Fig. 7: MFHR13 binds C5 and MAC components and blocks the terminal pathway.
From: A synthetic protein as efficient multitarget regulator against complement over-activation
a C5 binding was measured by ELISA. Nonlinear regression using the sigmoidal equation and comparison of IC50 showed similar binding profiles for MFHR13 and MFHR1I62 to C5 (P = 0.2565). Data represent mean values ± SD from 4 or 5 independent experiments. Absorbance values from one representative experiment are shown in Supplementary Fig. 8c. b MFHR1 binds to the individual TCC proteins; hFH does not. MFHR1 or hFH (25 nM) as well as the negative controls BSA and the purified extract from the parental line (Δxt/ft) were added to immobilized complement proteins in microtiter plates and detected by anti-FH polyclonal antibodies. Data represent mean values ± SD from 3 independent experiments. c MFHR13, MFHR1, and FHR1 bind to the terminal complement proteins involved in MAC formation. Regulators (25 or 50 nM) were added to immobilized terminal complement proteins and bound proteins were detected by anti-His-tag antibodies. Data represent mean values ± SD from 3 or 4 independent experiments. Absorbance values from one representative experiment from (b) and (c) are shown in Supplementary Fig. 9. d Binding of NT-647 RED-NHS-labeled MFHR13 to C3d, C5, C7, and C9 was evaluated by Microscale Thermophoresis (MST) in fluid phase. Normalized fluorescence (ΔFnorm) was plotted against the concentration of single complement components. e MFHR13 and FHR1 inhibit the formation of the MAC on sheep erythrocytes. The inhibition by MFHR13 and FHR1 was significantly better than hFH (P = 0.0018), eculizumab (P < 0.0001), BSA (P = 0.0012), or Δxt/ft. Hemolysis of sheep erythrocytes was induced with addition of C5b6, C7, C8, and C9 and release of hemoglobin was determined by measuring the absorbance at 405 nm. The average absorbance values without C5b6 and without C9 were subtracted from the positive control and samples. Lysis induced by TCC in absence of regulators was set to 100%. Data represent mean values ± SD from three independent experiments (one-way ANOVA, Bonferroni post hoc test). f MFHR13 (700 nM) inhibits MAC formation on C3b-opsonized sheep erythrocytes significantly better than FHR1 (P = 0.0415), hFH (P = 0.0018), eculizumab (P < 0.0001), BSA (P = 0.0012), cytochrome c (P = 0.0007), and Δxt/ft. Hemolysis of C3b-coated sheep erythrocytes was induced with a mix of C5-C9 and determined by absorbance of released hemoglobin at 405 nm. Lysis without regulators was set to 100%. The average absorbance values without C5 were subtracted from all samples. Data represent mean values ± SD from three independent experiments (one-way ANOVA, Bonferroni post hoc test). ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05, ns (no significant difference).
