Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom.
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.
Appel
Marcia Helena
MH
Department of Cell Biology, Federal University of Paraná, Jardim das Américas, 81531-990, Curitiba, Paraná, Brazil.
da Silveira
Rafael Bertoni
RB
Chaim
Olga Meiri
OM
Paludo
Kátia Sabrina
KS
Silva
Dilza Trevisan
DT
Chaves
Daniele M
DM
da Silva
Paulo Henrique
PH
Mangili
Oldemir C
OC
Senff-Ribeiro
Andrea
A
Gremski
Waldemiro
W
Nader
Helena B
HB
Veiga
Silvio Sanches
SS
D000595:Amino Acid Sequence; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D020405:Dermis / Q000187:drug effects* / Q000473:pathology; D004487:Edema / Q000139:chemically induced; D051379:Mice; D008969:Molecular Sequence Data; D009336:Necrosis / Q000139:chemically induced; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000633:toxicity; D010802:Phylogeny; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D013111:Spider Venoms / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics / Q000633:toxicity*; D013112:Spiders / Q000201:enzymology
D018076:DNA, Complementary; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013111:Spider Venoms; C516323:dermonecrotic toxin, Loxosceles intermedia; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D