Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (Brown spider) venom gland

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (Brown spider) venom gland

Author Silveira, Rafael Bertoni da Google Scholar
Pigozzo, Romine Bachmann Google Scholar
Chaim, Olga Meiri Google Scholar
Appel, Marcia Helena Google Scholar
Dreyfuss, Juliana Luporini Google Scholar
Toma, Leny Google Scholar
Mangili, Oldemir Carlos Google Scholar
Gremski, Waldemiro Google Scholar
Dietrich, Carl Peter Google Scholar
Nader, Helena B. Google Scholar
Veiga, Silvio Sanches Google Scholar
Institution Univ Fed Parana
Universidade Federal de São Paulo (UNIFESP)
Catholic Univ Parana
Abstract Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dennonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni2+ chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dennonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators. (c) 2006 Elsevier SAS. All rights reserved.
Keywords Brown spider
venom
dermonecrotic toxin
Language English
Date 2006-09-01
Published in Biochimie. Paris: Elsevier France-editions Scientifiques Medicales Elsevier, v. 88, n. 9, p. 1241-1253, 2006.
ISSN 0300-9084 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 1241-1253
Origin http://dx.doi.org/10.1016/j.biochi.2006.02.008
Access rights Closed access
Type Article
Web of Science ID WOS:000241481000015
URI http://repositorio.unifesp.br/handle/11600/29125

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