Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin.

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.

Photobiostimulation reduces edema formation induced in mice by Lys-49 phospholipases A2 isolated from Bothrops moojeni venom.

The prominent local myotoxic effects induced by Bothrops snake venom are due, in part, to myotoxins. This effect is not neutralized by antivenom, which is the main therapy for victims of snakebite. Two basic myotoxins named MjTX-I and MjTX-II were isolated from Bothrops moojeni venom. Both myotoxins have a Lys-49 phospholipase A2 structure devoid of enzymatic activity, but are highly myonecrotic and edema-inducing. In this study, we analyzed the effect of a low-level laser (LLL) at 685 nm, an energy density of 2.2 J cm(-2), and the irradiation time of 15 s, and a light emitting diode (LED) at 635 or 945 nm at energy densities of 4 and 3.8 J cm(-2), and irradiation times of 41 and 38 s, respectively, applied 30 min and 3 h after edema formation in mice caused by MjTX-I or MjTX-II. MjTX-I or MjTX-II caused a significant edema formation in envenomed paws. LLL and LED irradiation significantly reduced the edema formation by both myotoxins from 1 up to 6 hours after the injection. Both LLL and LEDs were similar in reducing the edema formation induced by myotoxins. The combined photobiostimulation with antivenom had the same effect in reducing edema as treatment with the LLL or LEDs alone. In conclusion, the results of this study indicate that photobiostimulation could be used in association with antivenom therapy for treatment of local effects of Bothrops species venom.

Two color morphs of the pelagic yellow-bellied sea snake, Pelamis platura, from different locations of Costa Rica: snake venomics, toxicity, and neutralization by antivenom.

The yellow-bellied sea snake, Pelamis platura, is the most broadly distributed snake species. Despite being endowed with a highly lethal venom, a proteomic analysis of its toxin composition was unavailable. The venoms of specimens collected in Golfo de Papagayo and Golfo Dulce (Costa Rica), where two distinctive color morphs occur, were chromatographically compared. The latter inhabits a fjord-like gulf where the transit of oceanic sea snakes into and from the basin is restricted, thus possibly affecting gene flow. RP-HPLC evidenced a conserved venom protein profile in both populations, despite their divergent color phenotypes. Following a trend observed in other sea snakes, P. platura venom is relatively simple, being composed of proteins of the three-finger toxin (3FTx), phospholipase A2 (PLA2), cysteine-rich secretory protein (CRISP), 5'-nucleotidase, and metalloproteinase families. The first three groups represent 49.9%, 32.9%, and 9.1% of total venom protein, respectively. The most abundant component (~26%) is pelamitoxin (P62388), a short-chain 3FTx, followed by a major basic PLA2 (~20%) and a group of three isoforms of CRISPs (~9%). Whereas isolated pelamitoxin was highly lethal to mice, neither the PLA2 nor the CRISP fraction caused death. However, the PLA2 rapidly increased plasma creatine kinase activity after intramuscular injection, indicating its myotoxic action. Differing from myotoxic PLA2s of viperids, this PLA2 was not cytolytic to murine myogenic cells in vitro, suggesting possible differences in its mechanism of action. The median lethal dose (LD50) estimates for P. platura crude venom in mice and in three species of fishes did not differ significantly. The sea snake antivenom manufactured by CSL Ltd. (Australia), which uses Enhydrina schistosa as immunogen, cross-recognized the three major components of P. platura venom and, accordingly, neutralized the lethal activity of crude venom and pelamitoxin, therefore being of potential usefulness in the treatment of envenomations by this species. BIOLOGICAL SIGNIFICANCE: Integrative analyses of animal venoms that combine the power of proteomics (venomics) with the characterization of their functional and immunological properties are significantly expanding knowledge on these remarkable bioweapons, both from a basic and a medical perspective. Costa Rica harbors a unique population of the yellow-bellied sea snake, Pelamis platura, that is restricted to a fjord-like gulf (Golfo Dulce). This population differs markedly from oceanic populations found elsewhere along the Pacific coast of this country, by presenting a patternless bright yellow coloration, instead of the typical bicolored or tricolored pattern of this species. It has been suggested that the dominance of this yellow-morph in Golfo Dulce might reflect gene flow restrictions, caused by the oceanographic conditions at this location. The present study demonstrates that the remarkable phenotypic variation between the two color morphs inhabiting Golfo Dulce and Golfo de Papagayo, respectively, is not associated with differences in the expression of venom components, as shown by their conserved RP-HPLC profiles. Proteomic analysis revealed the relatively simple toxin composition of P. platura venom, which contains three predominant types of proteins: three-finger toxins (protein abundance: 49.9%), phospholipases A2 (32.9%), and cysteine-rich secretory proteins (9.1%), together with few minor components. Further, the involvement of these most abundant proteins in the toxic effects of the venom, and their cross-recognition and neutralization by a sea snake antivenom produced against the venom of Enhydrina schistosa, were analyzed.

Evaluation of the genotoxicity of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes.

In the present study, experiments were carried out to evaluate the mutagenic potential and genotoxic effects of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes, using the micronucleus and comet assays. Significant damage to DNA was observed for crotoxin and crotapotin (CA). Basic phospholipase A(2) (CB) and crotamine did not present any mutagenic potential when evaluated by the micronucleus test. C. d. terrificus crude venom was able to induce the formation of micronuclei, similarly to the mutagenic drug used as a positive control. In the comet assay, all the toxins tested (crotamine, crotoxin, CB and CA) and C. d. terrificus venom presented genotoxic activity. Studies on the cytogenetic toxicology of animal venoms and their isolated proteins are still very scarce in the literature, which emphasizes the importance of the present work for the identification and characterization of potential therapeutic agents, as well as for the better understanding of the mechanisms of action of toxins on the human body.

Effects of Bothrops asper snake venom on lymphatic vessels: insights into a hidden aspect of envenomation.

BACKGROUND: Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied. METHODOLOGY/PRINCIPAL FINDINGS: B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A(2) (PLA(2)) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA(2) homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells. CONCLUSIONS/SIGNIFICANCE: Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA(2)s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations.

Systemic and local myotoxicity induced by snake venom group II phospholipases A2: comparison between crotoxin, crotoxin B and a Lys49 PLA2 homologue.

The patterns of myotoxicity induced in mice by crotoxin, crotoxin B and a Lys49 phospholipase A(2) (PLA(2)) homologue were compared. Lys49 PLA(2)-induced local myotoxicity is reflected by creatine kinase (CK) loss in injected gastrocnemius muscle, and by a profile of CK increase in plasma characterized by a rapid increment and drop after intramuscular injection, and by a lack of CK increase in plasma after intravenous injection. In contrast, crotoxin and crotoxin B, which induce local and systemic myotoxicity, provoked a more prolonged increment in plasma CK activity upon intramuscular injection, and induced increments in plasma CK after intravenous injection. The three toxins promoted a similar extent of local myotoxicity, assessed by the loss of CK in injected gastrocnemius. A method for the quantitative assessment of the ability of toxins to induce systemic myotoxicity is proposed, based on the estimation of the ratio between the area under the curve in the plasma CK activity (total myotoxicity) to the loss of CK in injected gastrocnemius (local myotoxicity). The highest ratio corresponded to crotoxin, and the lowest corresponded to Lys49 PLA(2), the former being a systemic myotoxin and the latter a local myotoxin. Neutralization by antivenoms also differed between the toxins: a drastic reduction in plasma CK, with very poor neutralization of local CK loss, was achieved in the case of crotoxin B when antivenom was injected intravenously, whereas no neutralization was achieved in the case of Lys49 PLA(2). When tested in undifferentiated myoblasts in culture, Lys49 PLA(2) induced cytotoxicity, whereas crotoxin and crotoxin B did not, evidencing that the latter are devoid of widespread cytolytic activity. Molecular modeling analysis showed that Lys49 PLA(2) has a conspicuous cationic face, which is likely to interact with diverse membranes. In contrast, crotoxin B, despite its overall basic pI, has a lower density of positively charged residues at this molecular region. It is suggested that Lys49 PLA(2)s homologues interact, through this cationic face, with many different cell types, thus lacking specificity for muscle cells. In contrast, crotoxin B has a more selective interaction with targets in the muscle cell membrane. This selectivity might be the basis for the ability of crotoxin and crotoxin B to induce systemic myotoxicity.

