American Society of Hematology

Figure 7

$Figure 7. PfRh4 disrupts CR1's decay accelerating activity. (A) Decay acceleration of C3bBb was inhibited on PfRh4 binding. SPR was used to monitor formation of the C3bBb convertase complex as factor D and factor B were flowed together over C3b that was amine-coupled to a CM5 sensor chip (i, C3bBb formation). The subsequent decline in response reflects decay of the complex as Bb is released from the chip surface (ii, spontaneous decay). The rate of decay was accelerated by initiating a flow of CCP1-3 or factor H CCP1-4 (FH1-4; iii, analyte injection), but not when CCP1-3 was in the presence of 3-fold molar excess of rPfRh4. In panels A-D, any convertases remaining were decayed by injecting FH1-4 at ∼ 800 seconds to aid regeneration (B) Decay-acceleration of CCP1-3 was affected in a dose-dependent manner by rPfRh4. The rate of decay was monitored in the presence of increasing concentrations of rPfRh4. A small fraction of inactive rPfRh4 present on the surface triggers nonspecific background binding when the first duplicate injection was assayed. (CCP1-3 in 3-fold molar excess of PfRh4) as manifested in the imperfect reproducibility of the sensorgrams. After saturation of this nonspecific binding capacity, injections were of acceptable reproducibility (all other constructs). (C) Decay-acceleration activity of sCR1 was affected by presence of rPfRh4. Shown are binding responses for sCR1, and for sCR1 in the presence of a 5-fold molar excess of PfRh4, of the convertase C3bBb (+C) or C3b alone (−C). Three C3b-binding sites in sCR1 mediate the overall high binding levels to both the convertase and C3b alone. Only sCR1 binding to the convertase (+C) shows a distinctive association curve that is consistent with an initially enhanced binding of sCR1 to the convertase, followed by 2 simultaneous, overlapping processes: accelerated decay of C3bBb into Bb and surface-bound C3b, and binding of sCR1 to C3b. (D) Decay acceleration by factor H CCP1-4 was not affected by rPfRh4. Assays shown in panels A and C, and in panels B and D were performed on identical biosensor surfaces. (E) Hemolysis assay for the classic pathway. The classic pathway C3 convertase was assembled on the surface of antibody-sensitized sheep erythrocytes. PfRh4 plus CCP1-3 were pre-incubated before mixing with sheep erythrocytes. Cellular lysis was induced by the addition of guinea pig serum and monitored by the O.D. of the supernatant at 414nM. CCP1-3 was used at a concentration of 6.8nM. (F) Hemolysis assay for the alternative pathway. The alternative pathway convertase was prepared using EAC14 cells by the addition of C2 and C3. PfRh4 plus CCP1-3 were preincubated and these mixtures were then added to EAC43 cells and the alternative pathway components factor B, factor D, and properdin and were added and lysis measured as in panel E. A convertase was not formed in the absence of factor B. CCP1-3 was used at a concentration of 34nM. For all panels, **P < .01; ***P < .001).$

PfRh4 disrupts CR1's decay accelerating activity. (A) Decay acceleration of C3bBb was inhibited on PfRh4 binding. SPR was used to monitor formation of the C3bBb convertase complex as factor D and factor B were flowed together over C3b that was amine-coupled to a CM5 sensor chip (i, C3bBb formation). The subsequent decline in response reflects decay of the complex as Bb is released from the chip surface (ii, spontaneous decay). The rate of decay was accelerated by initiating a flow of CCP1-3 or factor H CCP1-4 (FH1-4; iii, analyte injection), but not when CCP1-3 was in the presence of 3-fold molar excess of rPfRh4. In panels A-D, any convertases remaining were decayed by injecting FH1-4 at ∼ 800 seconds to aid regeneration (B) Decay-acceleration of CCP1-3 was affected in a dose-dependent manner by rPfRh4. The rate of decay was monitored in the presence of increasing concentrations of rPfRh4. A small fraction of inactive rPfRh4 present on the surface triggers nonspecific background binding when the first duplicate injection was assayed. (CCP1-3 in 3-fold molar excess of PfRh4) as manifested in the imperfect reproducibility of the sensorgrams. After saturation of this nonspecific binding capacity, injections were of acceptable reproducibility (all other constructs). (C) Decay-acceleration activity of sCR1 was affected by presence of rPfRh4. Shown are binding responses for sCR1, and for sCR1 in the presence of a 5-fold molar excess of PfRh4, of the convertase C3bBb (+C) or C3b alone (−C). Three C3b-binding sites in sCR1 mediate the overall high binding levels to both the convertase and C3b alone. Only sCR1 binding to the convertase (+C) shows a distinctive association curve that is consistent with an initially enhanced binding of sCR1 to the convertase, followed by 2 simultaneous, overlapping processes: accelerated decay of C3bBb into Bb and surface-bound C3b, and binding of sCR1 to C3b. (D) Decay acceleration by factor H CCP1-4 was not affected by rPfRh4. Assays shown in panels A and C, and in panels B and D were performed on identical biosensor surfaces. (E) Hemolysis assay for the classic pathway. The classic pathway C3 convertase was assembled on the surface of antibody-sensitized sheep erythrocytes. PfRh4 plus CCP1-3 were pre-incubated before mixing with sheep erythrocytes. Cellular lysis was induced by the addition of guinea pig serum and monitored by the O.D. of the supernatant at 414nM. CCP1-3 was used at a concentration of 6.8nM. (F) Hemolysis assay for the alternative pathway. The alternative pathway convertase was prepared using EAC14 cells by the addition of C2 and C3. PfRh4 plus CCP1-3 were preincubated and these mixtures were then added to EAC43 cells and the alternative pathway components factor B, factor D, and properdin and were added and lysis measured as in panel E. A convertase was not formed in the absence of factor B. CCP1-3 was used at a concentration of 34nM. For all panels, **P < .01; ***P < .001).

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