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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2014 Apr 10;25(11):2425–2433. doi: 10.1681/ASN.2013070732

Characterization of a Factor H Mutation That Perturbs the Alternative Pathway of Complement in a Family with Membranoproliferative GN

Edwin KS Wong *, Holly E Anderson *, Andrew P Herbert †, Rachel C Challis *, Paul Brown ‡, Geisilaine S Reis *, James O Tellez *, Lisa Strain *, Nicholas Fluck ‡, Ann Humphrey ‡, Alison Macleod ‡, Anna Richards §, Daniel Ahlert *, Mauro Santibanez-Koref *, Paul N Barlow †, Kevin J Marchbank ‖, Claire L Harris , Timothy HJ Goodship *, David Kavanagh *,✉
PMCID: PMC4214516  PMID: 24722444

Abstract

Complement C3 activation is a characteristic finding in membranoproliferative GN (MPGN). This activation can be caused by immune complex deposition or an acquired or inherited defect in complement regulation. Deficiency of complement factor H has long been associated with MPGN. More recently, heterozygous genetic variants have been reported in sporadic cases of MPGN, although their functional significance has not been assessed. We describe a family with MPGN and acquired partial lipodystrophy. Although C3 nephritic factor was shown in family members with acquired partial lipodystrophy, it did not segregate with the renal phenotype. Genetic analysis revealed a novel heterozygous mutation in complement factor H (R83S) in addition to known risk polymorphisms carried by individuals with MPGN. Patients with MPGN had normal levels of factor H, and structural analysis of the mutant revealed only subtle alterations. However, functional analysis revealed profoundly reduced C3b binding, cofactor activity, and decay accelerating activity leading to loss of regulation of the alternative pathway. In summary, this family showed a confluence of common and rare functionally significant genetic risk factors causing disease. Data from our analysis of these factors highlight the role of the alternative pathway of complement in MPGN.

Keywords: complement, membranoproliferative GN (MPGN), immunology

Membranoproliferative GN (MPGN) is a pathologic term describing thickening of the glomerular capillary wall and an increase in mesangial components.1 MPGN has historically been classified by electron microscopy according to the relationship of the electron dense deposits relative to the glomerular basement membrane (MPGN1, dense deposit disease [DDD], and MPGN3).2 Immunofluorescence reveals C3 staining in all types of MPGN; however, many cases also stain for Igs. An increased understanding of the role of complement in the pathogenesis of disease has led to a proposed reclassification of those cases with no or minimal Ig staining as C3 glomerulopathy (C3GN).3–6

Hypocomplementaemia has long been associated with MPGN, and in 1969, C3 nephritic factor (C3Nef) was discovered. It was subsequently shown to be an antibody that stabilized the alternative pathway (AP; C3bBb) convertase,7–10 leading to increased complement turnover. Acquired partial lipodystrophy (APL) has also been reported in patients with MPGN,11 often in association with C3Nef.

Familial forms of MPGN caused by complete deficiency of complement factor H (fH) have been reported.12 In a pedigree with DDD reported by Licht et al.,13 a homozygous mutation resulting in a secreted, functionally inactive fH protein has been described. Additionally, an activating C3 mutation has been reported in a family with DDD.14 More recently, a hybrid CFHR3-1 gene15 and a CFHR116 mutation have been described in familial C3GN. Genetic variants in the complement system have also been described in sporadic MPGN/C3GN in a large cohort of French patients with no family history.17

We examine a family with MPGN and APL previously described by Power et al.18 in 1990 (Figure 1, clinical details in Supplemental Material). Patients 1:2, 2:1, and 2:2 all developed nephrotic syndrome and ESRD, with individuals 1:2 and 2:1 undergoing renal biopsy to confirm MPGN (Figure 1, B–E). Given the family history of MPGN and the low C3 level, patient 2:2 was assumed to have MPGN and did not undergo renal biopsy, although he was diabetic. Patients 1:2, 2:1, and 2:4 developed APL. Historically, patients 1:2, 2:1, and 2:4 all had low C3 levels in keeping with contemporaneous C3Nef results (Supplemental Table 1).

Figure 1.

Figure 1.

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Family pedigree and renal pathology. (A) Pedigree showing segregation of biopsy-proven MPGN, ESRD, and APL. Patient 2:2 was assumed to have MPGN because of the familial nature of disease and the low C3 levels. He did not have a confirmatory renal biopsy, although he was also diabetic. Patients carrying the heterozygous CFH rare genetic variant (CFH R83S) are highlighted. Individuals 2:3 and 2:4 do not carry the R83S variant. The common, functionally significant haplotypes (CFH H3/H5) and SNPs (C3 R102G and C3 P314L) are shown where analyzed. No patient carried the MCPaaggt haplotype associated with C3GN and MPGN117 (Supplemental Table 1). C3Nef status is highlighted. R, reference sequence; V, variant sequence; +ve, positive; −ve, negative. (B) Renal biopsy of patient 1:2 at age 32 years showing double layering of the glomerular basement membrane (methenamine silver stain). (C) Postmortem kidney biopsy 9 years later showing diffuse global endocapillary proliferation and double layering of glomerular basement membrane (hematoxylin and eosin). (D) High-power view of part of the glomerular tuft on the right and Bowman’s capsule and the beginning of proximal tubule on the left showing double layering of the glomerular basement membrane (methenamine silver stain). (E) Electron microscopy of patient 2:1 showing subendothelial and mesangial deposits.

