Functional expression of a recombinant toxin - rPnTx2-6 - active in erectile function in rat.

In the current study, the putative cDNA for PnTx2-6 toxin of the Phoneutria nigriventer spider venom was cloned and expressed as tioredoxin fusion protein in the cytoplasm of Escherichia coli. The fusion protein was purified from the bacterial extracts by combination of immobilized Ni-ion affinity and gel filtration chromatographies. Then, it was cleaved by enterokinase and the generated recombinant PnTx2-6 (rPnTx2-6) was further purified by reverse-phase HPLC. Likewise the native toxin purified from the spider venom, rPnTx2-6 potentiates the erectile function when injected in rats. This result indicates that the production of functional recombinant PnTx2-6 might be an alternative to provide this basic and valuable tool for study, as well as for further understanding such complex physiological system, including its correlation with the central nervous system and local tissue factors.

Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms Implications for snake toxin repertoire evolution

Efforts to describe toxins from the two major families of venomous snakes (Viperidae and Elapidae) usually reveal proteins belonging to few structural types, particular of each family. Here we carried on an effort to determine uncommon cDNAs that represent possible new toxins from Lachesis muta (Viperidae). In addition to nine classes of typical toxins, atypical molecules never observed in the hundreds of Viperidae snakes studied so far are highly expressed: a diverging C-type lectin that is related to Viperidae toxins but appears to be independently originated; an ohanin-like toxin, which would be the third member of the most recently described class of Elapidae toxins, related to human butyrophilin and B30.2 proteins; and a 3FTx-like toxin, a new member of the widely studied three-finger family of proteins, which includes major Elapidae neurotoxins and CD59 antigen. The presence of these common and uncommon molecules suggests that the repertoire of toxins could be more conserved between families than has been considered, and their features indicate a dynamic process of venom evolution through molecular mechanisms, such as multiple recruitments of important scaffolds and domain exchange between paralogs, always keeping a minimalist nature in most toxin structures in opposition to their nontoxin counterparts.

Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: evolution of venom group II phospholipase A2's may imply gene duplications.

The sequence coding for a snake venom phospholipase A2 (PLA2), BJUPLA2, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA2's. A striking feature of the cDNA is the high sequence conservation of the 5' and 3' untranslated regions in cDNAs coding for PLA2's from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA2 with group II PLA2's in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA2's.

Sequence of the cDNA coding for the lethal neurotoxin Tx1 from the Brazilian "armed" spider Phoneutria nigriventer predicts the synthesis and processing of a preprotoxin.

A cDNA library was constructed from the venom glands of the Brazilian "armed" spider, Phoneutria nigriventer, and a clone coding for Tx1, a lethal toxin, was identified and sequenced. The sequence data derived from this cDNA clone combined with the previously determined amino acid sequence predict that Tx1 is initially synthesized as a preprotoxin. Four segments (comprising the signal sequence, a short, 15-amino acid, glutamate-rich sequence, the functional toxin, and 2 glycine residues) can be distinguished. The structure of the preprotoxin and the proposed processing steps required to form the mature Tx1 toxin show similarities with the synthesis and processing of omega-agatoxin IA.

Purification and properties of a kininogenin from the venom of Lachesis muta (bushmaster).

An acidic kininogenin from Lachesis muta snake venom was purified to apparent homogeneity by a combination of gel filtration, isoelectric focusing and preparative gel electrophoresis. It was shown to be a highly stable serine protease (mol. wt 27,900; pI 5.4) capable of releasing bradykinin from low mol. wt bovine kininogen and of cleaving some synthetic chromogenic peptides with the following catalytic efficiencies (Kcat/Km, M-1.sec-1): N-benzoyl-Phe-Val-Arg-p-nitroanilide (1.92 x 10(4)); H-D-Val-Leu-Arg-p-nitroanilide (1.55 x 10(4)); N-acetyl-Phe-Arg-p-nitroanilide (3.98 x 10(2)); no hydrolysis was observed with N-benzoyl-Arg-p-nitroanilide. A marked and sustained hypotensive effect was recorded following i.v. injection of purified kininogenin into rats. Tachyphylaxis was observed after repeated i.v. injection of the enzyme, a phenomenon accompanied by a decrease of only 15% in the total circulating rat kininogen. Both the in vivo action and the enzymatic properties of the L. muta kininogenin indicate that this enzyme might be helpful for understanding the kinin-kininogen system.

Biological activities of venoms from South American snakes.

Standard assay procedures for the characterization of snake venoms, developed by the World Health Organization (WHO) Collaborating Centre for the Control of Antivenoms (CCCA), were used to analyse 33 snake venoms including eight International Reference Venoms for the assessment of lethal, defibrinogenating, procoagulant, haemorrhagic and necrotizing properties. The International Reference Venoms were assayed as part of an International Collaborative Programme for the evaluation of Venoms and Antivenoms; the results showed a close relationship to those obtained by the CCCA. Twenty-five venoms from 13 different species of medically important snakes from South America were assayed as standardized by the WHO-CCCA. Additionally, evaluation of lethal activity by the i.p. and intra cerebroventricular routes, proteolytic effects and venom-induced edema were also determined. Venom yields from captive snakes are also presented. Among the venoms examined, from the subfamily Crotalinae, the members of the genera Bothrops and Lachesis had strong haemorrhagic, proteolytic and edema-inducing activities, whereas all Crotalus durissus species had none. The Elapinae, Micrurus frontalis showed no procoagulant, defibrinogenating, haemorrhagic, necrotizing or proteolytic activities. The results reflect differences among individual samples of the same species and of different geographical regions. The results suggest that there is little or no relationship between the properties of the different venoms and that the determination of one effect cannot predict the value of the others. Therefore, the characterization of the different activities of snake venoms is necessary if toxicity is to be properly evaluated and neutralized.