A Novel Non-Synonymous Polymorphism (p.Arg240His) in C4b-Binding Protein Is Associated with Atypical Hemolytic Uremic Syndrome and Leads to Impaired Alternative Pathway Cofactor Activity¹

Atypical hemolytic uraemic syndrome (aHUS)³ is characterized by the triad of a microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure in the absence of a preceding diarrheal illness. aHUS, although rare, is a severe disease with death rates up to 25% in the acute phase of the disease and with 50% patients requiring kidney transplantation (1). aHUS can be either sporadic or familial and defective complement regulation occurs in both forms. The disease is associated with mutations in complement inhibitors (2) factor H (CFH) (3, 4, 5), membrane cofactor protein (MCP) (6, 7) and factor I (CFI) (8, 9) as well as gain of function mutations in both factor B (FB) (10) and C3 (11). Mutations in the genes encoding CD55 (12) and C4b-binding protein (C4BP) (13) have not yet been implicated in aHUS, although they are part of a single nucleotide polymorphism haplotype block strongly associated with severity of the disease (14).

C4BP is a plasma protein consisting of seven identical α-chains and a unique β-chain (15). The α- and β-chains contain eight and three complement control protein (CCP) domains (or short consensus repeats), respectively. CCP domains are characteristic feature of complement inhibitors and consist of ∼60 amino acids forming a compact hydrophobic core that is surrounded by five or more β-strands organized into β-sheets (16). C4BP controls mainly C4b-mediated reactions thereby inhibiting the classical and lectin pathways (17) but, it does act as a factor I (FI) cofactor in degradation of C3b and therefore contributes to regulation of the alternative pathway although not as strongly as factor H (FH) (18). Each α-chain of C4BP contains a C4b/C3b binding site, but most likely due to sterical hindrance, only up to four C4b or C3b can bind to one C4BP molecule (19). We have previously localized the binding site for C4b to CCP1–3 (20), whereas binding of C3b in addition requires CCP4 (i.e., CCP1–4) (18). C4BP interacts not only with C4b (21) and C3b (18) but also with anticoagulant protein S (22), serum amyloid P component (23), heparin (24), DNA (25), and a number of bacterial proteins (26).

So far, no inherited defects in C4BP were described in man, and in this study, we have examined whether C4BP is an additional susceptibility factor for aHUS.

The genes encoding C4BP α- and β-chains (C4BPA and C4BPB) were sequenced in 40 aHUS patients. In a further 126 aHUS patients and 542 normal controls (from European Collection of Cell Cultures, Health Protection Agency, Porton Down), genotyping of a novel nonsynonymous C4BPA single nucleotide polymorphism (c.719G>A, p.Arg240His) was undertaken either by direct sequencing or SnaPshot procedure. The amino acid numbering includes the signal sequence and nucleotide numbering was according to the published cDNA sequence (NM_000715.3) with the first nucleotide of the first methionine assigned as 1. The study was approved by the Northern and Yorkshire Multicentre Research Ethics Committee.

Genomic DNA was prepared, amplified using primers for C4BPA and C4BPB (Table I), and sequenced (6). Alternatively genotyping was performed with the ABI PRISIM SnaPshot dNTP Primer extension kit (Applied Biosystems) with subsequent detection with an ABI PRISIM 3100 Genetic analyser. SnaPshot reactions were performed using a primer 5′TCTTTAGAAATCACCTGTC at 0.4 μM concentration. The difference in genotype frequency between patients and controls was tested using the χ² test. The findings were replicated in an independent cohort of 170 aHUS patients using direct sequencing.

cDNA coding for human C4BP α-chain was in pcDNA3 vector (Invitrogen) (20), and the mutation was introduced using the QuikChange kit (Stratagene): Arg240His (primers: 5′GAA AAA ATC ACC TGT CAC AAG CCA GAT GTT TCA3′ and the corresponding antisense primer). Changed nucleotides are italic. The mutation was confirmed by sequencing. Both wild type (wt) and p.Arg240His were expressed in eukaryotic cells and purified by affinity chromatography (20). For circular dichroism analysis, both proteins were dialyzed extensively against 150 mM NaF. Approximately 120 μg of each mutant were analyzed in the far UV-region (185–250 nm) using Jasco J-720 spectropolarimeter. The resolution was 1 nm, the sensitivity 20 mdeg, the speed was 10 nm/min, and every presented spectrum is the average of eight measurements.

Human C4BP (27) and FI (28) were purified from human plasma as described in the references. C3b, C4b, C3, and factors B and D were from Complement Technology. Protein concentrations were determined from absorbance at 280 nm using known extinction coefficients or from amino acid analysis following 24-h hydrolysis in 6 M HCl (for recombinant C4BP). C4b, C3b, and C4BP were labeled with ¹²⁵I at 0.4–0.5 MBq/μg of protein using the chloramine T method.

Microtiter plates (Maxisorp, Nunc) were incubated overnight at 4°C with 50 μl of solution containing 10 μg/ml C4b or C3b in 75 mM Na-carbonate (pH 9.6). The wells were washed three times with washing buffer (50 mM Tris-HCl, 0.15 M NaCl, and 0.1% Tween (pH 7.5)) and then incubated at room temperature with 200 μl of quench (washing buffer supplemented with 3% fish gelatin). After another three washes, the ¹²⁵I-labeled plasma purified C4BP was added (20 kcpm/well for C4b and 50 kcp/well for C3b) together with unlabeled wt or the p.Arg240His mutant diluted in 50 mM Tris-HCl and 150 mM NaCl (pH 7.5) for C4b and in 25 mM Tris-HCl and 10 mM NaCl (pH 8.0) for C3b. The samples were incubated for 4 h at room temperature, washed three times with cold washing buffer, and the amount of radioactivity associated with each well was measured in a γ-counter (18, 20).

C4BP at 25–200 nM was mixed with 750 nM C3b (or 250 nM C4b), 60 nM FI, and trace amounts of ¹²⁵I-labeled C3b (or ¹²⁵I-C4b) in 50 μl of 50 mM Tris-HCl (pH 7.4) supplemented with 150 mM NaCl. The samples were incubated for 1.5 h at 37°C, and the reaction was terminated by the addition of SDS/PAGE sample buffer with reducing agent (dithiothreitol). The samples were then incubated at 95°C for 3 min and applied on 7.5–10% gradient SDS/PAGE gels. The separated proteins were visualized by autoradiography or using Phosphorimager (Molecular Dynamics/GE Healthcare).

Sheep erythrocytes (0.5 × 10⁹ cells/ml) were washed three times in GVB-Ni (1 mM veronal buffer (pH 7.3), containing 100 mM NaCl, 0.1% gelatin, and 2.5 mM NiCl₂). Erythrocytes were then incubated in a thermomixer (Eppendorf Scientific) at 800 rpm for 30 min at 30°C with C3 (0.5 mg/ml), factor B (0.02 mg/ml), and factor D (0.4 μg/ml). After two washes with GVB-Ni, the cells were treated with factors B and D for 5 min at 30°C, washed twice with GVB-EDTA (1 mM veronal buffer (pH 7.3), containing 100 mM NaCl, 0.1% gelatin, and 2 mM EDTA) and incubated with C3 for 20 min at 30°C. After the third and last cycle of incubation with factors B, D, and C3, the cells were washed with DGVB²⁺ (2.5 mM veronal buffer (pH 7.3), containing 70 mM NaCl, 140 mM dextrose, 0.1% gelatin, 1 mM MgCl₂, and 0.25 mM CaCl₂) and incubated for 30 min at 37°C with 20 μg/ml FI and 0–400 nM of recombinant C4BP. To control the amount of deposited C3b as well as generated iC3b, the cells were incubated with mouse mAbs directed against human C3d or iC3b Ab (3.33 μg/ml each Ab; Quidel) in PBS supplemented with 1% BSA, 15 mM EDTA, and 30 mM NaN₃ followed by matched FITC-labeled secondary Abs (10 μg/ml; DakoCytomation) and analysis by flow cytometry (FACS Calibur, BD Biosciences).

Alternatively, a hemolytic assay was used in which sheep erythrocytes were coated with C3b by sequential incubation with amboceptor, C1, C4, C2 and C3 to obtain EAC3b (30). Approximately 10⁷ EAC3b were incubated in Mg-EGTA (2.5 mM veronal buffered saline containing 140 mM dextrose, 0.1% gelatin, 7 mM MgCl₂ and 40 mM EGTA) with 70 nM C4BP and 80 nM FI at 37°C. Under these experimental conditions, 20–50% EC3b cells were converted to EC3bi. At indicated time intervals, 120 μl samples were removed, centrifuged and 120 μl of Mg-EGTA containing Factor B (25 ng/ml), Factor D (50 ng/ml) and properdin (0.5 μg/ml) were added to the pellet for 20 min at 30°C to form the C3bBb convertase. Lytic sites were developed by removing 50 μl of samples and adding 150 μl of guinea-pig serum diluted at 1:45 in GVB-EDTA (2.5 mM veronal buffered saline, 0.1% gelatin, 40 mM EDTA). After 1h incubation the samples were centrifuged and the amount of lysis was determined spectrophotometrically.

All exons of C4BPA and C4BPB were sequenced in 40 aHUS patients and lead to identification of a novel heterozygous nonsynonymous change (c.719G>A, p.Arg240His) in exon 7 of C4BPA (encodes CCP4 of the α-chain of C4BP). To analyze prevalence of this change, a larger group of patients and healthy controls was analyzed. We found this heterozygous change in 6/166 aHUS patients vs 5/542 normal controls (χ2 = 6.021, p = 0.014). The clinical details of the six aHUS patients carrying p.Arg240His are given in Table II. Three of six patients carry other known susceptibility factors for aHUS such as p.Ser206Pro in MCP and p.Gln950His in FH. Patient 4 presented with an associated diarrheal illness at the time of his HUS but his stool culture was clearly negative and therefore he was diagnosed as the aHUS case. However, a subsequent convalescent serum sample was positive for Escherichia coli O157 Abs, but it is not possible to know whether the Abs were generated during the infection associated with the current illness or perhaps previously in life. All patients recovered without need for renal replacement therapy. This is in contrast to patients carrying mutations in other complement inhibitors and who most often require transplantation. For instance, in our cohort, as in others, patients known to have a factor H mutation have a poor long-term outcome with ∼40% requiring renal replacement therapy (2).

To confirm our findings in and independent cohort of aHUS patients, we determined by direct sequencing the frequency of the Arg240His change in the French aHUS cohort in collaboration with Dr. Fremeaux-Bacchi. We found that 5 of 170 (3%) aHUS patients carried the Arg240His change (χ2 = 3.808, p = 0.051). Of the five carriers in that cohort, one also had a C3 mutation, one a heterozygous MCP mutation, one a CFI mutation, and one autoantibodies against FH while in one patient we could not identify any known susceptibility risk.

To determine whether C4BP expression was affected by the polymorphism, we have expressed p.Arg240His and wt in a transient manner in a human HEK293 cell line. We found that both the p.Arg240His and the wt expressed well (Fig. 1,A). We have also observed that stably transfected clones used for protein purification expressed comparable amounts of p.Arg240His and the wt. Accordingly, the plasma level of C4BP was normal in the five tested patients (Table II).