Genetic analysis of this family revealed that all individuals with the renal phenotype (1:2, 2:1, and 2:2) carry a mutation in heterozygosity in the CFH gene. The mutation c.249G>T results in a nonsynonymous substitution in the N-terminal region of fH, p.R83S (Figures 1 and 2A). Patients 2:3 and 2:4 did not carry this mutation.

Figure 2.

Figure 2.

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Structural effects of R83S mutation. (A) R83S mutation displayed on the fH/C3b cocrystal structure. An x-ray–derived cocrystal structure of fH/C3b19 was used to model the mutation and displayed with Pymol (Delano Scientific). The location of the R83S mutation (red spheres) is shown within the cocrystal structure of an fH1–4 (light gray)–C3b (dark gray) complex. The R83 aa is in direct opposition to C3b (Protein Database ID code 2WII).19 (B) 15N-heteronuclear single quantum coherence spectra of fH1–2WT and fH1–2R83S were acquired at 37°C, and resonances were assigned where possible by comparison with previously assigned fH1–2WT spectra.22 Overlay of 15N-heteronuclear single quantum coherence spectra of fH1–2 WT (blue) and R83S (red). It is clear that both spectra show good chemical shift dispersion consistent with a well structured protein, implying that this mutation does not result in local unfolding of the protein. (C) A graphical representation of the combined 1H and 15N chemical shift differences of R83S with respect to WT chemical shifts. Residues for which no chemical shift difference could be ascribed have been given a value of −0.01. The majority of the residues exhibits only minor chemical shift differences (only 18 aa with combined chemical shift difference greater than the threshold of 0.05 ppm), indicating that the overall fold of the protein should remain largely unchanged as a result of this mutation. (D) Cartoon representation of the chemical shift difference; line thickness and color (blue to red with increasing chemical shift difference) indicate the degree of chemical shift difference. The positions of proline residues (for which it is not possible to assign chemical shifts) are displayed in black, and residues with chemical shift that could not be confidently assigned are displayed in white. It is clear from this representation that the mutation R83 results in only localized changes in the structure of the protein; however, these changes are located at the intermodular interface between CCPs 1 and 2.

To determine the structural effects of the R83S mutation, nuclear magnetic resonance (NMR) spectroscopy was used. The overlay of 15N-heteronuclear single quantum coherence spectra (Figure 2B) shows a well dispersed spectrum indicative of a well structured protein for both wild-type (WT; fH1–2WT) and fH1–2R83S. Moreover, differences between the two spectra were relatively few in number and clustered into the intermodular interface (Figure 2, C and D). These observations rule out the suggestion that the R83S substitution significantly disrupted the structure of fH and imply that the protein should be secreted normally in keeping with the normal fH serum level. They suggest that the R83S mutation could elicit its effects through either direct interaction of the R83 side chain with C3bBb or an alteration to the fH1–2 intermodular orientation. Modeling this mutation on a cocrystal structure of fH/C3b19 showed that amino acid (aa) R83 is in direct opposition to C3b, and it would seem most likely that this residue directly participates in the interaction of fH with C3b (Figure 2A).

To confirm functional significance of the genetic variant, WT (fH1–4WT) and mutant (fH1–4R83S) recombinant proteins, comprising the functional domains of fH (complement control protein modules 1–4 [CCPs 1–4]), were generated. Surface plasmon resonance (SPR) showed that the affinity of fH1–4R83S for C3b is weak compared with fH1–4WT. Using the quantity of fH1–4R83S available (40 μM in the solution flowed over the chip), it was not possible to accurately determine the Kd. It may be estimated that the Kd of the fH1–4R83S–C3b interaction is approximately 75 μM under the same conditions used to measure a Kd of 10.7 μM for fH1–4WT–C3b (Figure 3).

Figure 3.

Figure 3.

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WT and mutant (R83S) fH1–4 binding to C3b as measured by SPR. C3b was immobilized to a CM5 chip using standard amine coupling. Duplicate injections of WT and R83S were performed (concentrations of 0.02–40 µM) in 10 mM Hepes-buffered saline with 3 mM EDTA and 0.05% (vol/vol) surfactant p20. Overlaid sensograms show steady-state response (resonance units) showing binding of (A) WT (0.04–20 μM) and (B) R83S (0.04–40 µM) to C3b. Plots of steady-state response (resonance units) versus concentration used to estimate Kd values are shown for (C) WT and (D) R83S. The binding affinity curve is fitted, and dissociation constants were calculated using a steady-state fitted model. The R83S exhibits much weaker binding to C3b (Kd approximately 75 μM; Rmax=23.42 RU; chi-squared=0.0736) than the WT (Kd=10.7 µM; Rmax=65.23 RU; chi-squared=0.552). RU, resonance units.