Structural and pharmacological comparison of daboiatoxin from Daboia russelli siamensis with viperotoxin F and vipoxin from other vipers.

Russell's viper (Vipera russelli, also known as Daboia russelli) is one of the major causes of fatal snakebites. To date, five Daboia russelli subspecies have been recognized. Daboiatoxin (DbTx) is the main lethal phospholipase A(2) (PLA(2)) toxin in the venom of D. russelli siamensis (Myanmar viper) and has strong neurotoxic, myotoxic and cytotoxic activities. DbTx and its homologous neurotoxins viperotoxin F from D. russelli formosensis (Taiwan viper) and vipoxin from the Bulgarian sand viper V. ammodytes meridionalis consist of complexes between a nontoxic acidic PLA(2) protein and an enzymatically active basic PLA(2). DbTx and viperotoxin F are presynaptic toxins, while vipoxin is postsynaptic. The two chains of DbTx have been separated and their PLA(2) enzymatic activity has been measured using the secretory PLA(2) assay kit. The enzymatic activity of DbTx chain B is reduced by 30% of its original activity by chain A in a unimolar ratio, thus indicating that DbTx chain A acts as an inhibitor. The lethal activity of the two chains has also been studied in male albino mice and chain A is less lethal than chain B. The crystal structure of DbTx has also been determined and its structural details are compared with those of the two homologues. Furthermore, an attempt is made to correlate the sequence and structural determinants of these toxins with their enzymatic activities and their pharmacological effects.

Triterpenoid saponins, new metalloprotease snake venom inhibitors isolated from Pentaclethra macroloba.

We report here the antiproteolytic and antihemorrhagic properties of triterpenoid saponin inhibitors, named macrolobin-A and B, from Pentaclethra macroloba, against Bothrops snake venoms. The inhibitors were able to neutralize the hemorrhagic, fibrin(ogen)olytic, and proteolytic activities of class P-I and P-III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. Clotting and fibrinogenolytic activities induced by snake venoms and isolated thrombin-like enzymes were partially inhibited. Furthermore, the potential use of these inhibitors to complement antivenom therapy as an alternative treatment and/or used as molecular models for development of new therapeutical agents in the treatment of snake bite envenomations needs to be evaluated in future studies.

Effects of the myotoxic Lys49 phospholipase A2 from Agkistrodon contortrix laticinctus snake venom on water transport in the toad bladder epithelium: evidence for a role of microtubules and calmodulin.

ACLMT is a myotoxic Lys49 phospholipase A2 isolated from the venom of the snake Agkistrodon contortrix laticinctus. We have previously demonstrated that ACLMT affects the water transport in toad bladders through a mechanism partially mediated by an increase in the cytosolic calcium. This study aims to further investigate the sites and mechanisms involved in the effects of ACLMT on water transport in toad bladders by examining the role of microtubules and calmodulin. Water flow across the membrane was gravimetrically measured in bladder sac preparations. ACLMT increased basal water transport and inhibited water transport stimulated by vasopressin. Colchicine and trifluoperazine reduced the effect of the toxin on basal water transport and enhanced it on vasopressin-stimulated water transport. The results suggest that both microtubules and calmodulin may be involved in the effect of ACLMT on basal water transport. On the other hand, the effect of the toxin on vasopressin-stimulated water transport appears to be neither dependent on the microtubules integrity nor directly mediated by calmodulin. This study provides a deeper understanding of the effects of the Lys49 PLA2 myotoxins on membrane permeability, thus contributing to elucidate the toxicity mechanism of these myotoxins on biological tissues.

Neurotoxic activity of Gln49 phospholipase A(2) from Gloydius ussuriensis snake venom.

A novel neurotoxic protein phospholipase A(2) (PLA(2)), molecular weight 13 881.83 Da, has been isolated from snake venom of Gloydius ussuriensis, named as Gln49-PLA(2), which shows weak lethal toxic, myotoxic and apparent anticoagulant activity, but lacks phospholipase activity. The Gln49-PLA(2) obviously induced an increase of the pain threshold in intoxicated 615 mice compared with the control group, suggesting it is a neurotoxin. Hot-plate tests also showed that its analgesic activity was dose-dependent, and naloxone antagonized the analgesic effect, implying the mechanism of action of Gln49-sPLA(2) is correlated with opioid receptors. Electrophysiology studies revealed decreases in the action potential and the nerve conduction velocity in isolated hoptoad (Bufo bufo gargarizans Cantor) sciatic nerve, indicating Gln49-PLA(2) most probably had effects on ion channels.

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

BaTX PLA(2), a K49 phospholipase A(2) homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on mu-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 PLA(2). The complete amino acid sequence of BaTX PLA(2) contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. 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 LD(50) of BaTX was 7 mug/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 microM: 40+/-0.4 min and 0.07 microM: 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 PLA(2) family, which presents the typical bioactivities described for such proteins.

Biochemical, pharmacological and structural characterization of two PLA2 isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom.

Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on micro-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry. The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA2, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA2 family, which presents the typical bioactivities described for such proteins.

Cytotoxicity of Lachesis muta muta snake (bushmaster) venom and its purified basic phospholipase A2 (LmTX-I) in cultured cells.

Human envenoming by Lachesis muta muta venom, although infrequent, is rather severe, being characterized by pronounced local tissue damage and systemic dysfunctions. Studies on the pharmacological actions of L. m. muta venom are relatively scant and the direct actions of the crude venom and its purified phospholipase A(2) (PLA(2)) have not been addressed using in vitro models. In this work, we investigated the cytotoxicity of L. m. muta venom and its purified PLA(2) isoform LmTX-I in cultured Madin-Darby canine kidney (MDCK) and in a skeletal muscle (C2C12) cell lines. As revealed by neutral red dye uptake assay, the crude venom (10 or 100 microg/ml) induced a significant decrease in cell viability of MDCK cells. LmTX-I at the concentrations tested (70-270 microg/ml or 5-20 microM) displayed no cytotoxicity in both MDCK and C2C12 cell lines. Morphometric analysis of Feulgen nuclear reaction revealed a significant increase in chromatin condensation (pyknosis), apparent reduction in the number of mitotic nuclei and nuclear fragmentation of some MDCK cells after incubation with L. m. muta venom. Monolayer exposure to crude venom resulted in morphological changes as assessed by scanning electron microscopy. The staining with TRITC-labelled phalloidin showed a marked disarray of the actin stress fiber following L. m. muta venom exposure. In contrast, LmTX-I had no effect on nucleus and cell morphologies as well as on stress fiber organization. These results indicate that L. m. muta venom exerts toxic effects on cultured MDCK cells. The LmTX-I probably does not contribute per se to the direct venom cytotoxicity, these effects are mediated by metalloproteinases/disintegrins and other components of the venom.

Secretory phospholipases A2 stimulate mucus secretion, induce airway inflammation, and produce secretory hyperresponsiveness to neutrophil elastase in ferret trachea.

