Biological and structural comparison of recombinant phospholipase D toxins from Loxosceles intermedia (brown spider) venom

Biological and structural comparison of recombinant phospholipase D toxins from Loxosceles intermedia (brown spider) venom

Author Ribeiro, Rodrigo Otavio S. Google Scholar
Chaim, Olga Meiri Autor UNIFESP Google Scholar
Silveira, Rafael Bertoni da Google Scholar
Gremski, Luiza Helena Autor UNIFESP Google Scholar
Sade, Youssef Bacila Google Scholar
Paludo, Katia Sabrina Autor UNIFESP Google Scholar
Senff-Ribeiro, Andrea Google Scholar
Moura, Juliana de Google Scholar
Chavez-Olortegui, Carlos Google Scholar
Gremski, Waldemiro Google Scholar
Nader, Helena B. Autor UNIFESP Google Scholar
Veiga, Silvio Sanches Google Scholar
Institution Univ Fed Parana
Universidade Federal de São Paulo (UNIFESP)
Univ Estadual Ponta Grossa
Universidade Federal de Minas Gerais (UFMG)
Catholic Univ Parana
Abstract The clinical features of brown spider bites are the appearance of necrotic skin lesions, which can also be accompanied by systemic involvement, including weakness, vomiting, fever, convulsions, disseminated intravascular coagulation, intravascular hemolysis and renal disturbances. Severe systemic loxoscelism is much less common than the cutaneous form, but it may be the cause of clinical complications and even death following envenomation. Here, by using three recombinant dermonecrotic toxins, LiRecDT1, LiRecDT2 and LiRecDT3 (the major toxins found in the venom), we report the biological, immunological and structural differences for these members of this toxin family. Purified toxins evoked similar inflammatory reactions following injections into rabbit skin. Recombinant toxin treatments of MDCK cells with LiRecDT1 and LiRecDT2 changed cell viability, as evaluated by neutral red uptake and assessment of cell morphology through inverted microscopy, whereas LiRecDT3 caused only residual activity. Differences in cell cytotoxicity triggered by recombinant toxins were confirmed through a human red blood lysis assay, during which LiRecDT1 and LiRecDT2 caused a high degree of hemolysis compared to LiRecDT3, which induced only a small hemolytic effect. Additionally, biological differences for recombinant toxins were corroborated through mice lethality experiments, which showed animal mortality after LiRecDT1 and LiRecDT2 treatments, but an absence of lethality following LiRecDT3 exposure. Moreover, in experiments for edema, both the LiRecDT1 and the LiRecDT2 toxins evoked similar results, causing edema following toxin exposure, whereas LiRecDT3 caused only residual effects. Characterization of antigenic cross-reactivity using sera against crude venom toxins by immunoWestern blotting and immunodot blotting with recombinant LiRecDT1, LiRecDT2 and LiRecDT3 compared among themselves pointed to a higher cross-reactivity for LiRecDT1 compared to LiRecDT2 and LiRecDT3, corroborating structural and antigenic differences for these three toxins. Finally, evidence for structural differences among the recombinant toxins was strengthened by circular dichroism spectra, which suggested that the toxins were folded, and not aggregated or denatured proteins. (c) 2007 Elsevier B.V. All rights reserved.
Keywords brown spider
venom
phospholipase D
recombinant toxins
Language English
Date 2007-12-15
Published in Toxicon. Oxford: Pergamon-Elsevier B.V., v. 50, n. 8, p. 1162-1174, 2007.
ISSN 0041-0101 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 1162-1174
Origin http://dx.doi.org/10.1016/j.toxicon.2007.08.001
Access rights Closed access
Type Article
Web of Science ID WOS:000251557900014
URI http://repositorio.unifesp.br/handle/11600/30216

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