Fernandes Pedrosa Matheus de F, Junqueira de Azevedo Inácio de L M, Gonçalves-de-Andrade Rute M, van den Berg Carmen W, Ramos Celso R R, Ho Paulo Lee, Tambourgi Denise V
Laboratório de Imunoquímica, Instituto Butantan, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, SP, Brazil.
Biochem Biophys Res Commun. 2002 Nov 15;298(5):638-45. doi: 10.1016/s0006-291x(02)02521-4.
The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.
洛氏蛛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依赖性溶血。本研究的目的是从洛氏蛛的腺体中生成重组蛋白，以促进对洛氏蛛中毒机制的结构和功能研究。利用来自巴西游走蛛毒腺cDNA文库的随机克隆的“表达序列标签”策略，我们鉴定出了一些克隆，其插入片段编码的蛋白质与先前从巴西中间游走蛛毒液中获得的鞘磷脂酶N端具有显著相似性[1]。克隆H17被表达为一种在其N端含有6x组氨酸标签的融合蛋白，并产生了一种33kDa的蛋白质。该重组蛋白具有归因于整个巴西游走蛛毒液和巴西中间游走蛛鞘磷脂酶的所有生物学特性，包括皮肤坏死和补体依赖性溶血活性。针对该重组蛋白产生的抗血清识别出巴西游走蛛粗毒液中的一种32kDa蛋白质，并能够阻断由整个巴西游走蛛毒液引起的皮肤坏死反应。这项研究最终证明，整个毒液中的鞘磷脂酶活性是洛氏蛛蜘蛛毒液中毒主要病理效应的原因。
