Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

Abstract

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E(Man)) was examined. While the C4BP concentration for inhibiting 50% (IC(50)) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05nM), approximately 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81nM) and E(Man) (42.66nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000-431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven alpha-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79+/-0.60) when the C4b density on E(Man) was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a approximately 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.

Fig. 1

Inhibition of formation of the lectin pathway C3/C5 convertase by C4BP on zymosan and E_Man cells measured as the amount of C5 convertase activity remaining. (A) The C3 convertase was allowed to form on zymosan particles bearing 346,000 C4b/Zym while the C5 convertase was assembled on zymosan bearing 54,000 C4b and 362,000 C3b per zymosan in the presence of indicated concentration of C4BP. Inhibition of enzyme formation was measured as the amount of C5 convertase activity remaining by determining the amount of C5b,6 formed using hemolytic assay with E_C. C5b,6 formed in the absence of C4BP was considered 100%. To obtain IC₅₀, the data were fit by nonlinear regression using Grafit version 5.0 Erithacus software. Vertical bar indicates the normal concentration range of C4BP in plasma (250-400 nM). Symbols: soluble C3 convertase C4b,C2a (Δ); surface-bound C3 convertase, ZymM1,C4b,C2a (○); surface-bound C5 convertase, ZymM1,C3bC4b,C2a (●). (B). Inhibition of formation of the lectin pathway C3/C5 convertase on E_Man cells by C4BP. Experimental details are as described above in A except that E_Man cells bearing 471,000 C4b/E_Man were employed to assemble the C3 convertase while cells bearing 138,000 C4b and 110,000 C3b per E_Man were used for assembling the C5 convertase. Symbols: soluble C3 convertase, C4b,C2a(Δ); surface-bound C3 convertase, E_ManM1,C4b,C2a (○); surface-bound C5 convertase, E_ManM1,C3bC4b,C2a (●).

Fig. 2

Effect of C4b density on C4BP regulation of surface-bound C3 convertase assembly on zymosan and E_Man cells. Inhibition of C3 convertase (ZymM1,C4b,C2a and E_ManM1,C4b,C2a) assembly on zymosan and E_Man cells bearing different amounts of C4b/cell ranging from 22,000-346,000 C4b/Zym and 15,000-471,000 C4b/E_Man. The number of C4b/cell was measured using ¹²⁵I-labeled C4. Inhibition of enzyme formation was measured as the amount of C5 convertase activity remaining as described in Fig. 1. The IC₅₀ for each density of C4b/cell employed in the assays was calculated from individual inhibition plots. These IC₅₀ values were considered as one set of data and were fit by linear regression using Grafit version 5.0 Erithacus software. Symbols: ZymM1,C4b,C2a (○); E_ManM1,C4b,C2a (◊).

Fig. 3

Equilibrium binding of C4BP to surface-bound C4b alone, C3b alone, and C4b-C3b complexes on zymosan. (A). C4BP binding to surface-bound C4b was measured as the amount of ¹²⁵I-C4BP bound as cpm to ZymM1,C4b (○) under equilibrium conditions. ZymM1,C4b (0.44×10⁶) bearing 59,000 C4b/Zym was incubated for 45 min at 37 °C with indicated concentration of ¹²⁵I-C4BP (4.1×10⁵ cpm/μg). The y axis represents the amount of radiolabeled C4BP bound to ZymM1C4b as cpm. The data were fit by nonlinear regression to a one-site binding equation to obtain the apparent binding constant (K_d). (B). C4BP binding to surface-bound C4b-C3b complexes was analyzed as described in Fig. 3(A) except that 0.44×10⁶ ZymM1,C3bC4b bearing 54,000 C4b and 362,000 C3b/Zym was incubated with radiolabeled ¹²⁵I-C4BP (4.1×10⁵ cpm/μg). C4BP binding to zymosan bearing only C3b was also analyzed by incubating ZymC3b bearing 181,000 C3b/Zym with radiolabeled ¹²⁵I-C4BP (7.65×10⁵ cpm/μg). Data obtained with 0.84×10⁶ ZymC3b was normalized for 0.44×10⁶ ZymM1,C4b as well as for the specific activity of ¹²⁵I-C4BP used and are shown in the figure. Symbols: ZymM1,C4bC3b (●); ZymC3b (✯).

