Tambourgi D V, Magnoli F C, van den Berg C W, Morgan B P, de Araujo P S, Alves E W, Da Silva W D
Instituto Butantan, Universidade de São Paulo, São Paulo, 05508-900, Brazil.
Biochem Biophys Res Commun. 1998 Oct 9;251(1):366-73. doi: 10.1006/bbrc.1998.9474.
The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.
洛氏蝎属蜘蛛的叮咬可引发多种生物学效应，包括皮肤坏死和补体（C）依赖性溶血。本研究的目的是鉴定毒液中导致不同生物学效应的毒素。我们之前已经表明，从中间洛氏蝎毒液中纯化出的一种名为F35的35 kDa蛋白质可整合到人类红细胞膜中，并使其易受自身补体替代途径的作用。在此，我们进一步纯化了F35蛋白，该蛋白通过反相色谱法分离为三个紧密相邻的峰，分别称为P1、P2和P3。通过SDS-PAGE和N端氨基酸分析表明P1和P2是均一的，而P3由两种高度同源的蛋白质组成。对所有四种蛋白质的N端测序显示出高度同源性，这通过针对各个纯化蛋白产生的抗血清的交叉反应得到证实。P1和P2的功能表征表明存在鞘磷脂酶活性，单独的任何一种蛋白质都能够诱导出全蜘蛛毒液所产生的所有体内效应，包括C依赖性溶血和皮肤坏死。在所有试验中，P2比P1更具活性，而P3完全无活性。这些数据表明，中间洛氏蝎毒液的不同生物学效应可归因于两种高度同源蛋白质P1和P2的鞘磷脂酶活性。将这些蛋白质鉴定为全毒液诱导的主要病理效应的诱导剂，将有助于研究毒液的作用机制以及开发有效的治疗方法。