To assess the functional consequences of the impaired binding to C3b, fluid-phase cofactor activity assays were undertaken, showing minimal activity of the fH1–4R83S mutant (Figure 4, A and B). Sheep erythrocyte cell surface assays of cofactor activity confirmed minimal activity of fH1–4R83S compared with fH1–4WT (Figure 4C).

Figure 4.

Figure 4.

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Cofactor and decay acceleration activity. Limiting concentrations of fH1–4 WT and mutant (R83S) were incubated with factor I and the substrate, C3b, for increasing time points. (A) The products were run on SDS-PAGE/Western blot, scanned using laser densitometry, and analyzed by GELscan software. (B) The kinetic analysis shows the loss of C3b cleavage activity (ratio of α-chain to β-chain normalized to percentage of negative control) of R83S compared with WT. (C) C3b cofactor activity on sheep erythrocytes. WT and mutant (R83S) fH1–4 were tested for the ability to act as a cofactor for factor I–catalyzed inactivation of C3b deposited on the surfaces of sheep erythrocytes. The C3 convertase (C3bBb) was formed on residual C3b and lysis commences. Maximal lysis occurs in the presence of buffer only (0 μM fH). The addition of fH1–4 causes inactivation of C3b and therefore, less convertase formation, which is shown as increasing amounts of inhibition of lysis (expressed as percentage of maximal lysis) after incubation with factor I and fH1–4 WT (black circles) or R83S (white circles). The R83S mutant can be seen to be less active than WT. (D) Decay-accelerating SPR assay. Using a Biacore ×100 instrument, C3b was coupled to the CM5 sensorchip by standard amine coupling. Subsequently, a mixture of factor B (500 nM) and factor D (60 nM) was flowed over the surface to form the C3 convertase. WT and mutant (R83S) fH1–4 in running buffer (i.e., 10 mM Hepes-buffered 150 mM saline [pH 7.4], 0.05% [vol/vol] surfactant p20, and 1 mM MgCl2) was flowed over the chip, and convertase decay was visualized in real time. The R83S showed impaired decay acceleration compared with WT. (E) Decay acceleration activity on sheep erythrocytes. Alternative pathway convertase (C3bBb) was formed on sheep erythrocytes. Cells were incubated for 15 minutes with dilutions of WT and mutant (R83S) fH1–4 before instigating lysis with normal human serum depleted of factor B and fH. Maximal lysis occurs in buffer-only (0 μM fH) conditions. Addition of fH1–4 caused decay of the C3 convertase and therefore, results in inhibition of lysis. The R83S mutant was up to 75-fold less efficient at inhibiting lysis.

SPR-based decay acceleration assays were then performed, showing markedly reduced function of fH1–4R83S compared with the WT protein (Figure 4D). Sheep erythrocyte cell surface decay acceleration assays confirmed an approximately 75-fold reduction in activity of the mutant protein compared with WT. For fH1–4WT, 50% inhibition of lysis was seen at 5.98 nM, and for fH1–4R83S, 50% inhibition of lysis was seen at 447.5 nM (Figure 4E).

Thus, in this family, we describe a mutation in CFH that segregates with renal disease and results in minimal disruption of structure but leads to a profound loss of complement regulation. Complete fH deficiency or homozygous functionally inactivating mutations in CFH had been reported in cases of MPGN/C3GN previously. This family carries one normally functioning allele and the abnormal allele encoding R83S. More recently, several patients with MPGN1 carrying a heterozygous genetic variant have been reported,17 albeit without functional studies confirming a pathogenic role.

Genotyping of functional nonsynonymous single-nucleotide polymorphisms (SNPs) associated with MPGN20 was subsequently undertaken. In addition to a functionally significant CFH mutation, patients 2:1 and 2:2 had one copy of the CFH H3 haplotype and one CFH H5 haplotype.21 These CFH haplotypes both express a valine at aa 62, which has been shown to be less effective in complement regulation compared with the alternate isoleucine at aa 62.22,23 Likewise, all affected members carried SNPs in C3 (R102G [C3 slow/fast polymorphism]24 /P314L), which have previously been shown to bind fH less strongly, and thus, C3b is less efficiently inactivated.25,26 Both these SNPs, which result in increased AP activity, have been associated with an increased risk of DDD.20

