Structural and functional characterization of factor H mutations associated with atypical hemolytic uremic syndrome

Genetic studies have demonstrated the involvement of the complement regulator factor H in nondiarrheal, nonverocytotoxin (i.e., atypical) cases of hemolytic uremic syndrome. Different factor H mutations have been identified in 10%-30% of patients with atypical hemolytic uremic syndrome (aHUS), and most of these mutations alter single amino acids in the C-terminal region of factor H. Although these mutations are considered to be responsible for the disease, the precise role that factor H plays in the pathogenesis of aHUS is unknown. We report here the structural and functional characterization of three different factor H proteins purified from the plasma of patients with aHUS who carry the factor H mutations W1183L, V1197A, or R1210C. Structural anomalies in factor H were found only in R1210C carriers; these individuals show, in their plasma, a characteristic high-molecular-weight factor H protein that results from the covalent interaction between factor H and human serum albumin. Most important, all three aHUS-associated factor H proteins have a normal cofactor activity in the proteolysis of fluid-phase C3b by factor I but show very low binding to surface-bound C3b. This functional impairment was also demonstrated in recombinant mutant factor H proteins expressed in COS7 cells. These data support the hypothesis that patients with aHUS carry a specific dysfunction in the protection of cellular surfaces from complement activation, offering new possibilities to improve diagnosis and develop appropriate therapies.     

Atypical hemolytic uremic syndrome-associated variants and autoantibodies impair binding of factor h and factor h-related protein 1 to pentraxin 3

Atypical hemolytic uremic syndrome (aHUS) is a renal disease associated with complement alternative pathway dysregulation and is characterized by endothelial injury. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule expressed by endothelial cells and upregulated under inflammatory conditions. PTX3 activates complement, but it also binds the complement inhibitor factor H. In this study, we show that native factor H, factor H-like protein 1, and factor H-related protein 1 (CFHR1) bind to PTX3 and that PTX3-bound factor H and factor H-like protein 1 maintain their complement regulatory activities. PTX3, when bound to extracellular matrix, recruited functionally active factor H. Residues within short consensus repeat 20 of factor H that are relevant for PTX3 binding were identified using a peptide array. aHUS-associated factor H mutations within this binding site caused a reduced factor H binding to PTX3. Similarly, seven of nine analyzed anti-factor H autoantibodies isolated from aHUS patients inhibited the interaction between factor H and PTX3, and five autoantibodies also inhibited PTX3 binding to CFHR1. Moreover, the aHUS-associated CFHR1*B variant showed reduced binding to PTX3 in comparison with CFHR1*A. Thus, the interactions of PTX3 with complement regulators are impaired by certain mutations and autoantibodies affecting factor H and CFHR1, which could result in an enhanced local complement-mediated inflammation, endothelial cell activation, and damage in aHUS.     

New functional and structural insights from updated mutational databases for complement factor H,Factor I,membrane cofactor protein and C3

aHUS (atypical haemolytic uraemic syndrome), AMD (age-related macular degeneration) and other diseases are associated with defective AP (alternative pathway) regulation. CFH (complement factor H), CFI (complement factor I), MCP (membrane cofactor protein) and C3 exhibited the most disease-associated genetic alterations in the AP. Our interactive structural database for these was updated with a total of 324 genetic alterations. A consensus structure for the SCR (short complement regulator) domain showed that the majority (37%) of SCR mutations occurred at its hypervariable loop and its four conserved Cys residues. Mapping 113 missense mutations onto the CFH structure showed that over half occurred in the C-terminal domains SCR-15 to -20. In particular, SCR-20 with the highest total of affected residues is associated with binding to C3d and heparin-like oligosaccharides. No clustering of 49 missense mutations in CFI was seen. In MCP, SCR-3 was the most affected by 23 missense mutations. In C3, the neighbouring thioester and MG (macroglobulin) domains exhibited most of 47 missense mutations. The mutations in the regulators CFH, CFI and MCP involve loss-of-function, whereas those for C3 involve gain-of-function. This combined update emphasizes the importance of the complement AP in inflammatory disease, clarifies the functionally important regions in these proteins, and will facilitate diagnosis and therapy.     

