Laboratory of Immunopathology, Butantan Institute, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil.
Biochimie. 2013 Sep;95(9):1773-83. doi: 10.1016/j.biochi.2013.06.002. Epub 2013 Jun 14.
Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (65%) and dermonecrosis (100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.
狼蛛毒液包含多种毒素，会引起强烈的局部炎症反应、皮肤坏死损伤、血小板聚集、溶血性贫血和急性肾衰竭。在毒液中的几种毒素中，磷脂酶 D（PLD），也称为皮肤坏死毒素，是最重要和研究最多的，因为它们占狼蛛咬伤的主要影响。尽管它们很重要，但由于从毒液中纯化的 PLD 量很少，因此对其进行生物分析受到阻碍，因此已经做出了许多努力来克隆这些毒素。然而，迄今为止，尚未在异源系统中获得来自 Loxosceles gaucho 的 PLD。因此，在这项工作中，我们展示了从 L. gaucho 毒腺中克隆的一种 PLD，命名为 LgRec1，它在细菌系统中成功表达。LgRec1 引起局部反应（水肿、红斑、瘀斑和苍白）、皮肤坏死和溶血。它还能够水解神经鞘磷脂并促进血小板聚集。ELISA 和 Western blot 分析表明，LgRec1 被抗 L. gaucho 毒液血清、一种商业蛛形动物抗毒液以及针对 L. gaucho 毒液皮肤坏死部分的单克隆抗体识别。此外，LgRec1 表现出高度的免疫原性，针对这种重组毒素的抗体抑制了由 L. gaucho 全毒液引起的局部反应（65%）和皮肤坏死（100%）。由于 PLD 被认为是主要成分，可导致由 Loxosceles 属蜘蛛引起的局部和全身中毒作用，因此这里提供的信息可能有助于理解临床症状背后的机制。
