Figure 1.
CFHR2, CFHR2/1-2 and CFHR2/3-4 protein expression and purification.
(A) Domain composition of CFHR2 and CFHR2 fragments CFHR2/1-2 and CFHR2/3-4. Numbers indicate amino acids. (B) Recombinant purified full length CFHR2 and two CFHR2 fragments (CFHR2/1-2 and CFHR2/3-4) were separated by SDS-PAGE and detected by silver staining and Western blot using CFHR2 specific antiserum. CFHR2 appeared with mobility of ∼27 to 33 kDa, and both CFHR2 mutants of ∼16 to 18 kDa. (C) Determination of the solution oligomeric state of CFHR2 proteins. Recombinant CFHR2 proteins were purified by Ni2+-chelat chromatography, deglycosylated and purified by gel chromatography (Superdex 200 or Superdex 75). Analysis of the solution oligomeric state of CFHR2, (D) CFHR2/1-2 and (E) CFHR2/3-4 by static light scattering in combination with size exclusion chromatography. The light scattering signals (LS) are shown as the mass distribution (Mw) in each of the peaks in the elution profiles monitored by the absorbance at 280 nm (UV) and changes of the refractive index (dRI). The molar mass of each protein is indicated as well as the status of dimerization.
Figure 2.
CFHR2 binds via the C-terminus to C3b and C3d.
(A) CFHR2 SCR domains are aligned against the highly related SCR domains of CFHR1 and factor H. The number above each SCR indicates the identity to the corresponding domain in CFHR1 respectively factor H. The regulatory region (yellow), SCRs 6–7 (green), and the surface recognition site (blue) of factor H are marked. (B) CFHR2 binds to C3b, C3dg and C3d, but not to C3c. In parallel factor H binding was determined. Equimolar amounts of CFHR2 and factor H were used. Data represent mean values ± SD of four independent ELISA experiments. Background binding of the antibodies to ligand C3b alone (lig ctrl) or without ligand C3b (Ab ctrl) is shown. (C) CFHR2 binds via the two C-terminal domains to C3b and C3d. The proteins were used in equimolar amounts. Data represent mean values ± SD of three independent experiments. Background binding of the antibodies to ligand C3b or C3d alone (ctrl) is shown.
Figure 3.
CFHR2 binds to C3b and C3d and does not compete off factor H.
(A) Real-time in vitro SPR binding analysis of CFHR2 (left panel) and factor H (right panel) to sensor immobilized C3b. CFHR2 and factor H binding sensograms (black lines) are shown overlaid with the best fit derived from a 1∶1 interaction model including a mass transport term (red lines). As CFHR2 forms exclusively dimers, one CFHR2 dimer was regarded as one molecule. (B) CFHR2 slightly competes off factor H from binding to C3b (left panel) and does not effect factor H binding to C3d (right panel). Mean values ± SD of three independent experiments and the molar ratios of factor H (1 = 66 nM) to CFHR2 are shown. (D) factor H binds preferentially to C3b and to C3d in the presence of constant amounts of CFHR2 (1 = 66 nM). Constant amounts of CFHR2 and increasing concentrations of factor H were incubated with immobilized C3b or C3d. Binding rates of CFHR2 and factor H alone to C3b or C3d are represented by dashed lines. A representative experiment is shown. (E) C-terminal amino acid sequence alignment of CFHR2 SCR3 and SCR4 with factor H SCR19 and SCR20. Conserved cysteins (I–IV, brown), identical (grey) and non-identical amino acids (red) are marked. Charged amino acids that are relevant for C3b and heparin binding are indicated (green).
Figure 4.
CFHR2 regulates AP complement activation.
(A) CFHR2 inhibits AP activation in NHS. (B) CFHR2 had no effect on CP activation in NHS. TCC deposition in untreated NHS was set as 100%. C4bp is a classical pathway inhibitor. (C) CFHR2 and CFHR2/1–2 but not CFHR2/3–4 inhibited C3b deposition. (D) C3a generation in NHS activated via AP (ELISA). C3b depositon in untreated NHS was set as 100%. (E) CFHR2 together with factor H increased inhibition of C3b deposition in NHS as compared to CFHR2 or factor H alone. Data in (A–E) represent the mean values ± SD of three independent experiments (*p≤0.05, ***p≤0.001). Control (ctrl) indicates binding of the detection Ab to the plate.
Figure 5.
CFHR2 inhibits the AP C3 convertase.
CFHR2 inhibited in vitro assembled AP C3 convertase as measured by C3a generation. The corresponding densitometric analysis of the C3a bands of the Western blot is shown. C3a generation by C3 convertase alone was normalized to 100%. A representative analysis of three experiments is shown.
Figure 6.
CFHR2 exerts neither decay acceleration nor cofactor activity.
(A) CFHR2 can not dissociate factor B from the in vitro assembled convertase C3bBb, but factor H efficiently decays factor Bb fragment from C3b. (B) CFHR2 does not influence cleavage of C3b bound factor B by factor D. CFHR2 preincubated NHS, which was activated by surface immobilized IgM via the AP and Ba cleavage was followed by ELISA. A representative experiment is shown. (C) CFHR2 alone lacks cofactor activity for factor I to degrade C3b (lanes 6–8). CFHR2 does not interfere with factor H cofactor activity (lanes 2–5). Increasing amounts of CFHR2 (1–100 µg/ml) were incubated with C3b and factor I in the presence or absence of factor H. Reaction mixtures were separated by SDS PAGE and immunoblotted using a C3 polyclonal antibody.
Figure 7.
CFHR2 inhibits terminal pathway activation.
(A) CFHR2 (blue column), factor H (gray column) and vitronectin (Vn, gray column) were preincubated with purified TCC components C7, C8,C9 and added to C5b-6 loaded SRBC. TCC mediated erythrocyte lysis was monitored by measuring the absorbance of the supernatant at 414 nm. Reduced absorbance correlates with inhibition of the TCC formation on SRBC. (B) CFHR2 inhibited TCC formation via the N-terminal SCRs 1–2 in a dose dependent manner. CFHR2 proteins were preincubated with TCC components and lysis of SRBC was measured. Data in (A) and (B) represent mean values ± SD of three independent experiments (***p≤0.001).
Figure 8.
Scematic overview of CFHR2 functions in regulation of the AP.
CFHR2 inhibits the amplification loop by inhibiting C3 cleavage by C3 convertases and acts on the assembly of the terminal complement complex.