Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

• Published in: Biochimica et biophysica acta Vol. 1780; no. 2; pp. 167 - 178
• Main Authors: Appel, Marcia Helena, da Silveira, Rafael Bertoni, Chaim, Olga Meiri, Paludo, Kátia Sabrina, Silva, Dilza Trevisan, Chaves, Daniele M., da Silva, Paulo Henrique, Mangili, Oldemir C., Senff-Ribeiro, Andrea, Gremski, Waldemiro, Nader, Helena B., Veiga, Silvio Sanches
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2008
• Subjects: Amino Acid Sequence; Animals; Capillary Permeability - drug effects; Cloning, Molecular; Dermis - drug effects; Dermis - pathology; Dermonecrotic toxin; DNA, Complementary - genetics; Edema - chemically induced; Loxosceles; Mice; Molecular Sequence Data; Necrosis - chemically induced; Phospholipase D; Phospholipase D - chemistry; Phospholipase D - genetics; Phospholipase D - toxicity; Phosphoric Diester Hydrolases - genetics; Phosphoric Diester Hydrolases - toxicity; Phylogeny; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - toxicity; Rabbits; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - toxicity; Sphingomyelinase; Spider Venoms - chemistry; Spider Venoms - enzymology; Spider Venoms - genetics; Spider Venoms - toxicity; Spiders - enzymology; Venom; Sphingomyelinase; Loxosceles; Dermonecrotic toxin; Venom; Phospholipase D
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2007.11.007

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.