Secretory phospholipases A(2) (sPLA(2)) are increased in the bronchoalveolar lavage fluid of patients with asthma and acute respiratory distress syndrome. Intratracheal sPLA(2) instillation induces acute lung injury in the rat and guinea pig. We hypothesized that sPLA(2) would stimulate mucus secretion in vitro and that intratracheal sPLA(2) exposure would induce mucus hypersecretion and airway inflammation in the ferret trachea in vivo. In vitro, porcine pancreatic sPLA(2) at a concentration of 0.5 or 5 U/ml significantly increased mucous glycoconjugate (MG) secretion from the excised ferret trachea. P-bromophenacylbromide (a sPLA(2) inhibitor), quercetin (a lipoxygenase inhibitor), or MK-886 (a 5-lipoxygenase inhibitor), each at 10(-4) M, significantly reduced sPLA(2)-induced MG secretion. sPLA(2)-stimulated MG secretion was decreased in Ca(2+)-free medium. In vivo, ferrets were intubated for 30 min once per day for 3 days using an ETT coated with 20 units of porcine pancreatic sPLA(2) mixed in water-soluble jelly. Constitutive MG secretion increased 1 day after sPLA(2) exposure and returned to control 5 days later. Human neutrophil elastase (HNE) at 10(-8) M increased MG secretion in the sPLA(2)-exposed trachea compared with that in the control trachea, but methacholine at 10(-7) M did not. sPLA(2)-induced secretory hyperresponsiveness continued for at least 5 days after sPLA(2) exposure ended. sPLA(2) increased tracheal inflammation, MG secretion, and secretory hyperresponsiveness to HNE probably through enzymatic action rather than by activation of its receptor.

Interfacial surface charge and free accessibility to the PLA2-active site-like region are essential requirements for the activity of Lys49 PLA2 homologues.

Lys49 phospholipase A2 homologues are highly myotoxic and cause extensive tissue damage but do not display hydrolytic activity towards natural phospholipids. The binding of heparin, heparin derivatives and polyanionic compounds such as suramin result in partial inhibition (up to 60%) of the myotoxic effects due to a change in the overall charge of the interfacial surface. In vivo experiments demonstrate that polyethylene glycol inhibits more than 90% of the myotoxic effects without exhibiting secondary toxic effects. The crystal structure of bothropstoxin-I complexed with polyethylene glycol reveals that this inhibition is due to steric hindrance of the access to the PLA2-active site-like region. These two inhibitory pathways indicate the roles of the overall surface charge and free accessibility to the PLA2-active site-like region in the functioning of Lys49 phospholipases A2 homologues. Molecular dynamics simulations, small angle X-ray scattering and structural analysis indicate that the oligomeric states both in solution and in the crystalline states of Lys49 phospholipases A2 are principally mediated by hydrophobic contacts formed between the interfacial surfaces. These results provide the framework for the potential application of both clinically approved drugs for the treatment of Viperidae snakebites.

Purification, sequencing and structural analysis of two acidic phospholipases A2 from the venom of Bothrops insularis (jararaca ilhoa).

Bothrops snake venoms contain a variety of phospholipases (PLA(2)), some of which are myotoxic. In this work, we used reverse-phase HPLC and mass spectrometry to purify and sequence two PLA(2) from the venom of Bothrops insularis. The two enzymes, designated here as BinTX-I and BinTx-II, were acidic (pI 5.05 and 4.49) Asp49 PLA(2), with molecular masses of 13,975 and 13,788, respectively. The amino acid sequence and molecular mass of BinTX-I were identical to those of a PLA(2) previously isolated from this venom (PA2_BOTIN, SwissProt accession number ) while those of BinTX-II indicated that this was a new enzyme. Multiple sequence alignments with other Bothrops PLA(2) showed that the amino acids His48, Asp49, Tyr52 and Asp99, which are important for enzymatic activity, were fully conserved, as were the 14 cysteine residues involved in disulfide bond formation, in addition to various other residues. A phylogenetic analysis showed that BinTX-I and BinTX-II grouped with other acidic Asp49 PLA(2) from Bothrops venoms, and computer modeling indicated that these enzymes had the characteristic structure of bothropic PLA(2) that consisted of three alpha-helices, a beta-wing, a short helix and a calcium-binding loop. BinTX-I (30 microg/paw) produced mouse hind paw edema that was maximal after 1h compared to after 3h with venom (10 and 100 microg/paw); in both cases, the edema decreased after 6h. BinTX-1 and venom (40 microg/ml each) produced time-dependent neuromuscular blockade in chick biventer cervicis preparations that reached 40% and 95%, respectively, after 120 min. BinTX-I also produced muscle fiber damage and an elevation in CK, as also seen with venom. These results indicate that BinTX-I contributes to the neuromuscular activity and tissue damage caused by B. insularis venom in vitro and in vivo.

Structural and functional properties of Cr 5, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Calloselasma rhodostoma.

Cr 5 PLA(2) homologous (K49) was isolated from Calloselasma rhodostoma venom in only one chromatographic step in reverse phase HPLC (RP-HPLC) (on mu-Bondapack C-18). A molecular mass of 13.965 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that Cr 5 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical residues of a basic PLA(2). The complete amino acid sequence of Cr 5 PLA(2) contains 120 residues, resulting in a calculated pI value of 5.55. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. The sequence found was SLVELGKMIL QETGKNPAKS YGAYGCNCGV LGRHKPKDAT DRCCFVHKCC YKKLTGCDPK KDRYSYSWKD KTIVCGENNP CLKEMCECDK AVAICLRENL DTYNKKYRYL KPFCKKADDC. In mice, Cr 5 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr 5 was 0.070 mg/kg of the animal weight, by intracerebroventricular (i.c.v.) route. In vitro, the toxin caused rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. The isolation of this PLA(2) and the combined structural and functional information obtained classify Cr 5 as a new member of the K49 PLA(2) family, since it presents typical features from such proteins.

Reversible skeletal neuromuscular paralysis induced by different lysophospholipids.

Lysophosphatidylcholine rapidly paralyses the neuromuscular junction (NMJ), similarly to snake phospholipase A2 neurotoxins, implicating a lipid hemifusion-pore transition in neuroexocytosis. The mode and kinetics of NMJ paralysis of different lysophospholipids (lysoPLs) in high or low [Mg2+] was investigated. The following order of potency was found: lysophosphatidylcholine>lysophosphatidylethanolamine>lysophosphatidic acid>lysophosphatidylserine>lysophosphatidylglycerol. The latter two lysoPLs closely mimic the profile of paralysis caused by the toxins in high [Mg2+]. This paralysis is fully reversed by albumin washing. These findings provide novel insights on the mode of action of snake neurotoxins and qualify lysoPLs as novel agents to study neuroexocytosis.

Discovery of novel [Arg49]phospholipase A2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan.

Protobothrops (formerly Trimeresurus) elegans, a Crotalinae snake, inhabits Ishigaki and Iriomote islands of the Sakishima Islands of Japan which are located between Okinawa island of Japan and Taiwan. Two phospholipase A(2) (PLA(2)) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA(2) isozymes with Arg at position 49, that is, [Arg(49)]PLA(2) forms, named PeBP(R)-I and PeBP(R)-II. They are polymorphic at position 3, Val for PeBP(R)-I and Ile for PeBP(R)-II. The cDNAs encoding PeBP(R)-I and PeBP(R)-II were cloned. The cDNA encoding an [Asp(49)]PLA(2) named PePLA(2) was also obtained. In contrast to PLA(2) isozymes from Protobothrops genus with 122 amino acid residues, PeBP(R)-I and PeBP(R)-II are composed of 121 amino acid residues due to lack of Pro at position 90. They exhibited necrotic and edema-inducing activities but no hemorrhagic activity was detected. A phylogenetic tree constructed for venom PLA(2) isozymes of Protobothrops genus and of related genera in the southwestern islands of Japan and Taiwan revealed that PeBP(R)-I and PeBP(R)-II of P. elegans are evolutionarily much closer to PmK49PLA(2), a [Lys(49)]PLA(2), from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys(49)]PLA(2) forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp(49)]PLA(2) isozymes from the three Protobothrops species. Thus, P. elegans is the species much closer to P. mucrosquamatus than P. flavoviridis. Their evolutionary distances seem to be well related to geological history of the islands where they have lived. In addition, it was clearly noted that Ovophis okinavensis (Amami-Oshima), which had formerly belonged to the Trimeresurus genus, and Trimeresurus stejnegeri (Taiwan) are the species fairly distant from Protobothrops genus.

Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-phospholipase A2 with low catalytic activity from Bothrops jararacussu venom.

For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA2s.