Structure of complement factor H carboxyl-terminus reveals molecular basis of atypical haemolytic uremic syndrome

Factor H (FH) is the key regulator of the alternative pathway of complement. The carboxyl-terminal domains 19-20 of FH interact with the major opsonin C3b, glycosaminoglycans, and endothelial cells. Mutations within this area are associated with atypical haemolytic uremic syndrome (aHUS), a disease characterized by damage to endothelial cells, erythrocytes, and kidney glomeruli. The structure of recombinant FH19-20, solved at 1.8 A by X-ray crystallography, reveals that the short consensus repeat domain 20 contains, unusually, a short alpha-helix, and a patch of basic residues at its base. Most aHUS-associated mutations either destabilize the structure or cluster in a unique region on the surface of FH20. This region is close to, but distinct from, the primary heparin-binding patch of basic residues. By mutating five residues in this region, we show that it is involved, not in heparin, but in C3b binding. Therefore, the majority of the aHUS-associated mutations on the surface of FH19-20 interfere with the interaction between FH and C3b. This obviously leads to impaired control of complement attack on plasma-exposed cell surfaces in aHUS.     

Disease-associated N-terminal complement factor H mutations perturb cofactor and decay-accelerating activities

Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19-20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1-4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1-4(V62) and FH1-4(I62) bind immobilized C3b with similar affinities (K(D) = 10-14 μM), whereas FH1-4(I62) is slightly more effective than FH1-4(V62) as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1-4(V62) binds to C3b with comparable affinity (K(D) ～12 μM) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1-4(V62), binds poorly to immobilized C3b (K(D) >35 μM) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.     

Human Factor H-Related Protein 2 (CFHR2) Regulates Complement Activation

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.     

Clustering of missense mutations in the C-terminal region of factor H in atypical hemolytic uremic syndrome

Hemolytic-uremic syndrome (HUS) is a microvasculature disorder leading to microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Most cases of HUS are associated with epidemics of diarrhea caused by verocytotoxin-producing bacteria, but atypical cases of HUS not associated with diarrhea (aHUS) also occur. Early studies describing the association of aHUS with deficiencies of factor H suggested a role for this complement regulator in aHUS. Molecular evidence of factor H involvement in aHUS was first provided by Warwicker et al., who demonstrated that aHUS segregated with the chromosome 1q region containing the factor H gene (HF1) and who identified a mutation in HF1 in a case of familial aHUS with normal levels of factor H. We have performed the mutational screening of the HF1 gene in a novel series of 13 Spanish patients with aHUS who present normal complement profiles and whose plasma levels of factor H are, with one exception, within the normal range. These studies have resulted in the identification of five novel HF1 mutations in four of the patients. Allele HF1 Delta exon2, a genomic deletion of exon 2, produces a null HF1 allele and results in plasma levels of factor H that are 50% of normal. T956M, W1183L, L1189R, and V1197A are missense mutations that alter amino acid residues in the C-terminal portion of factor H, within a region--SCR16-SCR20--that is involved in the binding to solid-phase C3b and to negatively charged cellular structures. This remarkable clustering of mutations in HF1 suggests that a specific dysfunction in the protection of cellular surfaces by factor H is a major pathogenic condition underlying aHUS.     

Structure shows that a glycosaminoglycan and protein recognition site in factor H is perturbed by age-related macular degeneration-linked single nucleotide polymorphism

A common single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration. Factor H blocks the alternative pathway of complement on self-surfaces bearing specific polyanions, including the glycosaminoglycan chains of proteoglycans. Factor H also binds C-reactive protein, potentially contributing to noninflammatory apoptotic processes. The at risk sequence contains His (rather than Tyr) at position 402 (384 in the mature protein), in the seventh of the 20 complement control protein (CCP) modules (CCP7) of factor H. We expressed both His(402) and Tyr(402) variants of CCP7, CCP7,8, and CCP6-8. We determined structures of His(402) and Tyr(402) CCP7 and showed them to be nearly identical. The side chains of His/Tyr(402) have similar, solvent-exposed orientations far from interfaces with CCP6 and -8. Tyr(402) CCP7 bound significantly more tightly than His(402) CCP7 to a heparin affinity column as well as to defined-length sulfated heparin oligosaccharides employed in gel mobility shift assays. This observation is consistent with the position of the 402 side chain on the edge of one of two glycosaminoglycan-binding surface patches on CCP7 that we inferred on the basis of chemical shift perturbation studies with a sulfated heparin tetrasaccharide. According to surface plasmon resonance measurements, Tyr(402) CCP6-8 binds significantly more tightly than His(402) CCP6-8 to immobilized C-reactive protein. The data support a causal link between H402Y and age-related macular degeneration in which variation at position 402 modulates the response of factor H to age-related changes in the glycosaminoglycan composition and apoptotic activity of the macula.     

