected patients. Novel mutations explained the null phenotype in two cases, which was confirmed by family studies in one. In the third case, family studies showed that a known mutation was present on the Y allele. The cause of reduced expression of the H allele was not found, although the data suggested altered splicing. In each family, inheritance of low expression or null alleles for fH strongly associated with aHUS. Thus, our assays provide a rapid means to identify fH expression defects without resorting to gene sequencing or expression analysis.