Molecular Cloning and Expression of a Functional Dermonecrotic and Complement-Dependent Haemolytic Factor from Loxosceles laeta Spider Venom Gland

FERNANDES-PEDROSA, M.F.(1), JUNQUEIRA-DE-AZEVEDO, I. L. M.(2), GONÇALVES-DE ANDRADE, R. M.(1), VAN DEN BERG, C. W.(3), RAMOS, C.R.R.(2), HO, P.L.(2), TAMBOURGI, D.V.(1).

(1)Laboratório de Imunoquímica and (2)Centro de Biotecnologia, InstitutoButantan, Brazil, (3)Department of Pharmacology, Therapeutics and Toxicology, University of Wales, College of Medicine, UK.

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” (EST) 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 P1 and P2 from L. intermedia venom. The longest cDNA insert from clone H17, which shared a high percentage of identity with these toxins, was further subcloned and expressed in a bacterial system. The final construct encoded for a 33-kDa-fusion protein containing a 6xHis-tag at its N-terminus. The recombinant L. laeta spider venom protein was endowed with all biological properties ascribed for the whole L. laeta venom and purified toxins P1 and P2 from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities and ability of hydrolysing sphingomyelin. Considering its lipase activity, the recombinant L. laeta toxin expressed from clone H17 was named L. laeta Smase I. Antiserum raised against L. laeta Smase I recognised a 32-kDa protein in crude L. laeta venom and gland extract and was able to block the dermonecrotic reaction caused by the whole L. laeta venom. This is the first report of the complete primary structure of the dermonecrotic and complement dependent haemolytic factor from Loxosceles spider venom glands that was obtained through cDNA cloning and functionally expressed in E. coli. This study also demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.

Supported by The Wellcome Trust and CAPES.

CORRESPONDENCE TO:

FERNANDES-PEDROSA, M.F., Laboratório de Imunoquímica, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo, 05503-900, SP, Brazil, E.mail: mpedrosa@usp.br