Figure 4
Figure 4. Sialic acid enhances FH binding to EsC3b. (A-B) Binding of FH to sialic acid in the absence of C3b was studied by using the MST technique. Fluorescently labeled (A) MAL II or (B) FH was mixed with increasing concentrations of the sialic acid containing compound 3′sialyllactose followed by thermophoresis. Results of experiments performed in triplicate are represented as spheres, triangles, and squares. Binding data for MAL II was fitted with NanoTemper software to obtain the binding isotherm and the respective dissociation constant (Kd). (C) To study FH binding to sialic acid in the context of C3b, C3b was deposited on Es by using purified C3 and factors B and D. C3b deposition on Es was verified by showing increased binding of anti-C3c antibody using flow cytometry. (D) Erythrocytes with C3b deposition on them (EsC3b) were treated with sialidase to remove sialic acid from their surfaces. Efficiency of the sialidase treatment was verified by showing decreased binding of MAL II lectin to sialidase-treated cells by flow cytometry. (E) The level of C3b deposition on EsC3b was not affected by treatment with sialidase, as judged by similar binding of anti-C3c antibody to untreated and sialidase-treated EsC3b cells. (C-E) Representative histograms of assays performed 3 to 6 times. (F-I) Binding of FH to untreated and sialidase-treated EsC3b was competed with wt and mutant FH19-20 fragments. Binding of fluorescently labeled FH was analyzed by flow cytometry. (F) Removal of sialic acid from EsC3b decreased FH binding to the cells. The wt FH19-20 had a major inhibitory effect on FH binding to untreated EsC3b but only a small effect on FH binding to the sialidase-treated EsC3b. (G) FH binding to untreated EsC3b in the presence of wt and mutant FH19-20. (H) A dose-response study of the ability of various FH19-20 mutants to antagonize FH binding to untreated EsC3b. Some controls (FH19-20 mutants W1157L, R1182A, and R1215Q) were included in the assay. (I) FH binding to sialidase-treated EsC3b in the presence of wt and mutant FH19-20. The assays were performed (H) 3 or (F-G, I) 5 times. Shown are average GMFI values (± SD). One-way ANOVA with Tukey’s multiple comparison posttests was performed.*P < .05; **P < .01; ***P < .001.

Sialic acid enhances FH binding to EsC3b. (A-B) Binding of FH to sialic acid in the absence of C3b was studied by using the MST technique. Fluorescently labeled (A) MAL II or (B) FH was mixed with increasing concentrations of the sialic acid containing compound 3′sialyllactose followed by thermophoresis. Results of experiments performed in triplicate are represented as spheres, triangles, and squares. Binding data for MAL II was fitted with NanoTemper software to obtain the binding isotherm and the respective dissociation constant (Kd). (C) To study FH binding to sialic acid in the context of C3b, C3b was deposited on Es by using purified C3 and factors B and D. C3b deposition on Es was verified by showing increased binding of anti-C3c antibody using flow cytometry. (D) Erythrocytes with C3b deposition on them (EsC3b) were treated with sialidase to remove sialic acid from their surfaces. Efficiency of the sialidase treatment was verified by showing decreased binding of MAL II lectin to sialidase-treated cells by flow cytometry. (E) The level of C3b deposition on EsC3b was not affected by treatment with sialidase, as judged by similar binding of anti-C3c antibody to untreated and sialidase-treated EsC3b cells. (C-E) Representative histograms of assays performed 3 to 6 times. (F-I) Binding of FH to untreated and sialidase-treated EsC3b was competed with wt and mutant FH19-20 fragments. Binding of fluorescently labeled FH was analyzed by flow cytometry. (F) Removal of sialic acid from EsC3b decreased FH binding to the cells. The wt FH19-20 had a major inhibitory effect on FH binding to untreated EsC3b but only a small effect on FH binding to the sialidase-treated EsC3b. (G) FH binding to untreated EsC3b in the presence of wt and mutant FH19-20. (H) A dose-response study of the ability of various FH19-20 mutants to antagonize FH binding to untreated EsC3b. Some controls (FH19-20 mutants W1157L, R1182A, and R1215Q) were included in the assay. (I) FH binding to sialidase-treated EsC3b in the presence of wt and mutant FH19-20. The assays were performed (H) 3 or (F-G, I) 5 times. Shown are average GMFI values (± SD). One-way ANOVA with Tukey’s multiple comparison posttests was performed.*P < .05; **P < .01; ***P < .001.

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