Flavoxobin, a serine protease from Trimeresurus flavoviridis (habu snake) venom, independently cleaves Arg726-Ser727 of human C3 and acts as a novel, heterologous C3 convertase

Abstract

We have recently shown that crude Trimeresurus flavoviridis (habu snake) venom has a strong capability for activating the human alternative complement system. To identify the active component, the crude venom was fractionated and purified by serial chromatography using Sephadex G-100, CM-cellulose C-52, diethylaminoethyl-Toyopearl 650M, and Butyl-Toyopearl, and the active fractions were evaluated by the C3a-releasing and soluble membrane attack complex-forming activities. Two peak fractions with the highest activities were detected after gel filtration and ion exchange chromatography, and the first fraction was purified to homogeneity. The homogeneous protein was examined for its N-terminal amino acid sequence by Edman degradation. The determined sequence of 25 amino acids completely coincided with that of a previously reported serine protease with coagulant activity, flavoxobin, purified from the same snake venom. To elucidate the molecular mechanism of the complement activation, the reactive products of the mixture of the purified human C3 and flavoxobin were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The digesting pattern revealed that flavoxobin cleaves the alpha chain of the C3 molecule into two fragments. The N-terminal amino acid sequences for the remnant fragments of C3 disclosed that flavoxobin severs the human C3 at the Arg726-Ser727 site to form C3b and C3a the way C3bBb, the human alternative C3 convertase, does. In conclusion, flavoxobin acts as a novel, heterologous C3 convertase that independently cleaves human C3 and kick-starts the complement cascade.

Figure 1

Purification of complement activation component. (a) CM-cellulose C-52 elution profile of G-2 fraction from gel filtration. G-2 fraction (0·9 g) was applied to a column of CM-cellulose C-52 at a flow rate of 32 ml/ hr, and every 6·4 ml fraction was collected. The fractions incorporated in the shaded regions were combined and subjected to further purification of the enzyme. The peak 1 fraction was designated as G-2-1. (b) The active fraction (G-2-1, approximately 60 mg) purified by CM-cellulose C-52 was applied to a column (2·5×29 cm) of DEAE-Toyopearl 650M. The flow rate was 32 ml/ hr, and the fraction volume was 8·0 ml. (c) Butyl-Toyopearl elution profile of purified protein from the steep peak of DEAE-Toyopearl 650M (shaded region in b). Approximately 10 mg of protein was applied to a column (2·4×26 cm) of Butyl-Toyopearl 650M at a flow rate of 32 ml/ hr. A homogeneous peak of the active enzyme was obtained.

Figure 2

Fractional capability of complement activation. (a) C3a-releasing activity. (b) Soluble membrane attack complex-forming activity. The G-1 fraction displayed C3a-releasing activity but did not induce soluble membrane attack complex formation. Both activities were strong with the G-2-1 and G-2-8 fractions. All samples except for the control were proportioned at 0·5 mg/ml of the final protein concentration.

Figure 4

Cleaved products of C3 by flavoxobin. (a) Native PAGE after 60 min of incubation shows the digestion of the C3 into a relatively mobile fragment (C3b). Lane 1 represents isolated flavoxobin; lane 2 C3; and lane 3, mixture of C3 and flavoxobin. (b) SDS–PAGE with 2-ME-treatment after 15 min of incubation shows the partial dissociation of the larger chain (C3 α) into C3b α′ and C3a (hardly visible). Notice that intact C3 α band is still visible. Lane 1, flavoxobin; lane 2, C3; lane 3, mixture of C3 and flavoxobin; and lane 4, molecular weight markers. (c) After 60 min of incubation, C3 α chains are almost completely cleaved into C3b α′ and C3a fragments on SDS–PAGE with 2-ME-treatment. Lane 1, isolated flavoxobin; lane 2, mixture of C3 and flavoxobin; lane 3, C3; and lane 4, molecular weight markers.

Figure 3

Comparison of complement activation by flavoxobin, trypsin, and thrombin. Flavoxobin showed significantly stronger C3a-releasing and soluble membrane attack complex-forming activity than trypsin and thrombin. From molar concentrations of initial C3 and released C3a, it is estimated that approximately 41% of plasma C3 was cleaved by complement activation triggered by flavoxobin. (a) C3a release shown in molar concentration. (b) Soluble MAC formation.