title: Biological and structural comparison of recombinant phospholipase D toxins from Loxosceles intermedia (brown spider) venom.
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.
authors: Department of Cell Biology, Federal University of Paraná, Jardim das Américas 81531-990, Curitiba, Paraná, Brazil.
authors: Rodrigo Otávio S
authors: RO
authors: Ribeiro
authors: Olga Meiri
authors: OM
authors: Chaim
authors: Rafael Bertoni
authors: RB
authors: da Silveira
authors: Luiza Helena
authors: LH
authors: Gremski
authors: Youssef Bacila
authors: YB
authors: Sade
authors: Kátia Sabrina
authors: KS
authors: Paludo
authors: Andrea
authors: A
authors: Senff-Ribeiro
authors: Juliana
authors: J
authors: de Moura
authors: Carlos
authors: C
authors: Chávez-Olórtegui
authors: Waldemiro
authors: W
authors: Gremski
authors: Helena B
authors: HB
authors: Nader
authors: Silvio Sanches
authors: SS
authors: Veiga
mesh_terms: D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D003429:Cross Reactions; D004285:Dogs; D004487:Edema / Q000139:chemically induced; D006461:Hemolysis / Q000187:drug effects; D008297:Male; D010739:Phospholipase D / Q000737:chemistry / Q000633:toxicity*; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000633:toxicity; D013111:Spider Venoms / Q000737:chemistry / Q000633:toxicity*; D013112:Spiders
chemical_list: D011994:Recombinant Proteins; D013111:Spider Venoms; C516323:dermonecrotic toxin, Loxosceles intermedia; D010739:Phospholipase D