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. 2011 May 24;108(21):8761-6.
doi: 10.1073/pnas.1019338108. Epub 2011 May 9.

"VSports注册入口" Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk

Meike Heurich  1 , Ruben Martínez-Barricarte, Nigel J Francis, Dawn L Roberts, Santiago Rodríguez de Córdoba, B Paul Morgan, Claire L Harris
Affiliations

Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk (VSports注册入口)

Meike Heurich et al. Proc Natl Acad Sci U S A. .

Abstract

Common polymorphisms in complement alternative pathway (AP) proteins C3 (C3(R102G)), factor B (fB(R32Q)), and factor H (fH(V62I)) are associated with age-related macular degeneration (AMD) and other pathologies. Our published work showed that fB(R32Q) influences C3 convertase formation, whereas fH(V62I) affects factor I cofactor activity. Here we show how C3(R102G) (C3S/F) influences AP activity. In hemolysis assays, C3(102G) activated AP more efficiently (EC(50) C3(102G): 157 nM; C3(102R): 191 nM; P < 0 VSports手机版. 0001). fB binding kinetics and convertase stability were identical, but native and recombinant fH bound more strongly to C3b(102R) (K(D) C3b(102R): 1. 0 μM; C3b(102G): 1. 4 μM; P < 0. 0001). Accelerated decay was unaltered, but fH cofactor activity was reduced for C3b(102G), favoring AP amplification. Combining disease "risk" variants (C3(102G), fB(32R), and fH(62V)) in add-back assays yielded sixfold higher hemolytic activity compared with "protective" variants (C3(102R), fB(32Q), and fH(62I); P < 0. 0001). These data introduce the concept of a functional complotype (combination of polymorphisms) defining complement activity in an individual, thereby influencing susceptibility to AP-driven disease. .

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V体育官网 - Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Hemolytic activity of the C3 variants C3102G and C3102R. C3 was purified from plasma of two donors homozygous for C3102G (circles) and three donors homozygous for C3102R (squares/diamond) and was added to C3-depleted serum to deposit C3b on ShEA. C3 convertase was formed and lysis was developed using NHS-MA supplemented with C3 of each variant. Data points represent mean ± SD of triplicate measurements. Curves were fitted using nonlinear regression to calculate the EC50.
Fig. 2.
Fig. 2.
Binding of native fH and recombinant fH to C3bR102G variants. Native fH (A and B) or rfH1-4 (D and E) were flowed over immobilized C3b102G (A and D) or C3b102R (B and E) at concentrations between 45 nM and 2.9 μM (fH) and 80 nM–23 μM (rfH1–4) and equilibrium binding response was measured. Affinity (KD) was determined by steady-state analysis (Inset). Multiple analyses on different days with different donors confirmed consistent and significant differences in native fH (C) or rfH1-4 (F) binding to the C3bR102G variants.
Fig. 3.
Fig. 3.
Regulatory activities of native fH, rfH1–4, and sDAF on surface-bound convertase formed from C3R102G variants. (A–C) Identical amounts (1,500 RU) of C3b102R (gray dotted line) and C3b102G (black solid line) were deposited on the chip surfaces using thioester coupling. Convertase was formed by flowing (20 μL/min) fB and fD in the presence of Mg2+ and allowed to decay naturally for 150 s. At the arrow, either (A) sDAF (10, 30, 100, and 300 nM; from Upper to Lower curve), (B) fH (30, 100, and 300 nM), or (C) rfH1–4 (30, 100, 300, and 1,000 nM) was injected (90 s) and accelerated decay of the convertase was monitored. Inset shows whole sensorgram; expanded decay curves are illustrated. (D) Decay accelerating activity of fH was quantitated by incubating C3b-coated ShEA with fB and fD to form convertase and then treating with varying concentrations of fH to decay convertase before developing lysis. (E) Cofactor activity was analyzed by treating C3b-coated ShEA with fH and fI before forming convertase and developing lysis. In D and E, curves were fitted using nonlinear regression to calculate the IH50. Data points represent mean ± SD of three determinations. C3b102G-ShEA are shown as circles, C3b10R-ShEA as squares, fH62V as filled symbols, and fH62I as open symbols.
Fig. 4.
Fig. 4.
Polymorphic variations in C3, fB, and fH collaborate to control AP hemolytic activity. (A) C3b was deposited on ShEA using NHSΔC3B spiked with either variant of C3 (C3102R, squares; C3102G, circles); convertase was formed using fD and either fB32R (filled symbols) or fB32Q (open symbols); lysis was developed with NHSΔBH. (B) C3 variants were spiked into NHSΔC3BH and used to deposit C3b on cells, which were then treated with fI and a specific variant of fH. Convertase was formed by adding different concentrations of a specific fB variant with fD, and lysis was developed. The complotype predicted to yield highest AP activity (C3102G, fB32R, fH62V) is shown by circles, whereas that predicted to yield lowest AP activity (C3102R, fB32Q, fH62I) is represented by squares. Data points represent mean ± SD of three determinations (error bars depicted for each point); nonlinear regression was used to calculate the EC50. Inset shows SDS/PAGE of purified C3, fB, and fH variants.
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