Fig. 4

Effect of C4b density on the binding of C4BP to surface-bound C4b alone and C4b-C3b complexes on zymosan and E_Man cells. (A). Effect of C4b density on the binding of C4BP to ZymM1,C4b and ZymM1,C3bC4b. The apparent binding constant (K_d) obtained for binding interactions of C4BP with ZymM1,C4b (○) or ZymM1,C3bC4b (●) assembled with different amounts of C4b/Zym (ranging from 14,000-431,000 C4b/Zym) was measured under equilibrium conditions and calculated by nonlinear regression analysis of individual binding curves as described in Fig. 3. The apparent K_d value thus obtained from each binding curve were considered as one data set and was fit by linear regression using Grafit version 5.0 Erithacus software as shown in the figure. (B). Effect of C4b density on the binding of C4BP to E_ManM1,C4b and E_ManM1,C3bC4b cells. The apparent binding constant (K_d) obtained for binding interactions of C4BP with E_ManM1,C4b (○) and E_ManM1,C3bC4b (●) assembled with different densities of C4b/E_Man (ranging from 15,000-471,000 C4b/E_Man) was measured under equilibrium conditions and calculated by nonlinear regression analysis of individual binding curves as described in Fig. 3. The apparent K_d thus obtained from each binding curve was considered as one data set and was fit by linear regression using Grafit version 5.0 Erithacus software as shown in the figure.

Fig. 5

Effect of C3b density on the binding of C4BP to ZymM1,C3bC4b and E_ManM1,C3bC4b cells. The apparent binding constants (K_d) obtained for binding interactions of C4BP with ZymM1,C3bC4b (○) and E_ManM1,C3bC4b cells (●) assembled with different ratio of number of C3b:C4b was measured under equilibrium conditions. The number of C4b/cell was measured using ¹²⁵I-C4 and the number of C3b/cell was measured using ¹²⁵I-Factor B as described (Rawal et al., 1998). The data were fit by nonlinear regression to a one-site binding equation to obtain the apparent dissociation constant (K_d) as described in Fig. 3. The apparent K_d obtained from these individual plots was considered as one set of data and was fit by linear regression using Grafit version 5.0 Erithacus software as shown in the figure.

Fig. 6

Comparison of the effect of C4b density on the number of C4b molecules bound per C4BP at saturation on zymosan and E_Man cells. (A). The effect of C4b density on the number of C4b molecules bound per C4BP at saturation on zymosan. The number of C4b/cell was measured using ¹²⁵I-C4. The number of C4BP molecules bound to ZymM1,C4b (○) and ZymM1,C3bC4b (●) was calculated from the maximum amount of ¹²⁵I-C4BP bound to the cells under saturating conditions and from the specific activity of C4BP after subtracting the contribution from ¹²⁵I-C4b and correcting for nonspecific binding of both. The number of surface-bound C4b molecules bound to C4BP, determined from individual binding plots, was considered as one set of data and was fit by nonlinear regression using Grafit version 5.0 Erithacus software and is shown in the figure. (B). Effect of C4b density on the number of C4b molecules bound per C4BP at saturation on E_Man cells. Details are as described in Fig. 6A for zymosan except that E_ManM1,C4b (○) and E_ManM1,C3bC4b (●) cells were used. The number of surface-bound C4b molecules bound to C4BP, determined from individual binding plots, was considered as one set of data and was fit by linear regression using Grafit version 5.0 Erithacus software and is shown in the figure. Data obtained with E_ManM1,C4b cells bearing 2,300 C4b/E_Man were not included in the fit.