His-384 allotypic variant of factor H associated with age-related macular degeneration has different heparin binding properties from the non-disease-associated form

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.     

Antibody recognition of complement factor H reveals a flexible loop involved in atypical hemolytic uremic syndrome pathogenesis

Atypical hemolytic uremic syndrome (aHUS) is a disease associated with dysregulation of the immune complement system, especially of the alternative pathway (AP). Complement factor H (CFH), consisting of 20 domains called complement control protein (CCP1-20), downregulates the AP as a cofactor for mediating C3 inactivation by complement factor I. However, anomalies related to CFH are known to cause excessive complement activation and cytotoxicity. In aHUS, mutations and the presence of anti-CFH autoantibodies (AAbs) have been reported as plausible causes of CFH dysfunction, and it is known that CFH-related aHUS carries a high probability of end-stage renal disease. Elucidating the detailed functions of CFH at the molecular level will help to understand aHUS pathogenesis. Herein, we used biophysical data to reveal that a heavy-chain antibody fragment, termed VHH4, recognized CFH with high affinity. Hemolytic assays also indicated that VHH4 disrupted the protective function of CFH on sheep erythrocytes. Furthermore, X-ray crystallography revealed that VHH4 recognized the Leu1181–Leu1189_CCP20 loop, a known anti-CFH AAbs epitope. We next analyzed the dynamics of the C-terminal region of CFH and showed that the epitopes recognized by anti-CFH AAbs and VHH4 were the most flexible regions in CCP18-20. Finally, we conducted mutation analyses to elucidate the mechanism of VHH4 recognition of CFH and revealed that VHH4 inserts the Trp1183_CCP20 residue of CFH into the pocket formed by the complementary determining region 3 loop. These results suggested that anti-CFH AAbs may adopt a similar molecular mechanism to recognize the flexible loop of Leu1181-Leu1189_CCP20, leading to aHUS pathogenesis.     

Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein

Complement factor H (FH) is an important regulator of the alternative complement pathway. The Y402H polymorphism within the seventh short consensus repeat of FH was recently shown to be associated with age-related macular degeneration, the most common cause of irreversible blindness in the Western world. We examined the effects of this polymorphism on various FH functions. FH purified from sera of age-related macular degeneration patients homozygous for the FH(402H) variant showed a significantly reduced binding to C-reactive protein (CRP), an acute phase protein, as compared with FH derived from unaffected controls homozygous for the FH(402Y) variant. Strongly reduced binding to CRP was also observed with a recombinant fragment of FH (short consensus repeat 5-7) containing the same amino acid change. Because the interaction of CRP and FH promotes complement-mediated clearance of cellular debris in a noninflammatory fashion, we propose that the reduced binding of FH(402H) to CRP could lead to an impaired targeting of FH to cellular debris and a reduction in debris clearance and enhanced inflammation along the macular retinal pigmented epithelium-choroid interface in individuals with age-related macular degeneration.     

Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H-related 1 (CFHR1) and complement factor H-related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of approximately 84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.     

