title: Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom.
abstract: 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.
authors.affiliation: Laboratório de Imunoquímica, Instituto Butantan, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, SP, Brazil.
authors.forename: Matheus de F
authors.initials: Mde F
authors.lastname: Fernandes Pedrosa
authors.forename: Inácio de L M
authors.initials: Ide L
authors.lastname: Junqueira de Azevedo
authors.forename: Rute M
authors.initials: RM
authors.lastname: Gonçalves-de-Andrade
authors.forename: Carmen W
authors.initials: CW
authors.lastname: van den Berg
authors.forename: Celso R R
authors.initials: CR
authors.lastname: Ramos
authors.forename: Paulo Lee
authors.initials: PL
authors.lastname: Ho
authors.forename: Denise V
authors.initials: DV
authors.lastname: Tambourgi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003167:Complement Activation / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects; D015870:Gene Expression; D006021:Glycophorins / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009336:Necrosis; D011994:Recombinant Proteins / Q000235:genetics / Q000633:toxicity; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000633:toxicity; D001098:Spider Bites / Q000209:etiology; D013111:Spider Venoms / Q000201:enzymology / Q000235:genetics* / Q000633:toxicity; D013112:Spiders / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity
chemical_list: D018076:DNA, Complementary; D006021:Glycophorins; D011994:Recombinant Proteins; D013111:Spider Venoms; D013108:Sphingomyelin Phosphodiesterase