THESIS. K.C. Barbaro submitted this thesis for the degree of Doctor of Science in Immunology publicly examined at the Department of Immunology of the Institute of Biomedical Sciences of the University of São Paulo, São Paulo, Brazil, in 1996.
Advisor: Professor Ivan Mota
ABSTRACT. Envenoming by Loxosceles spiders usually causes a typical dermonecrotic lesion in patients, but it may also cause systemic effects that can be lethal. The biological activities of the venom of Loxosceles gaucho, L. laeta and L. intermedia were studied. The results showed that the dermonecrotic and lethal activities are shared by all three Loxosceles venoms. Only low levels of proteolytic, myotoxic and phospholipase A2 activities were detected even when a large amount of venom was used. No direct hemolytic activity was detected. L. intermedia venom was the most lethal (LD50 0.48 mg/kg), the L. laeta the least lethal (LD50 1.45 mg/kg), whereas the venom of L. gaucho showed an LD50 of intermediate quantity of 0.74 mg/kg. Separation by SDS-PAGE showed the existence of many common components in the three venoms. No individual antigen was observed. Antigenic cross-reactivity among the components of L. gaucho, L. laeta and L. intermedia was studied. Species specific antisera were prepared by immunization of rabbits with each loxoscelic venom. Anti-L. gaucho horse hyperimmune serum provided by the Butantan Institute for treatment of envenoming by these spiders was also used. Analysis of antisera by ELISA and immunoblotting showed cross-reactivity as well as several common bands among the three venoms. The horse anti-L. gaucho venom serum recognized many common proteins when antigens of the other two species were used. Antigens in the range of 35-30 kDa showed the most cross-reactivity. Both horse and rabbit anti-venom sera contained antibodies able to neutralize the lethal and dermonecrotic activities of the venom of the three species studied. Gel filtration on Sephadex G 100 of Loxosceles venoms resulted in three fractions: A, containing higher molecular mass components, B, containing intermediate molecular mass components and C, with lower molecular mass components. Dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein present in fraction A of the venoms had approximately 35 kDa in L. gaucho and L. intermedia but 32 kDa in L. laeta venom and was responsible for the dermonecrotic activity. These toxins were isolated from venoms of L. gaucho, L. laeta and L. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of the L. reclusa (North American spider) toxin. A multiple sequence alignment of the Loxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% between L. gaucho and L. reclusa venoms to 61.1% between L. intermedia and L. reclusa venoms. The purified toxins were also submitted to capillary electrophoresis peptide mapping after in situ partial hydrolysis of the blotted samples. The results obtained suggest that L. intermedia protein is similar to L. laeta toxin rather than L. gaucho toxin. Altogether, these findings suggest that the toxins responsible for the most important activities of venoms of Loxosceles species have a molecular mass of 35-32 kDa and are problably similar proteins
CORRESPONDENCE TO:
K. C. BARBARO - Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, CEP 05503-900, São Paulo, SP, Brasil.
Publication Dates
-
Publication in this collection
08 Jan 1999 -
Date of issue
1997
