Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: Endothelial cell membrane phospholipids as targets for toxicity

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: Endothelial cell membrane phospholipids as targets for toxicity

Author Chaim, Olga Meiri Autor UNIFESP Google Scholar
Da Silveira, Rafael Bertoni Google Scholar
Silva, Dilza Trevisan Google Scholar
Ferrer, Valéria Pereira Google Scholar
Sade, Youssef Bacila Google Scholar
Boia-Ferreira, Mariana Google Scholar
Gremski, Luiza Helena Autor UNIFESP Google Scholar
Gremski, Waldemiro Google Scholar
Senff-Ribeiro, Andrea Google Scholar
Takahashi, Helio Kiyoshi Autor UNIFESP Google Scholar
Toledo, Marcos Sergio Autor UNIFESP Google Scholar
Nader, Helena Bonciani Autor UNIFESP Google Scholar
Veiga, Silvio Sanches Autor UNIFESP Google Scholar
Institution Univ Fed Parana
Universidade Federal de São Paulo (UNIFESP)
Univ Estadual Ponta Grossa
Catholic Univ Parana
Abstract Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. the mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids. (C) 2010 Elsevier B.V. All rights reserved.
Keywords Venom
Loxosceles intermedia
Dermonecrotic toxin
Phospholipase-D
Endothelial cell
Inflammatory response
Language English
Sponsor Secretaria de Estado de Ciencia
Tecnologia e Ensino Superior (SETI) do Parana
Fundacao Araucaria-PR
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Date 2011-02-01
Published in Biochimica Et Biophysica Acta-molecular and Cell Biology of Lipids. Amsterdam: Elsevier B.V., v. 1811, n. 2, p. 84-96, 2011.
ISSN 1388-1981 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 84-96
Origin http://dx.doi.org/10.1016/j.bbalip.2010.11.005
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000286998500004
URI http://repositorio.unifesp.br/handle/11600/33367

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