Figure 7
Figure 7. Functional activity of fH and fH/CFHR3 using isolated C3b and C3b bound to the surface of sheep erythrocytes. (A-B) Cofactor activity was tested in the fluid phase by incubating C3b, fI with differing concentrations (1:2 serial dilution) of either fH or (fH/CFHR3 (Tyr402 variants, position on gel indicated by */**). Functional activity of fH is indicated by loss of the C3b α′ chain and cleavage to the 68-kDa and 41-kDa bands. Both fH and fH/CFHR3 have similar cofactor activity in the fluid phase indicating that the amino terminal domains are fully functional. (C) Cofactor activity was confirmed by immobilizing C3b on a biacore chip (1200 RU) and flowing fH (7nM, top) or fH/CFHR3 (6nM, bottom) over the surface with fI (5 μg/mL). The surface was regenerated, and the ability to form convertase was measured after (black line) treatment with cofactor and fI and compared with activity before (gray line) treatment (fH, top; fH/CFHR3, bottom). Both fH and fH/CFHR3 had cofactor activity as evidenced by decrease in ability of C3b to form convertase. (D) Decay accelerating ability for the AP convertase was tested by forming C3bBb on a Biacore chip surface (370 RU amine-coupled C3b). After 160 seconds of natural decay, fH (34nM, Tyr variant from control, gray line) or fH/CFHR3 mutant protein (34nM, black line) flowed across the surface (120 seconds). Both efficiently decayed the convertase as did the fH His402 variants from carrier or control (dashed lines). Background binding of fH or fH/CFHR3 to the C3b surface alone was subtracted, and these data are shown. (E) Decay accelerating activity of purified fH and the hybrid protein was tested in hemolysis assays. C3b-coated sheep erythrocytes bearing the AP C3 convertase were incubated with fH proteins for a set length of time. The extent of lysis developed using NHSΔBH reflected the residual convertase remaining after incubation with fH proteins. Proteins from both a control person and a carrier of the fH/CFHR3 hybrid protein were tested (▾ represents fH/CFHR3 carrierTyr402; ▴, carrier His402; ■, control Tyr402; and ●, control His402). (F) Cell surface cofactor activity of purified fH proteins was assessed by incubating with C3b-coated sheep erythrocytes and fI for a defined length of time. Cells were washed and AP convertase formed on the residual C3b by incubation with factor B and factor D. Lysis was developed using NHSΔBH and reflected the residual C3b remaining after incubation with fH proteins; symbols as in panel E.

Functional activity of fH and fH/CFHR3 using isolated C3b and C3b bound to the surface of sheep erythrocytes. (A-B) Cofactor activity was tested in the fluid phase by incubating C3b, fI with differing concentrations (1:2 serial dilution) of either fH or (fH/CFHR3 (Tyr402 variants, position on gel indicated by */**). Functional activity of fH is indicated by loss of the C3b α′ chain and cleavage to the 68-kDa and 41-kDa bands. Both fH and fH/CFHR3 have similar cofactor activity in the fluid phase indicating that the amino terminal domains are fully functional. (C) Cofactor activity was confirmed by immobilizing C3b on a biacore chip (1200 RU) and flowing fH (7nM, top) or fH/CFHR3 (6nM, bottom) over the surface with fI (5 μg/mL). The surface was regenerated, and the ability to form convertase was measured after (black line) treatment with cofactor and fI and compared with activity before (gray line) treatment (fH, top; fH/CFHR3, bottom). Both fH and fH/CFHR3 had cofactor activity as evidenced by decrease in ability of C3b to form convertase. (D) Decay accelerating ability for the AP convertase was tested by forming C3bBb on a Biacore chip surface (370 RU amine-coupled C3b). After 160 seconds of natural decay, fH (34nM, Tyr variant from control, gray line) or fH/CFHR3 mutant protein (34nM, black line) flowed across the surface (120 seconds). Both efficiently decayed the convertase as did the fH His402 variants from carrier or control (dashed lines). Background binding of fH or fH/CFHR3 to the C3b surface alone was subtracted, and these data are shown. (E) Decay accelerating activity of purified fH and the hybrid protein was tested in hemolysis assays. C3b-coated sheep erythrocytes bearing the AP C3 convertase were incubated with fH proteins for a set length of time. The extent of lysis developed using NHSΔBH reflected the residual convertase remaining after incubation with fH proteins. Proteins from both a control person and a carrier of the fH/CFHR3 hybrid protein were tested (▾ represents fH/CFHR3 carrierTyr402; ▴, carrier His402; ■, control Tyr402; and ●, control His402). (F) Cell surface cofactor activity of purified fH proteins was assessed by incubating with C3b-coated sheep erythrocytes and fI for a defined length of time. Cells were washed and AP convertase formed on the residual C3b by incubation with factor B and factor D. Lysis was developed using NHSΔBH and reflected the residual C3b remaining after incubation with fH proteins; symbols as in panel E.

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