Rubén Martínez-Barricarte

Factor H (FH) is an abundant serum glycoprotein that regulates the alternative pathway of complement-preventing uncontrolled plasma C3 activation and nonspecific damage to host tissues. Age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS), and membranoproliferative glomerulonephritis type II (MPGN2) are associated with polymorphisms or mutations in the FH gene (Cfh), suggesting the existence of a genotype-phenotype relationship. Although AMD and MPGN2 share pathological similarities with the accumulation of complement-containing debris within the eye and kidney, respectively, aHUS is characterized by renal endothelial injury. This pathological distinction was reflected in our Cfh association analysis, which demonstrated that although AMD and MPGN2 share a Cfh at-risk haplotype, the haplotype for aHUS was unique. FH-deficient mice have uncontrolled plasma C3 activation and spontaneously develop MPGN2 but not aHUS. We show that these mice, transgenically expressing a mouse FH protein functionally equivalent to aHUS-associated human FH mutants, regulate C3 activation in plasma and spontaneously develop aHUS but not MPGN2. These animals represent the first model of aHUS and provide in vivo evidence that effective plasma C3 regulation and the defective control of complement activation on renal endothelium are the critical events in the molecular pathogenesis of FH-associated aHUS.

Common polymorphisms in complement alternative pathway (AP) proteins C3 (C3(R102G)), factor B (fB(R32Q)), and factor H (fH(V62I)) are associated with age-related macular degeneration (AMD) and other pathologies. Our published work showed that fB(R32Q) influences C3 convertase formation, whereas fH(V62I) affects factor I cofactor activity. Here we show how C3(R102G) (C3S/F) influences AP activity. In hemolysis assays, C3(102G) activated AP more efficiently (EC(50) C3(102G): 157 nM; C3(102R): 191 nM; P < 0.0001). fB binding kinetics and convertase stability were identical, but native and recombinant fH bound more strongly to C3b(102R) (K(D) C3b(102R): 1.0 μM; C3b(102G): 1.4 μM; P < 0.0001). Accelerated decay was unaltered, but fH cofactor activity was reduced for C3b(102G), favoring AP amplification. Combining disease "risk" variants (C3(102G), fB(32R), and fH(62V)) in add-back assays yielded sixfold higher hemolytic activity compared with "protective" variants (C3(102R), fB(32Q), and fH(62I); P < 0.0001). These data introduce the concept of a functional complotype (combination of polymorphisms) defining complement activity in an individual, thereby influencing susceptibility to AP-driven disease.

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.

The factor H-related protein family (CFHR) is a group of minor plasma proteins genetically and structurally related to complement factor H (fH). Notably, deficiency of CFHR1/CFHR3 associates with protection against age-related macular degeneration and with the presence of anti-fH autoantibodies in atypical hemolytic uremic syndrome (aHUS). We have developed a proteomics strategy to analyze the CFHR proteins in plasma samples from controls, patients with aHUS, and patients with type II membranoproliferative glomerulonephritis. Here, we report on the identification of persons carrying novel deficiencies of CFHR1, CFHR3, and CFHR1/CFHR4A, resulting from point mutations in CFHR1 and CFHR3 or from a rearrangement involving CFHR1 and CFHR4. Remarkably, patients with aHUS lacking CFHR1, but not those lacking CFHR3, present anti-fH autoantibodies, suggesting that generation of these antibodies is specifically related to CFHR1 deficiency. We also report the characterization of a novel CFHR1 polymorphism, resulting from a gene conversion event between CFH and CFHR1, which strongly associates with aHUS. The risk allotype CFHR1*B, with greater sequence similarity to fH, may compete with fH, decreasing protection of cellular surfaces against complement damage. In summary, our comprehensive analyses of the CFHR proteins have improved our understanding of these proteins and provided further insights into aHUS pathogenesis.