Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom

Author Silveira, Rafael B.da Google Scholar
Wille, Ana C. M. Google Scholar
Chaim, Olga Meiri Autor UNIFESP Google Scholar
Appel, Marcia H. Google Scholar
Silva, Dilza T. Google Scholar
Franco, Celia R. C. Google Scholar
Toma, Leny Autor UNIFESP Google Scholar
Mangili, Oldemir C. Google Scholar
Gremski, Waldemiro Google Scholar
Dietrich, Carl Peter Autor UNIFESP Google Scholar
Nader, Helena Bonciani Autor UNIFESP Google Scholar
Veiga, Silvio S. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Univ Fed Parana
Univ Estadual Ponta Grossa
Catholic Univ Parana
Abstract 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 metal loproteases 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), phylogenefic 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.
Keywords astacin
Loxosceles astacin-like protease (LALP)
metalloprotease
spider
toxin
venom
Language English
Date 2007-09-01
Published in Biochemical Journal. London: Portland Press Ltd, v. 406, p. 355-363, 2007.
ISSN 0264-6021 (Sherpa/Romeo, impact factor)
Publisher Portland Press Ltd
Extent 355-363
Origin http://dx.doi.org/10.1042/BJ20070363
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000249181200018
URI http://repositorio.unifesp.br/handle/11600/29978

Show full item record




File

File Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)

Search


Browse

Statistics

My Account