To analyze whether the polymorphism affects any function of C4BP, both recombinant C4BP molecules were expressed in a stable manner in a human HEK293 cell line and purified from culture media by affinity chromatography using mAb mAb104 directed against CCP1 of α-chain. Both forms of C4BP were analyzed by SDS/PAGE under both reducing and nonreducing conditions (Fig. 1,B). Both proteins migrated with the same apparent velocity and both were correctly assembled into high m.w. complexes composed of six α-chains. Furthermore, both proteins had similar folding as assessed by circular dichroism (Fig. 1 C). Taken together, it does not appear that the mutation cause any gross folding problems.

The ability of C4BP variants to bind C4b was tested using a direct binding assay in which microtiter plates were covered with C4b and the ¹²⁵I-C4BP was added together with increasing concentrations of the wt and the p.Arg240His mutant. We found that purified wt and p.Arg240His C4BP bound C4b well with the p.Arg240His showing significant 1.6-fold increase in the binding (Fig. 2,A). The difference between the two curves was statistically significant at a concentration of C4BP yielding 50% binding as determined by Student’s t test. To assess the FI cofactor activity of C4BP in a fluid phase, C4BP variants were incubated with ¹²⁵I-labeled C4b and FI. Proteins were then separated and visualized by autoradiography (Fig. 2,B) followed by densitometric detection of C4d product. The p.Arg240His mutant was equally as good a cofactor in the cleavage of C4b as wt (Fig. 2 C).

C4BP interacts with C3b and is an efficient cofactor in the degradation of C3b by FI (18, 31). CCP4, in which p.Arg240His resides, is required for binding of C3b (18). We found that p.Arg240His bound C3b with a significantly 4-fold lower affinity than wt (Fig. 3,A). We have used Student’s t test at a concentration of C4BP yielding 50% binding to assess significance of the difference. Three assays were used to test whether this impaired affinity for C3b had functional consequences. First, C4BP, FI and ¹²⁵I-C3b were incubated in a fluid phase and in the presence of the wt C4BP, α-chain subunit of C3b disappeared and several degradation products of 68, 46, and 3 kDa became apparent (Fig. 3,B). We have performed densitometric analysis of α-chain and 43 kDa degradation products and found that there was a significant difference in cofactor activity between wt and p.Arg240His at all concentrations tested (Fig. 3, B and C).

C4BP acts also as a cofactor in degradation of deposited C3b. Erythrocytes coated with C3b were incubated with FI and C4BP followed by detection of C3b and degradation product iC3b with specific Abs. Although wt C4BP degraded C3b into iC3b in a dose-dependent manner, p.Arg240His was severely impaired (Fig. 4,A). This was confirmed by independent hemolytic assay in which the mutant could not support degradation of C3b as well as the wt (Fig. 4 B). Thus, p.Arg240His clearly has impaired ability to regulate the alternative pathway of complement.

The involvement of the complement system in the pathogenesis of aHUS was suggested in the early observation that aHUS patients exhibited complement activation as reflected by low plasma C3 levels and/or C3 deposition in the kidney (32). Accordingly, genetic abnormalities in several complement inhibitors have been shown to predispose to aHUS and are found in ∼50% cases. Often more than one mutation or a predisposing polymorphism is found in the same patient. The complement regulator most extensively studied has been FH, in which aHUS-associated mutations affect binding to polyanionic glycosaminoglycans (33) and C3b (34), which decreases the protective effect of FH on endothelial cells. Mutations in MCP reduce its surface expression or/and ability to inhibit complement yet again predisposing renal endothelial cells vulnerable (35, 36). FH and MCP are both cofactors to FI, and therefore, it was not surprising that alterations in FI are also found in aHUS. The outcome of transplantation is poor in patients with either a CFH or FI mutation while patients known to have only a MCP mutation have a satisfactory transplantation outcome. This is expected because MCP is a transmembrane regulator and allografts will therefore be protected by wt MCP. Taken together, only two complement inhibitors have not been implicated in aHUS so far–decay accelerating factor (CD55) and C4BP. The present report is the first one to describe an alteration in the C4BP α-chain associated with aHUS. This novel non-synonymous polymorphism p.Arg240His was found in 6 of 166 investigated aHUS patients and in 5 of 177 patients in independent cohort. The p.Arg240His polymorphism was, however, also identified in 5 of 542 healthy controls. In two (siblings) of the aHUS patients carrying p.Arg240His polymorphism, we had previously identified a change in MCP (S206P), which severely impairs binding to C3b (36). In another patient, we found additional FH polymorphism that was in some (37) but not all studies (38) associated with aHUS. However, in the three remaining, not related, patients no additional predisposing factors were identified yet. We, therefore, suggest that C4BPA and in particular p.Arg240His may be another susceptibility factor for the development of aHUS. The phenotype of the six aHUS patients carrying this change is unusual in that all recovered renal function and suggests that the effect of p.Arg240His in C4BP is not as pronounced as in many patients carrying a CFH or CFI mutation.

The polymorphism does not appear to affect C4BP level in serum as observed by measurement of C4BP in the carriers and normal controls. Furthermore, both wt and p.Arg240His variant expressed well in eucaryotic cells that were transfected in a stable or transient manner. We have also analyzed whether p.Arg240His is equally stable in human serum as the wt. To this end equal, physiological concentrations of the two variants were added to serum depleted from C4BP and incubated for up to 24 h. The functional activity of the two variants was then assessed in a C4b-degradation assay and no differences were found (not shown).

Because p.Arg240His was also found in ∼1% of normal subjects, we have assessed the functional significance of this change. Therefore, recombinant p.Arg240His was expressed, purified, and compared with the wt. Interestingly, we did not detect any defect in binding of C4b and its ensuing degradation implying that p.Arg240His does not affect the ability of C4BP to regulate the classical and the lectin pathways of complement. This is in full agreement with our previous data showing that only CCP1–3 of the C4BP α-chain are needed for binding and degradation of C4b (20). The p.Arg240His polymorphism is located in CCP4 and should not affect interaction with C4b. However, the p.Arg240His mutant displayed statistically significant decreased binding to C3b and supported C3b degradation both in solution and on surface significantly less efficient than the wt. This observation is in good agreement with the fact that CCP1–4 are known to be required for full binding of C3b and its degradation (18). Analysis of 3D model of C4BP (39) shows that Arg240 is solvent exposed and forms a salt bridge with Asp284. The mutation, found in a domain interacting with C3b, could thus impede C3b binding either directly or indirectly, through minor local structural changes. Both Arg and His are classified as positively charged amino acids but there are significant differences between these two: His is a much shorter amino acid and it has much lower pKa value than Arg. Therefore, His may not always be positively charged depending on surrounding amino acids. Interestingly, arginines are often part of hot spots on protein surfaces, i.e., areas that are involved in interactions (40). In fact Arg240 forms the crucial part of an epitope for a mAb (MK67) directed against C4BP CCP4 (data not shown). This suggests that this Arg240 could definitively contribute to binding of C3b as its side chain is accessible for protein-protein interaction.

The association of C4BP with aHUS is not as strong as for most defects in CFH/MCP/CFI, and our findings suggest that C4BP is unlikely to be as significant a predisposing factor but they do support the hypothesis that C4BP is an additional susceptibility factor for the development of aHUS and that uncontrolled activation of the alternative pathway on cellular surfaces is pivotal for the disease (41). The relative importance of C4BP for the regulation of the alternative pathway in comparison with FH is not clear. In the quantitative in vitro comparison, we found that C4BP appears to be a comparable cofactor in degradation of C3b in a solution to FH but that C4BP is much weaker when C3b is to be degraded on surfaces (18). Considering the abundance of FH, the polymorphism in C4BP is unlikely to be a primary predisposing factor but a rather a secondary modifier which has an additive effect. Moreover, that this polymorphism was found in three patients with mutations in MCP and CFH adds weight to the hypothesis that individuals may carry multiple susceptibility factors in the complement system and that these have an additive effect on the risk of developing the disease.

The authors have no financial conflict of interest.