Maria Elena de Lima

Tityus serrulatus venom peptidomics: assessing venom peptide diversity.

MALDI-TOF-TOF and de novo sequencing were employed to assess the Tityus serrulatus venom peptide diversity. Previous works has shown the cornucopia of molecular masses, ranging from 800 to 3000Da, present in the venom from this and other scorpions species. This work reports the identification/sequencing of several of these peptides. The majority of the peptides found were fragments of larger venom toxins. For instance, 28 peptides could be identified as fragments from Pape proteins, 10 peptides corresponded to N-terminal fragments of the TsK beta (scorpine-like) toxin and fragments of potassium channel toxins (other than the k-beta) were sequenced as well. N-terminal fragments from the T. serrulatus hypotensins-I and II and a novel hypotensin-like peptide could also be found. This work also reports the sequencing of novel peptides without sequence similarities to other known molecules.

Phoneutria nigriventer Toxin 1: A Novel, State-Dependent Inhibitor of Neuronal Sodium Channels That Interacts with μ Conotoxin Binding Sites

A toxin was purified to homogeneity from the venom of the South American armed spider Phoneutria nigriventer and found to have a molecular mass of 8600 Da and a C-terminally amidated glycine residue. It appears to be identical to Toxin 1 (Tx1) isolated previously from this venom. Tx1 reversibly inhibited sodium currents in Chinese hamster ovary cells expressing recombinant sodium (Nav1.2) channels without affecting their fast biophysical properties. The kinetics of inhibition of peak sodium current varied with membrane potential, with on-rates increasing and off-rates decreasing with more depolarized holding potentials in the –100 to –50 mV range. Thus, the apparent affinity of Tx1 for the channel increases as the membrane is depolarized. A mono[125I]iodo-Tx1 derivative displayed high-affinity binding to a single class of sites (KD = 80 pM, Bmax = 0.43 pmol/mg protein) in rat brain membranes. Solubilized binding sites were immunoprecipitated by antibodies directed against a conserved motif in sodium channel α subunits. 125I-Tx1 binding was competitively displaced by μ conotoxin GIIIB (IC50 = 0.5 μM) but not by 1 μM tetrodotoxin. However, the inhibition of 125I-Tx1 binding by μ conotoxin GIIIB was abrogated in the presence of tetrodotoxin (1 μM). Patch-clamp and binding data indicate that P. nigriventer Tx1 is a novel, state-dependent sodium-channel blocker that binds to a site in proximity to pharmacological site 1, overlapping μ conotoxin but not tetrodotoxin binding sites.

Purification, amino-acid sequence and partial characterization of two toxins with anti-insect activity from the venom of the South American scorpion Tityus bahiensis (Buthidae).

We report here the isolation by a two-step chromatographic procedure of two new toxins from the South American scorpion Tityus bahiensis. Their amino-acid sequences and some of their biological features were established. The two toxins have different biological properties. Toxin TbIT-I had almost no activity or pharmacological effects in vertebrate tissues whereas it was lethal to house flies (LD50 80.0 ng/house fly). In contrast, Tb2-II was active against both mammals (intracerebroventricular injection of 100 ng/mouse was lethal) and insects (LD50 40.0 ng/house fly). The amino-acid sequences of these toxins were established and found to be similar (60-95%) to previously described beta-toxins from the Tityus genus. Based on the available comparative information, this study attempts identify possible structure-function relationships that may be responsible for the differences in bioactivity displayed by these toxins.

PhTx4, a new class of toxins from Phoneutria nigriventer spider venom, inhibits the glutamate uptake in rat brain synaptosomes.

We report the characterization of a new class of glutamate uptake inhibitors isolated from Phoneutria nigriventer venom. Glutamate transport activity was assayed in rat cerebrocortical synaptosomes by using [(3)H]-L-glutamate. PhTx4 inhibited glutamate uptake in a dose dependent manner. The IC(50) value obtained was 2.35+/-0.9 microg/ml which is in the observed range reported for glutamate uptake blockers. Tx4-7, one of PhTx4 toxins, showed the strongest inhibitory activity (50.3+/-0.69%, n=3).

Animal toxins: state of the art - perspectives in health and biotechnology

This single-volume edition presents for the first time results of several studies with different perspectives on the real possibilities of the use of animal venoms and toxins in the biotechnology industry. Animal Toxins consists of 39 articles, signed by renowned experts of various nationalities. The state of the art in compounds derived from venoms of marine animals, spiders and scorpions, lizards, snakes, among others, are the focus of this publication which aims to meet scientists, students and university researchers, biotechnologists interested in toxicology as well as the pharmaceutical industry. Animal venoms and toxins have been selected over millions of years of evolution to act quickly and effectively on the victim body, which results in a massive repertoire of molecules able to bind to specific targets. The possibility of using these toxins in biotechnological processes means that these venoms and toxins are regarded as one of the most promising sources of bioactive natural compounds.

Venomic analysis and evaluation of antivenom cross-reactivity of South American Micrurus species.

Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), β-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.

The Toxins Purified from Tityus Serrulatus (Lutz & Mello)Venom

AbstractThe toxicity of Tityus serrulatus venom is mainly due to a complex mixture of basic proteins of low molecular weight (MW< 8000 Da) which are active on the voltage-sensitive Na+ channel of excitable cells. One group of toxins, the α-toxins, delays inactivation of the Na+ channel. A second group, the β- toxins, produces a transient shift in the voltage-dependence of Na+ channel activation and increases the tendency of the cells to fire repetitively. The two groups bind specifically to two different binding sites, sites 3 and 4, of Na+ channels present in rat brain synaptosomes. The primary structure of the main toxins has been determined and consists of a single amino acid chain of 61 to 66 residues cross-linked by four disulfide bridges. Some secondary structural elements have also been determined. More recently, using molecular biological techniques, cDNAs encoding the precursors of α and β-toxins have been cloned from a cDNA library of Tityus serrulatus venom gland. The precursors contain a signa...

Structure and Activity Analysis of Two Spider Toxins That Alter Sodium Channel Inactivation Kinetics

In this work, Phoneutria nigriventer toxins PnTx2-5 and PnTx2-6 were shown to markedly delay the fast inactivation kinetics of neuronal-type sodium channels. Furthermore, our data show that they have significant differences in their interaction with the channel. PnTx2-6 has an affinity 6 times higher than that of PnTx2-5, and its effects are not reversible within 10-15 min of washing. PnTx2-6 partially (59%) competes with the scorpion alpha-toxin AaHII, but not with the scorpion beta-toxin CssIV, thus suggesting a mode of action similar to that of site 3 toxins. However, PnTx2-6 is not removed by strong depolarizing pulses, as in the known site 3 toxins. We have also established the correct PnTx2-5 amino acid sequence and confirmed the sequence of PnTx2-6, in both cases establishing that the cysteines are in their oxidized form. A structural model of each toxin is proposed. They show structures with poor alpha-helix content. The model is supported by experimental and theoretical tests. A likely binding region on PnTx2-5 and PnTx2-6 is proposed on the basis of their different affinities and sequence differences.

Novel structural class of four disulfide-bridged peptides from Tityus serrulatus venom

A new structural class of short peptides folded by four disulfide-bridges was found in the venom of the Brazilian scorpion Tityus serrulatus. Peptides were put on evidence independently by means of two different approaches of structurally guided prospection. First, a cDNA sequence was obtained using a degenerate primer constructed according to the C-terminal sequence of kaliotoxin (KTx2), from the Androctonus australis venom. Second, MALDI-TOF mass spectrometry analyses of toxic fraction FIII from T. serrulatus venom revealed a family of molecules ranging approximately from 2900 to 3000 Da. Three new peptides were isolated and named TsPep1, TsPep2, and TsPep3. Biochemical characterization showed that they are 29 amino acids long, constrained by a new pattern of four disulfide-bridges. These results enable us to classify these new molecules as part of a novel structural class of short peptides from scorpion venoms.

Nitric Oxide-Induced Vasorelaxation in Response to PnTx2-6 Toxin from Phoneutria nigriventer Spider in Rat Cavernosal Tissue

INTRODUCTION Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na(+) ) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. AIM We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. MAIN OUTCOME MEASURES PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. METHODS Rat cavernosal strips were incubated with bretylium (3 × 10(-5) M) and contracted with phenylephrine (PE; 10(-5) M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10(-8) M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10(-6) M), a muscarinic receptor antagonist, N-type Ca(2+) channel blockers (ω-conotoxin GVIA, 10(-6) M) and sildenafil (3 × 10(-8) M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. RESULTS Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca(2+) channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. CONCLUSION We show that PnTx2-6 slows Na(+) channels inactivation in nitrergic neurons, allowing Ca(2+) influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction.