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

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 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.

Factor Xa-like and fibrin(ogen)olytic activities of a serine protease from Hippasa agelenoides spider venom gland extract

“…Thus, the spider venoms appear to exhibit restricted proteolytic activity on the human fibrinogen and degrade readily the Aa-chain and later the Bb-chain but never the c-chain. An astacin-like metalloprotease from L. intermedia venom that specifically degrade Aa-and Bb-chains but not the c-chain was cloned and expressed in the recent past [4,27,28]. Hither to, the reported fibrinogenolytic activity from spider venoms was sensitive to metal chelators such as EDTA and 1,10-phenanthroline [4] except for the Hagprotease, which is sensitive to PMSF [10].…”

Klebsiella pneumoniae yggG Gene Product: A Zinc-Dependent Metalloprotease

“…Laborious refolding step was required to obtain the proteases in their active and stable forms [19,23,31]. The full length and 21 amino acids N-terminal truncated versions of YggG protein from E. coli were also membrane-associated [15].…”

Pattern of inflammatory response to Loxosceles intermedia venom in distinct mouse strains: A key element to understand skin lesions and dermonecrosis by poisoning

“…L. intermedia venom is liquid, colorless, crystalline and extremely rich in proteases, alkaline phosphatase, hyaluronidases, phospholipases, metalloproteases among other components (Barbaro et al, 2005;Chaim et al, 2011;da Silveira et al, 2007a;da Silveira et al, 2007b;Tambourgi et al, 2000). The spread of the venom and consequent dermonecrosis observed in humans and rabbits are attributed to hyaluronidase (Futrell, 1992) and phospholipase D (also referred as sphingomyelinase D) activities (Appel et al, 2008;Kalapothakis et al, 2007;Tambourgi et al, 1998).…”