It is interesting to note that, in addition to a functionally significant mutation in CFH and functionally significant SNPs, C3Nef was also detected. C3Nef was first described in 1969 in patients with MPGN and low C3 levels.10 DDD is classically associated with APL, although a recent review suggested that one half of the patients had MPGN1.11 In one series of 26 patients with C3Nef, 18 patients developed MPGN alone, 2 patients had APL alone, and 6 patients had both diseases.24 C3Nef is also found in normal control individuals, which has led to questions about its role in pathogenesis.27 The coexistence of a CFH mutation and C3Nef was also described by Licht et al.13 in DDD and in a case series of MPGN/C3GN by Servais et al.17

It may be hypothesized that C3Nef is partly responsible for the development of renal disease by working in concert with R83S to impair C3 convertase inactivation. However, in the family described herein, C3Nef segregated with APL and not the renal phenotype, which segregates with R83S. We cannot rule out the possibility that individual 2:2 may have been C3Nef-positive at some stage and subsequently, became C3Nef-negative. Clearly, the presence of C3Nef on its own does not result in MPGN in this family, because individual 2:4 does not have renal disease. The familial nature of the APL and C3Nef suggests an underlying genetic predisposition. No genetic variants were found in lipodystrophy-associated genes (Supplemental Table 2); however, all individuals with APL and C3Nef carried the C3 R102G polymorphism previously associated with C3Nef.24 This SNP may have predisposed to generation of C3Nef and consequently, APL. Factor D is highly expressed in adipose tissue and required for formation of the C3 convertase. It has been suggested that loss of fat may be because of C3Nef-induced lysis of adipocytes expressing factor D.28

In the initial report of this family, the term MPGN1 was used because of the heavy staining of IgM and less intense IgG staining in patient 1:2.18 Contemporary immunofluorescence is not available to reassess this pathology in the light of new classifications.6,29 Immune complexes may have triggered complement activation, unmasking the latent genetic complement defect. Alternatively, Igs may have deposited nonspecifically in areas of sclerosis and scarring.30 Genetic variants in AP genes have been detected among individuals with MPGN1.17 In this family, having definitively established the abnormal control of complement activation in disease, the term C3GN could be used, which was suggested by Pickering et al.6

In summary, in this family, we describe a mutation in CFH, which results in minimal disruption of structure but leads to a profound loss of complement regulation and segregates with renal disease. Members of the family with MPGN also carry functionally significant C3GN risk polymorphisms. It is likely that the cosegregation of these risk factors causes insufficient control of the AP, resulting in MPGN.

Concise Methods

The study was approved by Newcastle and North Tyneside 1 Research Ethics Committee, and informed consent was obtained in accordance with the Declaration of Helsinki.

Complement Assays

C3 and C4 levels were measured by rate nephelometry (Beckman Coulter Array 360). fH and factor I levels were measured by radioimmunodiffusion (Binding Site).

Genetic Analysis

Mutation screening of CFH, CFI, CFB, MCP, and C3 was undertaken using Sanger sequencing.31–35 Genotyping of the following SNPs, CFH 2332C>T (rs3753394); CFH c.184G>A, isoleucine at aa 62 V (rs800292); CFH c.1204C>T, H402Y (rs1061170); CFH c.2016A>G, Q672Q (rs3753396); and CFH c.2808G>T, E936D (rs1065489), was used to determine the CFH haplotypes. Screening for genomic disorders affecting CFH, CFHR1, CFHR2, CFHR3, and CFHR5 was undertaken using multiplex ligation-dependent probe amplification as previously described.36 For investigation of APL genetics, whole-exome sequencing was used (Supplemental Material).

Production and Purification of Proteins

Clones of Pichia pastoris strain KM71H producing WT (fH1–4WT) and mutant (fH1–4R83S) fH in the setting of CCPs 1–4 were generated as described previously.37 In brief, the R83S point mutation was generated in a pPICZαB (Invitrogen) vector containing residues 19–263 of fH, with a C-terminal 6× His tag and an N-terminal myc tag (EQKLISEEDL), using the QuikChange site-directed mutagenesis kit (Stratagene) with the following primers: (f) gggttgctcttaatccattaaggaaatgtcagaaaagTccctgtggacatcctggagatactcc; (r)ggagtatctccaggatgtccacagggActtttctgacatttccttaatggattaagagcaaccc. Fidelity was confirmed by bidirectional Sanger sequencing. KM71H cells were transformed using electroporation, selected by zeocin, and screened for protein expression.

Protein expression was carried out in a 3-L BioFlo 115 Biofermenter (New Brunswick). A starter culture was transferred into 1 L basal fermentor salts (0.095% [wt/vol] calcium sulfate, 1.82% [wt/vol] potassium sulfate, 1.5% [wt/vol] magnesium sulfate heptahydrate, 0.42% [wt/vol] potassium hydroxide, 2.7% [vol/vol] phosphoric acid, and 2.5% [vol/vol] glycerol) enriched with 1% (wt/vol) casein aa, 0.5% (wt/vol) PTM1 salts, and 0.5% (vol/vol) antifoam A (Sigma-Aldrich). After the initial batch-fed glycerol was exhausted, glycerol feeds were maintained for 24 hours at 30°C. The cells were allowed to starve for 4 hours before recombinant expression was induced with 0.75% methanol containing 0.5% (wt/vol) PTM1 salts. After 3 days at 15°C with methanol feeds, the supernatant was removed and filtered, and its pH was adjusted to 7.4.

