Molecular Cloning, Expression, Function, Structure and Immunoreactivities of a Sphingomyelinase D from Loxosceles adelaida, a Brazilian Brown Spider from Karstic Areas

Loxosceles is the most poisonous spider in Brazil and, at least, three different species of medical importance are known in Brazil (L. intermedia, L. gaucho, L. laeta), with more than 5000 cases of envenomation reported each year. In South Africa, L. parrami and L. spinulosa are responsible for cutaneous loxoscelism (Newlands et al., 1982). In Australia, a cosmopolitan species, L. rufescens, is capable of causing ulceration in humans. In the USA, at least five Loxosceles species, including L. reclusa (brown recluse), L. apachea, L. arizonica, L. unicolor and L. deserta are known to cause numerous incidents (Ginsburg W Gendron, 1990; Bey et al., 1997; Desai et al., 2000). Several studies have indicated that sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the main component responsible for the local and systemic effects observed in loxoscelism (Forrester et al., 1978; Kurpiewski et al., 1981; Tambourgi et al., 1998, 2000, 2002, 2004, 2005, 2007; van den Berg et al., 2002, 2007; Fernandes Pedrosa et al., 2002; Paixao Cavalcanti et al., 2006, Tambourgi et al., 2010). SMases D hydrolyze sphingomyelin resulting in the formation of ceramide-1-phosphate and choline (Forrester et al., 1978; Kurpiewski et al., 1981; Tambourgi et al., 1998) and, in the presence of Mg2+, are able to catalyze the release of choline from lysophosphatidylcholine (van Meeteren et al., 2004). All spider venom SMases D sequenced to date display a significant level of sequence similarity and thus likely possess the same (┙/┚)8 or TIM barrel fold (Murakami et al., 2005, 2006). Based on sequence identity, biochemical activity and molecular modelling, a scheme for classification of spider venom SMases D was proposed (Murakami et al., 2006). The class 1 enzymes include SMase I and H13, SMases D from L. laeta, which possess a single disulphide bridge and contain an extended hydrophobic loop or variable loop (Murakami et