title: Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity.
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.
authors.affiliation: Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil.
authors.forename: Olga M
authors.initials: OM
authors.lastname: Chaim
authors.forename: Rafael B
authors.initials: RB
authors.lastname: da Silveira
authors.forename: Dilza
authors.initials: D
authors.lastname: Trevisan-Silva
authors.forename: Valéria P
authors.initials: VP
authors.lastname: Ferrer
authors.forename: Youssef B
authors.initials: YB
authors.lastname: Sade
authors.forename: Mariana
authors.initials: M
authors.lastname: Bóia-Ferreira
authors.forename: Luiza H
authors.initials: LH
authors.lastname: Gremski
authors.forename: Waldemiro
authors.initials: W
authors.lastname: Gremski
authors.forename: Andrea
authors.initials: A
authors.lastname: Senff-Ribeiro
authors.forename: Hélio K
authors.initials: HK
authors.lastname: Takahashi
authors.forename: Marcos S
authors.initials: MS
authors.lastname: Toledo
authors.forename: Helena B
authors.initials: HB
authors.lastname: Nader
authors.forename: Silvio S
authors.initials: SS
authors.lastname: Veiga
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D007249:Inflammation / Q000139:chemically induced*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D050356:Lipid Metabolism; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D010743:Phospholipids / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000633:toxicity; D013111:Spider Venoms / Q000235:genetics / Q000633:toxicity*
chemical_list: D019476:Insect Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D013111:Spider Venoms; C516323:dermonecrotic toxin, Loxosceles intermedia; D010739:Phospholipase D; D002794:Choline