Purification and characterization of a fibrinogenolytic and hemorrhagic metalloproteinase isolated from Vipera lebetina venom

Serious clinical problems such as hemorrhage, edema and tissue necrosis are observed following viperid envenoming. A proteinase (VLH2) was isolated from Vipera lebetina by combination of two chromatographic steps of gel filtration on Sephadex G-75 followed by DEAE Sephadex A-50. This acidic proteinase, with a molecular mass of about 55 kDa and isoelectric point of 5.4, displayed a fibrinogenolytic and hemorrhagic activities. VLH2 hydrolyses rapidly the Aα-chain of fibrinogen, followed, more slowly, by the Bβ-chain, leaving the γ-chain unaffected. The proteolytic and hemorrhagic activities of VLH2 were inhibited by EDTA, EGTA and 1–10 phenanthroline. However, these activities were not affected by AEBSF, Aprotinine, and E64, suggesting that VLH2 is a metalloproteinase with an α-fibrinogenase activity, requiring calcium and zinc for its activity. The enzyme VLH2 did not have proteolytic activity towards extracellular components gelatin, laminin and fibronectin. The hemorrhagic metalloproteinase VLH2 has a myotoxic activity, as determined by serum CK level and histological observation of muscle tissue. Furthermore, VLH2 is able to induce apoptosis of C2C12 myotubes. These results indicate that VLH2 is implicated in the local and systemic bleeding, contributing thus in the toxicity of V. lebetina venom.     

R25 is an intracellular membrane receptor for a snake venom secretory phospholipase A(2)

Ammodytoxin is a presynaptically neurotoxic (beta-neurotoxic) snake venom secretory phospholipase A(2) (sPLA(2)). We detected a 25 kDa protein which binds the toxin with very high affinity (R25) in porcine cerebral cortex. Here we show that R25 is an integral membrane protein with intracellular localisation. It is the first sPLA(2) receptor known to date that localises to intracellular membranes. Centrifugation on sucrose gradients was used to fractionate porcine cerebral cortex. The subcellular composition of the fractions was determined by following the distribution of organelle-specific markers. The distribution of R25 in the fractions matched the distribution of the mitochondrial marker succinate dehydrogenase, but not the markers for plasma membrane, lysosomes, endoplasmic reticulum, synaptic and secretory vesicles. R25 most likely resides in mitochondria, which are known to be targets for sPLA(2) neurotoxins in the nerve ending and are potentially implicated in the process of beta-neurotoxicity.     

Biochemical and enzymatic characterization of BpMP-I, a fibrinogenolytic metalloproteinase isolated from Bothropoides pauloensis snake venom

Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SDS-PAGE and showed a single chain polypeptide of 20 kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and β-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme.     

Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara

Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. In this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrinogenolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate d-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca²⁺ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators.     

The neurotoxic phospholipase A2 associates,through a non-phosphorylated binding motif,with 14-3-3 protein gamma and epsilon isoforms

Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A(2) from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Using tandem mass spectrometry, the isolated acceptors were identified as 14-3-3 gamma and epsilon isoforms, highly conserved cytoplasmic proteins involved in the regulation of numerous physiological processes. The interaction between ammodytoxin C and 14-3-3 proteins is direct and not mediated by calmodulin, a high-affinity acceptor for both ammodytoxin C and 14-3-3 proteins, as demonstrated in pull-down experiments and by surface plasmon resonance. The latter technique gave an apparent dissociation constant of 1.0+/-0.2 microM for the interaction between chip-immobilized 14-3-3 and ammodytoxin C. 14-3-3 usually interacts with proteins through specific phospho-Ser/Thr motifs. Ammodytoxin C is not a phospho-protein, therefore the interaction must occur through a non-phosphorylated binding site, most probably the KEESEK sequence at its C-terminal end. The interaction we describe suggests an explanation for the pathophysiological effects evoked by some secreted phospholipases A(2), such as the inhibition of protein phosphorylation, of terminal ion currents, and of neurotransmission, as well as the initiation of neuronal cell death, all processes regulated by 14-3-3 proteins.     

Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom

Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. The proteinase has an apparent molecular mass of 55 kDa as revealed by the reduced SDS-PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. The partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methionine-containing turn of similar conformation (“Met-turn”), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. The enzyme only cleaves the Ala₁₄-Leu₁₅ peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the Aα-chain of fibrinogen and the α-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. In addition, its enzymatic activity was stimulated by the divalent cations Ca²⁺ and Mg²⁺ but inhibited by Zn²⁺ and Cu²⁺. The catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD = 30 ng in rabbit), with alterations in platelet function. In summary, here we report the isolation and the structure-function relationship of a P-III snake venom metalloproteinase.     

BJ-PI2, A non-hemorrhagic metalloproteinase from Bothrops jararaca snake venom

Background

Envenoming by Bothrops jararaca can result in local pain, edema, hemorrhage and necrosis, partially mediated by snake venom metalloproteinases (SVMPs). Here, we describe the characterization of BJ-PI2, a P-I class SVMP from B. jararaca venom, and its local tissue actions.

Methods

BJ-PI2 was purified by a combination of gel filtration, anion-exchange chromatography and reverse phase HPLC, and identified by mass spectrometry. Clotting and fibrin(ogen)olytic activities were assayed using conventional methods. Hemorrhagic activity and changes in vascular permeability were examined in rat dorsal skin. Myonecrosis and inflammatory activity were examined in mouse gastrocnemius muscle.

Results

BJ-PI2 was a 23.08 kDa single-chain polypeptide. Tryptic fragments showed highest homology with SVMP insularinase A from Bothrops insularis, but also with B. jararaca SVMP bothrojaractivase; less similarity was observed with B. jararaca SVMPs BJ-PI and jararafibrases II and IV. BJ-PI2 did not clot fibrinogen or rat citrated plasma but had α- and β-fibrinogenolytic activity (inhibited by EDTA and 1,10-phenanthroline but not by PMSF) and attenuated coagulation after plasma recalcification. BJ-PI2 had fibrinolytic activity. BJ-PI2 increased the vascular permeability of rat dorsal skin (inhibited by 1,10-phenanthroline). BJ-PI2 was not hemorrhagic or myonecrotic but caused migration of inflammatory cells. In contrast, venom was strongly hemorrhagic and myonecrotic but caused less infiltration of inflammatory cells.

Conclusions

BJ-PI2 is a non-hemorrhagic, non-myonecrotic, non-coagulant P-I class SVMP that may enhance vascular permeability and inflammatory cell migration in vivo.

General significance

BJ-PI2 contributes to enhanced vascular permeability and inflammatory cell migration after envenoming, but not to venom-induced hemorrhage and necrosis.     

Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.     

Binding to the high-affinity M-type receptor for secreted phospholipases A(2) is not obligatory for the presynaptic neurotoxicity of ammodytoxin A

R180, isolated from porcine brain cortex, is a high-affinity membrane receptor for ammodytoxin A (AtxA), a secreted phospholipase A(2) (sPLA(2)) and presynaptically active neurotoxin from venom of the long-nosed viper (Vipera ammodytes ammodytes). As a member of the M-type sPLA(2) receptors, present on the mammalian plasma membrane, R180 has been proposed to be responsible for one of the first events in the process of presynaptic neurotoxicity, the binding of the toxin to the nerve cell. To test this hypothesis, we prepared and analyzed three N-terminal fusion proteins of AtxA possessing a 12 or 5 amino acid residue peptide. The presence of such an additional "propeptide" prevented interaction of the toxin with the M-type receptor but not its lethality in mouse and neurotoxic effects on a mouse phrenic nerve-hemidiaphragm preparation. In addition, antibodies raised against the sPLA(2)-binding C-type lectin-like domain 5 of the M-type sPLA(2) receptor were unable to abolish the neurotoxic action of AtxA on the neuromuscular preparation. The specific enymatic activities of the fusion AtxAs were two to three orders of magnitude lower from that of the wild type, yet resulting in a similar but less pronounced neurotoxic profile on the neuromuscular junction. This is in accordance with other data showing that a minimal enzymatic activity suffices for presynaptic toxicity of sPLA(2)s to occur. Our results indicate that the interaction of AtxA with the M-type sPLA(2) receptor at the plasma membrane is not essential for presynaptic activity of the toxin. Interaction of AtxA with two intracellular proteins, calmodulin and the R25 receptor, was affected but not prevented by the presence of the N-terminal fusion peptides, implying that these proteins may play a role in the sPLA(2) neurotoxicity.     