It was applied to a 5-ml His trap column (GE Healthcare) at 4°C, and the protein was eluted with 500 mM imidazole followed by size exclusion chromatography on Superdex 200 (GE Healthcare). Protein identity was confirmed by mass spectrometry. Protein concentrations were calculated using absorbance at 280 nm and calculated extinction coefficients (47,870 M−1⋅cm−1).

NMR analysis was performed in the context of a previously generated fragment of fH22 (CCPs 1 and 2; residues 19–142) using QuikChange with the primers above as previously described; 15N-labeled samples of both WT and mutant were prepared by using (15NH4)2SO4 (Isotec) as the sole source of nitrogen during cell growth. Protein expression was carried out using baffled shaker flasks. A starter culture was transferred into three flasks (each containing 1 L buffered minimal glycerol) and incubated at 30°C for 48 hours. Protein expression was then induced daily with 0.5% methanol feeds for 72 hours. Purification of this fragment from supernatant after centrifugation of the cell pellet was achieved by cation-exchange chromatography at pH 4.0 (SP Sepharose Fast Flow; Sigma-Aldrich). Additional purification steps followed using size exclusion chromatography on Superdex 200 (GE Healthcare) and reverse-phase high-performance liquid chromatography (Supelco Discovery BIO Wide Pore C5 Column; Supelco, Inc.).

NMR Spectroscopy

NMR spectra were acquired on an 800-MHz Bruker Avance III spectrometer fitted with a TCI cryogenic probe. Two-dimensional 1H-15N-heteronuclear single quantum coherence spectra were acquired at 37°C with 100 µM samples of the fH1–2 proteins in 20 mM deuterated sodium acetate (pH 4.5) containing 10% (vol/vol) D2O and 0.02% (wt/vol) NaN3. Resonance assignments were determined where possible by comparison with the previously assigned WT spectrum.22 Chemical shift differences between the fH1–2WT and fH1–2R83S proteins were determined using the CCPNMR Analysis Suite of Programs. Normalized log values of the chemical shift differences were entered into the B factor field of the Protein Data Bank file (2RLP) and plotted onto cartoon representations of the representative structure of fH1–2 WT using Pymol (http://www.pymol.org/).38

Binding Affinity for C3b by SPR

The binding affinities of the fH1–4WT and fH1–4R83S were monitored by SPR using a Biacore ×100 instrument (GE Healthcare); 500 resonance units of human C3b (CompTech) were immobilized on a Biacore series CM5 sensor chip (GE Healthcare) using standard amine coupling.37 The reference surface of the chip was prepared by performing a mock coupling in the absence of any protein. Experiments were performed at 25°C and a 30-μl/min flow rate. Duplicate injections (concentrations of 0.5–40 μM) were performed in 10 mM Hepes-buffered saline, 3 mM EDTA, and 0.05% (vol/vol) surfactant p20 (GE Healthcare). A contact time of 90 seconds was used (sufficient for achieving steady-state conditions for all samples) followed by a dissociation period of 600 seconds. Chips were regenerated between sample injections with two 45-second injections of 1 M NaCl (pH 7.0). Data were processed using the BIAevaluation 4.1 software (GE Healthcare). Data from the reference cell and a blank (buffer) injection were subtracted, and dissociation constants were calculated using a steady-state affinity model from the background-subtracted traces.

Measurement of Decay Acceleration Activity by SPR

Decay accelerating activity was measured in real time using a Biacore ×100 instrument as described previously.37 Briefly, 2300 resonance units C3b were immobilized using standard amine coupling to the CM5 sensor chip. Subsequently, a mixture of factor B (500 nM) and factor D (60 nM) was flowed (10 μl/min) over the surface for 120 seconds to form the AP convertase.

The convertase was allowed to decay naturally for 210 seconds, fH1–4WT or fH1–4R83S (in running buffer of Hepes-buffered saline containing 0.5% [vol/vol] surfactant P20 and 1 mM MgCl2 [temperature at 25°C]) was flowed across the surface for 60 seconds, and convertase decay was visualized in real time. Between injections, surfaces were regenerated using a 45-second injection of 1 μM purified fH (CompTech) followed by a 45-second injection of 1 M NaCl (pH 7.0). Data were evaluated using BIAevaluation 4.1 (GE Healthcare). As a control, the construct was also flowed over the bare surface, and binding data were subtracted from the decay sensogram.

