Risk assessment of the antifungal and insecticidal peptide Jaburetox and its parental protein the Jack bean (Canavalia ensiformis) urease.

Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.

Functional and structural study comparing the C-terminal amidated ß-neurotoxin Ts1 with its isoform Ts1-G isolated from Tityus serrulatus venom.

Mature Ts1, the main neurotoxin from Tityus serrulatus venom, has its C-terminal Cys amidated, while the isolated isoform of Ts1, named Ts1-G, keeps the non-amidated Gly residue at the C-terminal region, allowing the study of the comparative functional importance of amidation at the C-terminal between these two native toxins. Voltage dependent sodium current measurements showed that the affinity of Ts1-G for sodium channels is smaller than that of the mature Ts1, confirming the important role played by the C-terminal amidation in determining Ts1 activity.

Frontoxins, three-finger toxins from Micrurus frontalis venom, decrease miniature endplate potential amplitude at frog neuromuscular junction.

Neurotoxicity is a major symptom of envenomation caused by Brazilian coral snake Micrurus frontalis. Due to the small amount of material that can be collected, no neurotoxin has been fully sequenced from this venom. In this work we report six new three-finger like toxins isolated from the venom of the coral snake M. frontalis which we named Frontoxin (FTx) I-VI. Toxins were purified using multiple steps of RP-HPLC. Molecular masses were determined by MALDI-TOF and ESI ion-trap mass spectrometry. The complete amino acid sequence of FTx II, III, IV and V were determined by sequencing of overlapping proteolytic fragments by Edman degradation and by de novo sequencing. The amino acid sequences of FTx I, II, III and VI predict 4 conserved disulphide bonds and structural similarity to previously reported short-chain alpha-neurotoxins. FTx IV and V each contained 10 conserved cysteines and share high similarity with long-chain alpha-neurotoxins. At the frog neuromuscular junction FTx II, III and IV reduced miniature endplate potential amplitudes in a time-and concentration-dependent manner suggesting Frontoxins block nicotinic acetylcholine receptors.

Nephrotoxicity caused by brown spider venom phospholipase-D (dermonecrotic toxin) depends on catalytic activity.

Bites from brown spiders (Loxosceles genus) have clinical manifestations including skin necrosis with gravitational spreading, and systemic involvement that may include renal failure, hemolysis, and thrombocytopenia. Mice were exposed to recombinant wild-type phospholipase-D, or to an isoform with a mutation in the catalytic domain resulting in no phospholipasic activity. Renal biopsies from mice treated with the wild-type toxin showed glomerular edema, erythrocytes and collapse of Bowman's space, edema and deposition of proteinaceous material within the tubular lumen. Ultrastructural analyses confirmed cytotoxicity by demonstrating disorders of glomerulus at foot processes and at fenestrated endothelium. Tubule alterations include deposits of amorphous material and edema, as well as an increase of epithelial cytoplasmic multivesicular bodies and electron-dense structures. There was an absence of nephrotoxicity in mice treated with the mutated toxin. Analyses of urine and blood showed that wild type toxin induced hematuria and elevation of blood urea, while treatment with mutated toxin caused no changes. Mouse lethality experiments also showed oliguria and mortality after treatment with wild-type toxin, but not following exposure to the mutated toxin. Immunofluorescence using antibodies to phospholipase-D toxin showed deposition of both toxins along the renal tubular structures as detected by confocal microscopy. Immunoblots of urine showed a 30 kDa band in samples from animals treated with wild-type toxin, but no band from mice exposed to mutated toxin. Wild-type toxin treatment caused cytoplasmic vacuolization, impaired spreading, reduction of cellular viability, and cell-cell and cell-substratum detachment in MDCK cells, while treatment with mutated isoform had no effect. Finally, there is a direct correlation between toxin activity on cell membrane phospholipids generating choline and cytotoxicity. We have defined for the first time a molecular mechanism for Loxosceles venom nephrotoxicity that is dependent on the catalytic activity of phospholipase-D toxin.

Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds.

Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways.

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom.

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.

Individual venom variability in Crotalus durissus ruruima snakes, a subspecies of Crotalus durissus from the Amazonian region.

Venoms of six specimens of Crotalus durissus ruruima snakes from the same geographical site in the Brazilian State of Roraima, were individually assayed for their main pharmacological properties. Quantitative and qualitative differences were found and the presence of crotoxin-like isoforms in these venoms was indicated. Our findings corroborate the existence of a considerable intrapopulational variability in C. d. ruruima venoms, and the importance of using a pool of venoms for antivenom production, in general, in order to assure the neutralization of the maximum possible number of toxins from a given species.

Long-sarafotoxins: characterization of a new family of endothelin-like peptides.

Sarafotoxins (SRTXs) constitute a family of vasoactive peptides that were initially isolated from the venom of Atractaspis engaddensis, and that are structurally and functionally related to endothelins (ETs). Analysis of the venom of Atractaspis microlepidota microlepidota revealed several new SRTX molecules manifesting some new structural and functional characteristics. These novel SRTXs are longer by three amino acids than the previously described SRTXs, and are designated here "long-SRTXs". Six isoforms, derived from new poly-cistronic precursors, have been identified so far in the venom of this snake. One of these isoforms, designated SRTX-m, was chemically synthesized and its biological properties were studied. Our results show that SRTX-m induces toxicity in mice, mostly due to vasoconstriction, and also that it has a lower toxicity and potency than the more potent SRTX described up to now: sarafotoxin-b (SRTX-b) from A. engaddensis.

Primary structure and electrophysiological characterization of two almost identical isoforms of toxin from Isometrus vittatus (family: Buthidae) scorpion venom.

Two almost identical proteins with 70 amino acid residues each, closely packed by four disufide bridges, and molecular masses of 7899.5 and 7884.7 were isolated and sequenced from the venom of the scorpion Isometrus vittatus from Pakistan. They differ by an acidic amino acid residue (glutamic or aspartic) at the same position 55 of the peptide chain, however, they exhibit the same length, the same charge and are undistinguishable when separated by C(18) reverse phase HPLC. The mixture of the two proteins called IsomTx1 depolarizes the cockroach isolated axon; artificial repolarization is followed by sustained repetitive activity, artificial hyperpolarization facilitates bursting activity observed as an answer to rapid depolarization to -60 mV. The depolarization is antagonized by TTX. In voltage-clamp experiments IsomTx1 increases axonal sodium permeability which has a particular importance between resting and threshold potentials and moderately slows down the fast inactivation. These characteristics closely resemble those of other anti-insect scorpion toxins classified as contractive toxins from Androctonus and Buthotus venoms.