Mutations of Factor H Impair Regulation of Surface-bound C3b by Three Mechanisms in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19–20 (FH19–20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19–20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19–20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19–20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19–20 to C3b/C3d is essential for target discrimination by the alternative pathway.Atypical hemolytic uremic syndrome (aHUS)2 is a familial disease characterized by erythrocyte fragmentation and hematuria, damaged renal endothelium, vascular microthrombi, and thrombocytopenia (1). The syndrome leads ultimately to end-stage renal disease with a high mortality rate (2). In aHUS cases point mutations have been found in complement components C3, factor B, CD46, factor I, and factor H (FH), all of which play a role in the activation or control of the alternative pathway (3–8). More than half of the mutations have been found to originate in the HF1 gene that encodes FH and FH-like protein 1.The alternative pathway is initiated spontaneously by hydrolysis of C3 to C3_H2O that forms the C3-convertase C3_H2OBb (9, 10). This enzyme complex converts numerous C3 molecules to C3b that are covalently bound onto practically any nearby surface (11). On a so-called activator surface, such as a microbe, the surface-bound C3b molecules are not efficiently eliminated and therefore new C3bBb complexes are formed leading to more C3b depositions and eventually effective opsonization or damage of the target cell. On non-activator surfaces, such as viable self (host) cells, factor I cleaves C3b to inactive C3b (iC3b) in the presence of one of the cofactors (CD46, CD35, FH, and FHL-1) (12–16). FH is the only one of these cofactors that mediates recognition of self-surfaces making the alternative pathway capable of discriminating between activating and non-activating surfaces (17–19).The two main functions of FH are to prevent the alternative pathway activation in plasma and on self-surfaces. This 150-kDa glycoprotein consists of 20 tandemly arranged short consensus repeat domains that are composed of ∼60 amino acids. Domains 1–4 are essential for the cofactor and decay accelerating activity (20). In the middle region of FH (domains 5–15) there are two binding sites for C-reactive protein (21), one or two sites for glycosaminoglycans (GAGs) (22–25), and one site for C3c part of C3b (C3b/C3c) (25, 26). The C-terminal domains 19–20 (FH19–20) possess binding sites for the thiol ester domain of C3b (C3d or C3dg, TED domain) and GAGs (26, 27).The most common types of mutations found in aHUS are FH missense mutations located within FH19–20 that was recently solved as crystal and NMR structures (2, 28, 29). The C terminus of FH is crucial in self-cell protection as demonstrated by the severity of the aHUS cases and also in a recent mouse model of aHUS where domains 16–20 had been deleted (30, 31). Histopathology of aHUS in these mice had all the characteristics of human aHUS being concordant with the similarity of binding sites for C3b, heparin, and human umbilical vein endothelial cells between human and mouse FH domains 18–20 (32). Binding of mouse or human FH to glomerular endothelial cells has not been characterized despite the fact that in aHUS damage occurs mainly in the small vessels, especially in the glomeruli.The molecular pathogenesis leading to the clinical aHUS in patients with FH mutations remains elusive. The suggested molecular mechanisms for some aHUS-associated mutations include defective binding of the mutated FH to GAGs, endothelial cells, or C3b/C3d (28, 29, 33, 34). The aim of this study was to define the effects of nine aHUS-associated FH mutations and five other structurally closely located mutations on binding of FH19–20 to C3b, C3d, mouse glomerular endothelial cells, and heparin. We identified three primary defects of the mutants: impaired C3b/C3d binding, enhanced mGEnC-1/heparin binding, and impaired mGEnC-1/heparin binding that could lead via three mechanisms to incapability of FH to eliminate C3b on plasma-exposed self-cells. The results clarify the mechanism of target discrimination of the alternative pathway by the C terminus of FH.     

Deletion of Complement Factor H–Related Genes CFHR1 and CFHR3 Is Associated with Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H–related 1 (CFHR1) and complement factor H–related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of ~84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.     

Two genetic pathways for age-related macular degeneration

The discovery of strong associations of the His402 variant of complement factor H (CFH) and the change in the promoter region of HtrA serine peptidase 1 (HTRA1) with age-related macular degeneration (AMD) have altered our conception of the pathophysiology of this disease. The complement system has been placed at the center of a flurry of research interest, and a similar growth in attention to the serine proteases is not far behind. The specific role of these variants in causing AMD is unknown, but they will undoubtedly lead to a deeper understanding of the biological mechanisms and will point to new avenues for pharmacologic management. Furthermore, these variants will enable clinicians and investigators to identify people at high risk for this condition, thereby establishing the preconditions for preventing the disease.     

Atypical hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is defined by the triad of mechanical hemolytic anemia, thrombocytopenia and renal impairment. Atypical HUS (aHUS) defines non Shiga-toxin-HUS and even if some authors include secondary aHUS due to Streptococcus pneumoniae or other causes, aHUS designates a primary disease due to a disorder in complement alternative pathway regulation. Atypical HUS represents 5 -10% of HUS in children, but the majority of HUS in adults. The incidence of complement-aHUS is not known precisely. However, more than 1000 aHUS patients investigated for complement abnormalities have been reported. Onset is from the neonatal period to the adult age. Most patients present with hemolytic anemia, thrombocytopenia and renal failure and 20% have extra renal manifestations. Two to 10% die and one third progress to end-stage renal failure at first episode. Half of patients have relapses. Mutations in the genes encoding complement regulatory proteins factor H, membrane cofactor protein (MCP), factor I or thrombomodulin have been demonstrated in 20-30%, 5-15%, 4-10% and 3-5% of patients respectively, and mutations in the genes of C3 convertase proteins, C3 and factor B, in 2-10% and 1-4%. In addition, 6-10% of patients have anti-factor H antibodies. Diagnosis of aHUS relies on 1) No associated disease 2) No criteria for Shigatoxin-HUS (stool culture and PCR for Shiga-toxins; serology for anti-lipopolysaccharides antibodies) 3) No criteria for thrombotic thrombocytopenic purpura (serum ADAMTS 13 activity > 10%). Investigation of the complement system is required (C3, C4, factor H and factor I plasma concentration, MCP expression on leukocytes and anti-factor H antibodies; genetic screening to identify risk factors). The disease is familial in approximately 20% of pedigrees, with an autosomal recessive or dominant mode of transmission. As penetrance of the disease is 50%, genetic counseling is difficult. Plasmatherapy has been first line treatment until presently, without unquestionable demonstration of efficiency. There is a high risk of post-transplant recurrence, except in MCP-HUS. Case reports and two phase II trials show an impressive efficacy of the complement C5 blocker eculizumab, suggesting it will be the next standard of care. Except for patients treated by intensive plasmatherapy or eculizumab, the worst prognosis is in factor H-HUS, as mortality can reach 20% and 50% of survivors do not recover renal function. Half of factor I-HUS progress to end-stage renal failure. Conversely, most patients with MCP-HUS have preserved renal function. Anti-factor H antibodies-HUS has favourable outcome if treated early.     

Inhibiting alternative pathway complement activation by targeting the factor D exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.     

Localization of the heparin-binding site on complement factor H.

Factor H is a regulator of complement activation and, in this capacity, it prevents activation of the alternative pathway on host cells and tissues when it recognizes markers on these surfaces. This report describes the binding characteristics and location of the site on factor H that is responsible for host recognition. Factor H was found to bind a variety of polyanions, including heparin, heparan sulfate, dextran sulfate, and clusters of sialic acid. In heparin-agarose binding assays it exhibited an affinity for heparin only 2-fold weaker than that of antithrombin III. Factor H exhibited little or no affinity for polyaspartic acid or bacterial colominic acid (polysialic acid). Factor H (Mr 150,000 with approximate dimensions of 30 x 600 A) is composed of 20 highly homologous domains (SCRs) that are arranged as beads on a string. Polyanions were found to block a tryptic cleavage site in domain 15, and a photoaffinity-tagged heparin probe labeled the region between domains 12 and 15. Affinity chromatography of tryptic fragments on heparin-Sepharose confirmed that this region contained the heparin-binding site. CNBr cleavage at Met787 located between SCRs 13 and 14 split the photoaffinity-tagged region. Sequence analysis strongly suggests that domain 13 contains the primary site of polyanion binding. Factor H expresses its complement regulatory function through a site located in domains 4-6 where C3b binds. Thus, the polyanion-binding site that regulates the affinity of factor H for C3b appears to reside more than 200 A away from the C3b-binding site.     

Complement Factor H Binds at Two Independent Sites to C-reactive Protein in Acute Phase Concentrations

Factor H (FH) regulates the activation of C3b in the alternative complement pathway, both in serum and at host cell surfaces. It is composed of 20 short complement regulator (SCR) domains. The Y402H polymorphism in FH is a risk factor for age-related macular degeneration. C-reactive protein (CRP) is an acute phase protein that binds Ca²⁺. We established the FH-CRP interaction using improved analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and synchrotron x-ray scattering methods. Physiological FH and CRP concentrations were used in 137 mm NaCl and 2 mm Ca²⁺, in which the occurrence of denatured CRP was avoided. In solution, AUC revealed FH-CRP binding. The FH-CRP interaction inhibited the formation of higher FH oligomers, indicating that CRP blocked FH dimerization sites at both SCR-6/8 and SCR-16/20. SPR confirmed the FH-CRP interaction and its NaCl concentration dependence upon using either immobilized FH or CRP. The SCR-1/5 fragment of FH did not bind to CRP. In order of increasing affinity, SCR-16/20, SCR-6/8 (His-402), and SCR-6/8 (Tyr-402) fragments bound to CRP. X-ray scattering showed that FH became more compact when binding to CRP, which is consistent with CRP binding at two different FH sites. We concluded that FH and CRP bind at elevated acute phase concentrations of CRP in physiological buffer. The SCR-16/20 site is novel and indicates the importance of the FH-CRP interaction for both age-related macular degeneration and atypical hemolytic uremic syndrome.     

The Major Autoantibody Epitope on Factor H in Atypical Hemolytic Uremic Syndrome Is Structurally Different from Its Homologous Site in Factor H-related Protein 1, Supporting a Novel Model for Induction of Autoimmunity in This Disease

Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH_19–20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR1_4–5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH_19–20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH_19–20 and CFHR1_4–5. The crystal structure of CFHR1_4–5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH_19–20 and CFHR1_4–5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH_19–20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS.