Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization.
Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(2+)-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.
da Silveira
Rafael Bertoni
RB
Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil.
Pigozzo
Romine Bachmann
RB
Chaim
Olga Meiri
OM
Appel
Marcia Helena
MH
Silva
Dilza Trevisan
DT
Dreyfuss
Juliana Luporini
JL
Toma
Leny
L
Dietrich
Carl Peter
CP
Nader
Helena B
HB
Veiga
Silvio Sanches
SS
Gremski
Waldemiro
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002199:Capillary Permeability / Q000187:drug effects; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D010740:Phospholipases / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D010974:Platelet Aggregation / Q000187:drug effects; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000187:drug effects / Q000473:pathology; D013111:Spider Venoms / Q000235:genetics* / Q000378:metabolism*; D013112:Spiders / Q000235:genetics* / Q000378:metabolism; D014118:Toxins, Biological / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013111:Spider Venoms; D014118:Toxins, Biological; D010740:Phospholipases