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.     

Protein disulphide isomerase binds ammodytoxin strongly: possible implications for toxin trafficking

Ammodytoxin, a group IIA secreted phospholipase A(2) from the venom of the long-nosed viper (Vipera ammodytes ammodytes), is a potent presynaptically acting neurotoxin. It blocks the secretion of neurotransmitter from the nerve cell, thus hindering the communication with the neighbouring neuron or muscle cell. To express the neurotoxicity, ammodytoxin should interact with specific receptors in the axon terminal and express phospholipase activity. Our previous results indicate that, following the association with a receptor on the external side of the presynaptic membrane, the toxin penetrates into the cytosol of the nerve cell. Here, we show that the toxin associates specifically with protein disulphide isomerase, a protein in the lumen of endoplasmic reticulum, which may be crucial for the retention and concentration of the toxin in this cellular compartment and for its subsequent transport across the membrane of endoplasmic reticulum into the cytosol of the nerve cell.     

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 23 kDa 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 Ca⁺² but inhibited by EDTA, dithiothreitol, excessive Zn⁺² and α2-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 α-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 α₇β₁ and α₁β₁ were cleaved by atroxlysin. Even without catalytic activity atroxlysin-I inhibited collagen- and ADP-triggered platelet aggregation.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

High molecular mass kininogen inhibits metalloproteinases of Bothrops jararaca snake venom

High molecular mass kininogen (HK) purified from Bothrops jararaca (Bj) plasma was tested on activities of the Bj venom in vivo and in vitro. Results showed that, when incubated with BjHK, the Bj venom presented inhibition on hemorrhagic, edema forming, myotoxic, and coagulant activities. It is well known that metalloproteinases are directly or indirectly involved in these activities. Similarly, human HK inhibits the hemorrhagic effect of the Bj venom as well as hemorrhagic and enzymatic effects of jararhagin, a hemorrhagic metalloproteinase isolated from Bj venom. Complex between HK and jararhagin was not detected by gel filtration. Nevertheless, the inhibitory effect of the hemorrhagic activity of the venom was only partial when HK was pre-incubated with 0.4mM ZnCl(2) or with 0.45mM CaCl(2). These data suggest that the inhibitory effect depends, at least partially, on the competition for ions between kininogen and metalloproteinases of the venom.     

Alkaloids in the venom of Messor ants

Tobacco alkaloids of the anabasine type have been found or confirmed in the venom of five species of arid-dwelling Messor ants. They are frequently accompanied by alkylpyrazines. Messor mediorubra contains four alkaloids, with anabasine the major component and also minor pyrazines. Anabasine was found alone in the venom of Messor semirufus and confirmed in Messor ebeninus. Messor rugosus from Tel Aviv contained a mixture of alkaloids and pyrazines, but those from Ein Yahav contained 3-ethyl-2,5-dimethylpyrazine, some 2-phenylethylamine, and N-ethylidene 2-phenylethylamine. Messor arenarius is confirmed as having a complex but variable mixture of alkaloids and pyrazines.     

Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40?318 AMU for DM40 and 42?373 and 43?010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS–PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.     