Cofactor Assay in Fluid Phase

Kinetic fluid-phase assays were used to measure cofactor activity for factor I–mediated proteolytic cleavage of C3b.37 Factor I, fH, and C3b were all purchased from CompTech; 3 μl C3b (5.68 μM), 4.5 μl factor I (1.14 μM), and 5 μl fH1–4 (0.75 μM) were made up to a final volume of 15 μl in PBS. For the positive control reaction, 5 μl purified fH (6.45 μM) was used, and for the negative control, 5 μl PBS was used. The mixture was incubated at 37°C for 15, 30, 60, and 120 minutes, and the reaction was stopped by the addition of 2× lamelli reducing buffer to a final volume of 30 μl and heated to 95°C for 5 minutes. The products of the reaction were then separated by SDS-PAGE and transferred to nitrocellulose membrane. The C3b α- and β-chain fragments were visualized using rabbit anti-C3 antibody (1:5000; Abcam, Inc.) and goat anti-rabbit horseradish peroxidase (1:5000; Abcam, Inc.) secondary and chemoluminescence substrate (Thermo Fisher Scientific).

Measuring Decay Acceleration on Sheep Erythrocytes

C3b-coated sheep erythrocytes were prepared as described previously.23 Cells were resuspended to 2% (vol/vol) in AP buffer (5 mM sodium barbitone [pH 7.4], 150 mM NaCl, 7 mM MgCl2, and 10 mM EGTA). The AP convertase was formed on the cell surface by incubating 50 μl cells with an equal volume of AP buffer containing factor B (40 μg/ml) and factor D (0.4 μg/ml; CompTech) at 37°C for 15 minutes. Cells (100 μl) were incubated with 50 μl concentration range fH1–4WT and fH1–4R83S in PBS/20 mM EDTA for 15 minutes. Lysis was developed by adding 50 μl 4% (vol/vol) normal human serum depleted of factor B and fH23 in PBS/20 mM EDTA and incubating at 37°C for 60 minutes. To determine the amount of lysis, cells were pelleted by centrifugation, and hemoglobin release was measured at 410 nm (A410). Controls included 0% lysis (buffer only) and 100% lysis (0.1% [vol/vol] Nonidet P-40). Percentage of inhibition from lysis was calculated by the formula (A410[buffer only]−A410[fH])/A410[buffer only]×100%.

Measuring Cofactor Activity on Sheep Erythrocytes

To test cofactor activity,37 washed C3b-coated sheep erythrocyte cells were resuspended to 2% (vol/vol) in AP buffer and incubated with an equal volume of a range of concentrations of fH1–4WT and fH1–4R83S and 2.5 μg/ml factor I (CompTech) for 8 minutes at 25°C. After three washes in AP buffer, a 50-μl aliquot of cells (2%) was mixed with 50 μl AP buffer containing factor B (40 μg/ml) and factor D (0.4 μg/ml) and then incubated for 15 minutes at 25°C to form AP convertase on the remaining C3b. Lysis was developed by adding 50 μl 4% (vol/vol) normal human serum depleted of factor B and fH in PBS/20 mM EDTA and incubating at 37°C for 10 minutes. Percentage of inhibition from lysis was calculated by the formula (A410[buffer only]−A410[fH])/A410[buffer only]×100%.

Disclosures

A.R., T.H.J.G., and D.K. have received honoraria for consultancy work from Alexion Pharmaceuticals.

Supplementary Material

Supplemental Data
supp_25_11_2425__index.html (892B, html)

Acknowledgments

The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under Grant 305608 (EURenOmics). This work was supported by Kidney Research UK Grant RP29/2010, including a grant from the Rosetrees Trust. E.K.S.W. is a Medical Research Council clinical training fellow. G.S.R. is funded by Instituto Nacional de Ciência e Tecnologia de Medicina Molecular—Universidade Federal de Minas Gerais. A.R. and D.K. are Wellcome Trust intermediate clinical fellows. D.A. was funded by the Wellcome Trust.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

