Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland.
Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.
Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil.
Rafael Bertoni
RB
da Silveira
Romine Bachmann
RB
Pigozzo
Olga Meiri
OM
Chaim
Marcia Helena
MH
Appel
Juliana Luporini
JL
Dreyfuss
Leny
L
Toma
Oldemir Carlos
OC
Mangili
Waldemiro
W
Gremski
Carl Peter
CP
Dietrich
Helena B
HB
Nader
Silvio Sanches
SS
Veiga
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D051379:Mice; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D010802:Phylogeny; D010974:Platelet Aggregation / Q000187:drug effects; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D016415:Sequence Alignment; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013111:Spider Venoms / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D013112:Spiders / Q000737:chemistry / Q000235:genetics*
D020033:Protein Isoforms; D011994:Recombinant Proteins; D013111:Spider Venoms; C516323:dermonecrotic toxin, Loxosceles intermedia; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase