Suely G. Figueiredo

Purification and amino acid sequences of six Tx3 type neurotoxins from the venom of the Brazilian ‘armed’ spider Phoneutria Nigriventer (keys.)

Six neurotoxic peptides (Tx3-1 to Tx3-6) were purified from the venom of the spider Phoneutria nigriventer by a combination of gel filtration, reverse phase FPLC on PEP-RPC and PRO-RPC columns, reverse phase HPLC on Vydac C18, and ion exchange HPLC on cationic and anionic columns. These toxins caused different neurological symptoms in mice after intracerebroventricular injection. At dose levels of 5 micrograms/mouse, Tx3-3 and Tx3-4 caused rapid general flaccid paralysis followed by death in 10-30 min; Tx3-2 induced immediate clockwise gyration and flaccid paralysis after 6 hr; Tx3-1, Tx3-5 and Tx3-6 produced paralysis only in the posterior limbs and gradual decreases in movement and aggression during 24 hr. The mol. wt of these cystine-rich peptides were found to be in the range of 3500-8500 by mass spectroscopy and SDS-PAGE. The complete amino acid sequences of the neurotoxins Tx3-1 (40 residues), Tx3-2 (34 residues) and Tx3-6 (55 residues), and the N-terminal sequences of Tx3-3 (34 res.), Tx3-4 (40 res.) and Tx3-5 (36 res.) were established by direct automated Edman degradation, and manual DABITC/PITC microsequence analyses of peptides obtained from digests with various proteases. The structures of these Tx3 neurotoxins from Phoneutria nigriventer exhibited sequence similarities to one another and to the neurotoxins from the venoms of the spiders Hololena curta and Agelenopsis aperta, which were most evident in the location of the Cys residues.

The novel metalloproteinase atroxlysin-I from Peruvian Bothrops atrox (Jergón) snake venom acts both on blood vessel ECM and platelets

We report the isolation and structure-function relationship of a 23kDa metalloproteinase named atroxlysin-I from the venom of the Peruvian Bothrops atrox (Jergón). Atroxlysin is a P-I metalloproteinase and contains 204 residues. Its proteolytic activity towards dimethylcasein is enhanced by Ca2+ but inhibited by EDTA, dithiothreitol, excessive Zn2+ and alpha2-macroglobulin. Unlike other structurally homologous P-I metalloproteinases, atroxlysin-I causes hemorrhages. To examine its hemorrhagic activity mechanistically, we studied its function in vitro and in vivo. It cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B-chain and specifically hydrolyzed the alpha-chains of fibrin(ogen) in a dose- and time-dependent manner. Atroxlysin-I cleaved plasma fibronectin and other extracellular matrix proteins (collagens I and IV) and the triple-helical fragment CB3 of collagen IV, but did not degrade laminin-111. Complementarily, the laminin and collagen binding integrins alpha7beta1 and alpha1beta1 were cleaved by atroxlysin. Even without catalytic activity atroxlysin-I inhibited collagen- and ADP-triggered platelet aggregation.

Purification and amino acid sequence of a highly insecticidal toxin from the venom of the Brazilian spider Phoneutria nigriventer which inhibits NMDA-evoked currents in rat hippocampal neurones

A new insecticidal toxin Tx4(5-5) was isolated from the fraction PhTx4 of the venom of the spider Phoneutria nigriventer by reverse phase high performance liquid chromatography (HPLC) and anion exchange HPLC. The complete amino acid sequence determined by automated Edman degradation showed that Tx4(5-5) is a single chain polypeptide composed of 47 amino acid residues, including 10 cysteines, with a calculated molecular mass of 5175 Da. Tx4(5-5) shows 64% of sequence identity with Tx4(6-1), another insecticidal toxin from the same venom. Tx4(5-5) was highly toxic to house fly (Musca domestica), cockroach (Periplaneta americana) and cricket (Acheta domesticus ), producing neurotoxic effects (knock-down, trembling with uncoordinated movements) at doses as low as 50 ng/g (house fly), 250 ng/g (cockroach) and 150 ng/g (cricket). In contrast, intracerebroventricular injections (30 microg) into mice induced no behavioural effects. Preliminary electrophysiological studies carried out on whole-cell voltage-clamped rat hippocampal neurones indicated that Tx4(5-5) (at 1 microM) reversibly inhibited the N-methyl-D-aspartate-subtype of ionotropic glutamate receptor, while having little or no effect on kainate-, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid- or gamma-aminobutyric acid-activated currents.

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).

Plumieribetin, a Fish Lectin Homologous to Mannose-binding B-type Lectins, Inhibits the Collagen-binding α1β1 Integrin

Recently, a few fish proteins have been described with a high homology to B-type lectins of monocotyledonous plants. Because of their mannose binding activity, they have been ascribed a role in innate immunity. By screening various fish venoms for their integrin inhibitory activity, we isolated a homologous protein from the fin stings and skin mucus of the scorpionfish (Scorpaena plumieri). This protein inhibits α1β1 integrin binding to basement membrane collagen IV. By protein chemical and spectroscopic means, we demonstrated that this fish protein, called plumieribetin, is a homotetramer and contains a high content of anti-parallel β strands, similar to the mannose-binding monocot B-lectins. It lacks both N-linked glycoconjugates and common O-glycan motifs. Despite its B-lectin-like structure, plumieribetin binds to α1β1 integrin irrespective of N-glycosylation, suggesting a direct protein-protein interaction. This interaction is independent of divalent cations. On the cellular level, plumieribetin failed to completely detach hepatocarcinoma HepG2 cells and primary arterial smooth muscle cells from the collagen IV fragment CB3. However, plumieribetin weakened the cell-collagen contacts, reduced cell spreading, and altered the actin cytoskeleton, after the compensating α2β1 integrin was blocked. The integrin inhibiting effect of plumieribetin adds a new function to the B-lectin family, which is known for pathogen defense.

Kallikrein-like proteinase from bushmaster snake venom.

A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (0.8 microg/g) was shown to lower blood pressure in experimental rats. In vitro, the isolated proteinase was shown to have neither fibrin(ogeno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity=13.0 and 31.5 U/mg, respectively; crude venom=0.25 and 6.0 U/mg) but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB and PMSF, indicating that the enzyme is a serine proteinase. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen was one of the activities exhibited by LV-Ka.

Cardiovascular effects of scorpionfish (Scorpaena plumieri) venom

The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri venom in both in vivo and in vitro models. In anesthetized rats, doses of the venom (14-216 microg protein/kg) induced a transient increase in the mean arterial pressure. However at higher dose (338 microg protein/kg) this effect was followed by a sudden hypotension and the animal evolved to death. The heart rate was temporarily increased and followed by bradycardia using doses > or =108 microg/kg. In isolated rat hearts the crude venom (5-80 microg protein) produced dose-dependent positive ventricular chronotropic, inotropic, lusitropic and coronary vasoconstriction responses. Partial purification of an active fraction (CF, cardiovascular fraction) which reproduced the cardiovascular effects induced by crude venom on isolated hearts was achieved by conventional gel filtration chromatography. Adrenergic blockades, prazosin and propranolol, significantly attenuated these responses. The coronary vasoconstriction response to CF was also attenuated by chemical endothelium denudation. In conclusion, the data showed that S. plumieri fish venom induces disorders in the cardiovascular system. It also suggests that alpha(1) and beta-adrenergic receptors, and the vascular endothelium, are involved at least partially, in these cardiac effects.

Systemic response induced by Scorpaena plumieri fish venom initiates acute lung injury in mice.

Scorpaena plumieri venomous fish inflicted severe injuries in humans characterized by systemic effects and cardiovascular abnormalities. Although cardiotoxic and hypotensive effects induced in rats by this venom have been studied, little is known about their effect on bronchial epithelial permeability and airway inflammation in mice. The primary goal of this study was to determine whether the intraplantar or intraperitoneal injection of S. plumieri venom results in systemic response, and whether this event initiates acute lung injury. We found that BALB/c mice developed neutrophilic infiltrates, areas of lung hemorrhage and alveolar macrophage activation within 24h after injection with S. plumieri venom. These histopathological changes were associated with an early increase in BAL fluid protein and early induction of cytokines, chemokines and matrix metalloproteinases, followed by a later increase in BAL fluid neutrophils. These findings provide clear evidence that the injection of S. plumieri venom in footpad or peritoneal cavity of mice results in venom deposition in the airway and initiates a sustained inflammatory response in the lungs.

Proteomic analysis of the soluble proteomes of miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates obtained from Brazilian patients with different treatment outcomes

UNLABELLED The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in experimental resistant lines; however, studies using clinical isolates with different miltefosine susceptibilities are still needed. In our study, we used a proteomic 2D-DIGE/MS approach to study different protein abundances in miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The high-resolution proteome obtained from these isolates showed 823 matched spots and 46 spots exhibited different abundances between the isolates. Out of these differentially expressed spots, 26 (56.5%) showed greater and 20 (43.5%) showed lower expression of the resistant isolate compared to the sensitive isolate. MALDI/TOF-TOF mass spectrometry allowed the identification of 32 spots with unique protein identification correspondent to 22 non-redundant proteins. Most of the proteins up-regulated in the proteome miltefosine-resistant isolates were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. These differentially expressed proteins are likely involved in miltefosine natural resistance and suggest that the miltefosine-resistance mechanism in Leishmania is multifactorial. BIOLOGICAL SIGNIFICANCE Visceral leishmaniasis (VL) is a serious disease with a challenging treatment plan requiring the prolonged and painful applications of poorly tolerated toxic drugs. Therefore, the identification of miltefosine, an effective and safe oral drug, was considered a significant advancement in leishmaniasis therapy. However, different sensitivities to miltefosine in Leishmania have been observed in clinically relevant species, and the biological mechanism by which clinical isolates of Leishmania acquire drug resistance is poorly understood. Our work aims to elucidate the mechanism of natural resistance to miltefosine in Leishmania by studying the isolates from VL patients who displayed different miltefosine treatment outcomes.