Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom.
Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.
da Silveira
Rafael B
RB
Department of Biochemistry, Federal University of São Paulo, Rua 3 de maio, 100 5 andar, São Paulo, Brazil.
Wille
Ana C M
AC
Chaim
Olga M
OM
Appel
Marcia H
MH
Silva
Dilza T
DT
Franco
Célia R C
CR
Toma
Leny
L
Mangili
Oldemir C
OC
Gremski
Waldemiro
W
Dietrich
Carl P
CP
Nader
Helena B
HB
Veiga
Silvio S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D008969:Molecular Sequence Data; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013111:Spider Venoms / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics / Q000633:toxicity; D013112:Spiders / Q000737:chemistry* / Q000145:classification / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D005353:Fibronectins; D013111:Spider Venoms; D005780:Gelatin; D005340:Fibrinogen; D008666:Metalloendopeptidases; C071288:astacin