BaG,a new dimeric metalloproteinase/disintegrin from the Bothrops alternatus snake venom that interacts with alpha5beta1 integrin

The alpha(5)beta(1) integrin is one of the major fibronectin receptors which plays an essential role in the adhesion of normal and tumor cells to extracellular matrix. Here, we describe the isolation and characterization of a novel dimeric metalloproteinase/disintegrin, which is an inhibitor of fibronectin binding to the alpha(5)beta(1) integrin. This protein (BaG) was isolated from the venom of the South American snake Bothrops alternatus by gelatin-Sepharose affinity and anion exchange chromatography. The molecular mass of BaG was approximately 130 kDa under non-reducing conditions and 55 kDa under reducing conditions by SDS-PAGE. BaG shows proteolytic activity on casein that was inhibited by EDTA. 1,10-phenanthroline-treated BaG (BaG-I) inhibits ADP-induced platelet aggregation with an IC(50) of 190 nM. BaG-I inhibits fibronectin-mediated K562 cell adhesion with an IC(50) of 3.75 microM. K562 cells bind to BaG-I probably through interaction with alpha(5)beta(1) integrin, since anti-alpha(5)beta(1) antibodies inhibited K562 cell adhesion to BaG-I. In addition, BaG-I induces the detachment of K562 cells that were bound to fibronectin. In summary, we have purified a novel, dimeric snake venom metalloproteinase/disintegrin that binds to the alpha(5)beta(1) integrin.     

Toxoplasma gondii: effects of neuwiedase, a metalloproteinase from Bothrops neuwiedi snake venom, on the invasion and replication of human fibroblasts in vitro

The major aim to the present study was to determine the effects of neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom, on invasion and replication of Toxoplasma gondii in human fibroblasts in vitro. Neuwiedase treatment was done on host cells previously infected with T. gondii or on parasite before fibroblast infection. When treatments were done after or before infection, infection rates were inhibited in 71% and 61%, respectively. Considering that therapy protocols currently used in T. gondii infection cause considerable side effects, particularly in immunocompromised individuals and pregnant women, the results of neuwiedase treatment described herein could be taken into account for the development of new synthetic therapeutic agents, mainly due to the capacity of this enzyme to degrade extracellular matrix components, such as laminin, fibronectin and type I collagen, which is important to interfere in T. gondii host cell invasion.     

Identification, cloning and expression of a second gene (vpr1) from the venom of the endoparasitic wasp, Pimpla hypochondriaca that displays immunosuppressive activity

Previously, we biochemically isolated an immunosuppressive protein (VPr3) from the venom of Pimpla hypochondriaca and cloned and expressed the gene in bacteria. The deduced amino acid sequence for VPr3 shares 63% identity with a second P. hypochondriaca protein, venom protein one (VPr1). We have now cloned and expressed the gene for vpr1. The expression of His-tagged recombinant VPr1 (rVPr1) in E. coli BL21 Star™ (DE3) cells was induced by the addition of 0.5 mM IPTG. Cultures were grown at 24 and 37 °C, and VPr1 more readily partitioned into the soluble fraction at 24 °C. Soluble rVPr1 was purified using the MagneHis purification system and a modified elution buffer to allow the protein to be directly tested for activity against haemocytes. It was observed that rVPr1 prevented the ability of haemocytes to spread and form aggregates in vitro in a dose-dependent manner. Furthermore, comparable levels of activity were observed when similar concentrations of rVPr1 and rVPr3 were tested. In addition, the encapsulation of Sephadex beads in vivo was reduced by the presence of rVPr1 and beads were unencapsulated (negative) or only weakly encapsulated. The functional and physio-chemical properties of rVPr1 and rVPr3 are compared and discussed.     

Structural and functional properties of BaTX,a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus

BaTX PLA₂, a K49 phospholipase A₂ homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on μ-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA₂. The complete amino acid sequence of BaTX PLA₂ contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA₂s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA₂s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD₅₀ of BaTX was 7 μg/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 μM: 40 ± 0.4 min and 0.07 μM: 35 ± 0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA₂ family, which presents the typical bioactivities described for such proteins.