References

  • 1.Sethi S, Fervenza FC: Membranoproliferative glomerulonephritis—a new look at an old entity. N Engl J Med 366: 1119–1131, 2012 [DOI] [PubMed] [Google Scholar]
  • 2.Anders D, Thoenes W: Basement membrane-changes in membranoproliferative glomerulonephritis: A light and electron microscopic study. Virchows Arch A Pathol Anat Histol 369: 87–109, 1975 [DOI] [PubMed] [Google Scholar]
  • 3.Bomback AS, Appel GB: Pathogenesis of the C3 glomerulopathies and reclassification of MPGN. Nat Rev Nephrol 8: 634–642, 2012 [DOI] [PubMed] [Google Scholar]
  • 4.Sethi S, Nester CM, Smith RJ: Membranoproliferative glomerulonephritis and C3 glomerulopathy: Resolving the confusion. Kidney Int 81: 434–441, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Fakhouri F, Frémeaux-Bacchi V, Noël LH, Cook HT, Pickering MC: C3 glomerulopathy: A new classification. Nat Rev Nephrol 6: 494–499, 2010 [DOI] [PubMed] [Google Scholar]
  • 6.Pickering MC, D’Agati VD, Nester CM, Smith RJ, Haas M, Appel GB, Alpers CE, Bajema IM, Bedrosian C, Braun M, Doyle M, Fakhouri F, Fervenza FC, Fogo AB, Frémeaux-Bacchi V, Gale DP, Goicoechea de Jorge E, Griffin G, Harris CL, Holers VM, Johnson S, Lavin PJ, Medjeral-Thomas N, Paul Morgan B, Nast CC, Noel LH, Peters DK, Rodríguez de Córdoba S, Servais A, Sethi S, Song WC, Tamburini P, Thurman JM, Zavros M, Cook HT: C3 glomerulopathy: Consensus report. Kidney Int 84: 1079–1089, 2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Scott DM, Amos N, Sissons JG, Lachmann PJ, Peters DK: The immunogloblin nature of nephritic factor (NeF). Clin Exp Immunol 32: 12–24, 1978 [PMC free article] [PubMed] [Google Scholar]
  • 8.Daha MR, Fearon DT, Austen KF: C3 nephritic factor (C3NeF): Stabilization of fluid phase and cell-bound alternative pathway convertase. J Immunol 116: 1–7, 1976 [PubMed] [Google Scholar]
  • 9.Thompson RA: C3 inactivating factor in the serum of a patient with chronic hypocomplementaemic proliferative glomerulo-nephritis. Immunology 22: 147–158, 1972 [PMC free article] [PubMed] [Google Scholar]
  • 10.Spitzer RE, Vallota EH, Forristal J, Sudora E, Stitzel A, Davis NC, West CD: Serum C’3 lytic system in patients with glomerulonephritis. Science 164: 436–437, 1969 [DOI] [PubMed] [Google Scholar]
  • 11.Misra A, Peethambaram A, Garg A: Clinical features and metabolic and autoimmune derangements in acquired partial lipodystrophy: Report of 35 cases and review of the literature. Medicine (Baltimore) 83: 18–34, 2004 [DOI] [PubMed] [Google Scholar]
  • 12.Pickering MC, Cook HT: Translational mini-review series on complement factor H: Renal diseases associated with complement factor H: Novel insights from humans and animals. Clin Exp Immunol 151: 210–230, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Licht C, Heinen S, Józsi M, Löschmann I, Saunders RE, Perkins SJ, Waldherr R, Skerka C, Kirschfink M, Hoppe B, Zipfel PF: Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel pathomechanism for dense deposit disease (MPGN II). Kidney Int 70: 42–50, 2006 [DOI] [PubMed] [Google Scholar]
  • 14.Martínez-Barricarte R, Heurich M, Valdes-Cañedo F, Vazquez-Martul E, Torreira E, Montes T, Tortajada A, Pinto S, Lopez-Trascasa M, Morgan BP, Llorca O, Harris CL, Rodríguez de Córdoba S: Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation. J Clin Invest 120: 3702–3712, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Malik TH, Lavin PJ, Goicoechea de Jorge E, Vernon KA, Rose KL, Patel MP, de Leeuw M, Neary JJ, Conlon PJ, Winn MP, Pickering MC: A hybrid CFHR3-1 gene causes familial C3 glomerulopathy. J Am Soc Nephrol 23: 1155–1160, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Tortajada A, Yébenes H, Abarrategui-Garrido C, Anter J, García-Fernández JM, Martínez-Barricarte R, Alba-Domínguez M, Malik TH, Bedoya R, Cabrera Pérez R, López Trascasa M, Pickering MC, Harris CL, Sánchez-Corral P, Llorca O, Rodríguez de Córdoba S: C3 glomerulopathy-associated CFHR1 mutation alters FHR oligomerization and complement regulation. J Clin Invest 123: 2434–2446, 2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Servais A, Noël LH, Roumenina LT, Le Quintrec M, Ngo S, Dragon-Durey MA, Macher MA, Zuber J, Karras A, Provot F, Moulin B, Grünfeld JP, Niaudet P, Lesavre P, Frémeaux-Bacchi V: Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int 82: 454–464, 2012 [DOI] [PubMed] [Google Scholar]
  • 18.Power DA, Ng YC, Simpson JG: Familial incidence of C3 nephritic factor, partial lipodystrophy and membranoproliferative glomerulonephritis. Q J Med 75: 387–398, 1990 [PubMed] [Google Scholar]
  • 19.Wu J, Wu YQ, Ricklin D, Janssen BJ, Lambris JD, Gros P: Structure of complement fragment C3b-factor H and implications for host protection by complement regulators. Nat Immunol 10: 728–733, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Abrera-Abeleda MA, Nishimura C, Frees K, Jones M, Maga T, Katz LM, Zhang Y, Smith RJ: Allelic variants of complement genes associated with dense deposit disease. J Am Soc Nephrol 22: 1551–1559, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Pickering MC, de Jorge EG, Martinez-Barricarte R, Recalde S, Garcia-Layana A, Rose KL, Moss J, Walport MJ, Cook HT, de Córdoba SR, Botto M: Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains. J Exp Med 204: 1249–1256, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hocking HG, Herbert AP, Kavanagh D, Soares DC, Ferreira VP, Pangburn MK, Uhrín D, Barlow PN: Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations. J Biol Chem 283: 9475–9487, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Tortajada A, Montes T, Martínez-Barricarte R, Morgan BP, Harris CL, de Córdoba SR: The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity. Hum Mol Genet 18: 3452–3461, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Finn JE, Mathieson PW: Molecular analysis of C3 allotypes in patients with nephritic factor. Clin Exp Immunol 91: 410–414, 1993 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Harris CL, Heurich M, Rodriguez de Cordoba S, Morgan BP: The complotype: Dictating risk for inflammation and infection. Trends Immunol 33: 513–521, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Heurich M, Martínez-Barricarte R, Francis NJ, Roberts DL, Rodríguez de Córdoba S, Morgan BP, Harris CL: Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk. Proc Natl Acad Sci U S A 108: 8761–8766, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Spitzer RE, Stitzel AE, Tsokos G: On the origin of C3 nephritic factor (antibody to the alternative pathway C3 convertase): Evidence for the Adam and Eve concept of autoantibody production. Clin Immunol Immunopathol 64: 177–183, 1992 [DOI] [PubMed] [Google Scholar]
  • 28.Mathieson PW, Würzner R, Oliveria DB, Lachmann PJ, Peters DK: Complement-mediated adipocyte lysis by nephritic factor sera. J Exp Med 177: 1827–1831, 1993 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Hou J, Markowitz GS, Bomback AS, Appel GB, Herlitz LC, Barry Stokes M, D’Agati VD: Toward a working definition of C3 glomerulopathy by immunofluorescence. Kidney Int 85: 450–456, 2014 [DOI] [PubMed] [Google Scholar]
  • 30.Bolton WK, Benton FR, Maclay JG, Sturgill BC: Spontaneous glomerular sclerosis in aging Sprague-Dawley rats. I. Lesions associated with mesangial IgM deposits. Am J Pathol 85: 277–302, 1976 [PMC free article] [PubMed] [Google Scholar]
  • 31.Richards A, Buddles MR, Donne RL, Kaplan BS, Kirk E, Venning MC, Tielemans CL, Goodship JA, Goodship TH: Factor H mutations in hemolytic uremic syndrome cluster in exons 18-20, a domain important for host cell recognition. Am J Hum Genet 68: 485–490, 2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Richards A, Kemp EJ, Liszewski MK, Goodship JA, Lampe AK, Decorte R, Müslümanoğlu MH, Kavukcu S, Filler G, Pirson Y, Wen LS, Atkinson JP, Goodship TH: Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc Natl Acad Sci U S A 100: 12966–12971, 2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kavanagh D, Kemp EJ, Richards A, Burgess RM, Mayland E, Goodship JA, Goodship TH: Does complement factor B have a role in the pathogenesis of atypical HUS? Mol Immunol 43: 856–859, 2006 [DOI] [PubMed] [Google Scholar]
  • 34.Kavanagh D, Kemp EJ, Mayland E, Winney RJ, Duffield JS, Warwick G, Richards A, Ward R, Goodship JA, Goodship TH: Mutations in complement factor I predispose to development of atypical hemolytic uremic syndrome. J Am Soc Nephrol 16: 2150–2155, 2005 [DOI] [PubMed] [Google Scholar]
  • 35.Frémeaux-Bacchi V, Miller EC, Liszewski MK, Strain L, Blouin J, Brown AL, Moghal N, Kaplan BS, Weiss RA, Lhotta K, Kapur G, Mattoo T, Nivet H, Wong W, Gie S, Hurault de Ligny B, Fischbach M, Gupta R, Hauhart R, Meunier V, Loirat C, Dragon-Durey MA, Fridman WH, Janssen BJ, Goodship TH, Atkinson JP: Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome. Blood 112: 4948–4952, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Francis NJ, McNicholas B, Awan A, Waldron M, Reddan D, Sadlier D, Kavanagh D, Strain L, Marchbank KJ, Harris CL, Goodship TH: A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome. Blood 119: 591–601, 2012 [DOI] [PubMed] [Google Scholar]
  • 37.Pechtl IC, Kavanagh D, McIntosh N, Harris CL, Barlow PN: Disease-associated N-terminal complement factor H mutations perturb cofactor and decay-accelerating activities. J Biol Chem 286: 11082–11090, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Herbert AP, Kavanagh D, Johansson C, Morgan HP, Blaum BS, Hannan JP, Barlow PN, Uhrín D: Structural and functional characterization of the product of disease-related factor H gene conversion. Biochemistry 51: 1874–1884, 2012 [DOI] [PubMed] [Google Scholar]

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