Myotoxic PLA Research Articles

Identification and characterization of a phospholipase A 2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A 2 (PLA 2), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA 2, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA 2, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA 2s from other snake venoms. The purified PLA 2 EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei ( KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against ( B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA 2 (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA(2) crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for approximately 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted ( approximately 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed.

Isolation and characterization of two new Lys49 PLA 2s with heparin neutralizing properties from Bothrops moojeni snake venom

Among the proteins and peptides already characterized in Bothrops moojeni venom, two novel phospholipases A 2 (PLA 2) have been purified and fully sequenced by ESI-MS/MS techniques. Both of them belong to the enzymatically non-active Lys49 variants of PLA 2. They consist of 122 amino acids and share a characteristic sequence in their C-terminal region composed of clusters of basic amino acids known to interact with heparin. Thus, as already established, heparin can be used as an antidote to antagonize some myotoxic PLA 2s from venoms of Bothrops genus. The two PLA 2 variants were shown to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH), neutralizing their anticoagulant properties. Although the influences of PLA 2s from snake venoms on the blood coagulation system are known, their use to antagonize the anticoagulant effect of heparin in vitro or in vivo has never been proposed. These finding recommend diagnostic and therapeutic applications, which are currently investigated.

Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake

We report the purification and biochemical/pharmacological characterization of two myotoxic PLA 2 (BbTX-II K49 PLA 2 homologue and BbTX-III PLA 2) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing M r ∼14 kDa for both myotoxins, showing high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BbTX-II K49 PLA 2 homologue and BbTX-III PLA 2, had a sequence of 121 amino acid residues. BbTX-II: SLFELGKMILQETGKNPAKSYGAYGCYCGVLGRGKPKDATDRCCYVHKCCYKLTGCDNKKKDRYSYSWKDKTIVCGENNPCLKELCECDKAVAICLRENLNTYNKKYRYHLKPLCKKADAC with p I value 8.73. BbTX-III: SLWEWGQMILKETGKNPFPYYGAYGCYCGWGGRRKPKDATDRCCFVHDCCRYKKLTGCPKTNDRYSYSRLDYTIVCGEDDPCKEICECDKAAAVCFRENLRTYNKKYMAHLRVLCKKDKPC with a p I value of 8.46. BbTX-III presented PLA 2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35–45 °C. Maximum PLA 2 activity required Ca 2+. In vitro, BbTX-II K49 PLA 2 homologue and BbTX-III PLA 2 caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In mice, BbTX-II K49 PLA 2 homologue and BbTX-III PLA 2 induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA 2 homologue, occur in the absence of a measurable PLA 2 activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.

Effects of Bothrops asper Snake Venom on Lymphatic Vessels: Insights into a Hidden Aspect of Envenomation

BackgroundEnvenomations 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 A2 (PLA2) 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 PLA2 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/SignificanceOur observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA2s 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.

A new acidic myotoxic, anti-platelet and prostaglandin I 2 inductor phospholipase A 2 isolated from Bothrops moojeni snake venom

Phospholipase A 2 (PLA 2, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl- sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A 2 from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE–Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl–Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and p I 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA 2s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA 2s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.

Myotoxic phospholipases A 2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A 2 (PLA 2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M r ∼ 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA 2s from snake venoms, MTX-I belonging to Asp49 PLA 2 class, enzymatically active, and MTX-II to Lys49 PLA 2s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA 2 and anticoagulant activities, corroborating the importance of residue His48 and Ca 2+ ions for the enzymatic catalysis. Both PLA 2s induced myotoxic activity and dose–time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115–129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA 2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer.

Purification, characterization and bactericidal activities of basic phospholipase A 2 from the venom of Agkistrodon halys (Chinese pallas)

Agkistrodon snake venoms contain a variety of phospholipases (PLA 2), some of which are myotoxic. In this study, we used reverse-phase HPLC to purify PLA 2 from the venom of Agkistrodon halys. The enzyme named as AgkTx-II, a basic Asp49 PLA 2, has a molecular masses of 13,869.05. The amino acid sequence and molecular mass of AgkTx-II was identical to those of an Asp49 basic myotoxic PLA 2 previously isolated from this venom. Antibacterial activities were tested by susceptibility and broth-dilution assays. AgkTx-II exerted a potent antibacterial activity against Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Burkholderia pseudomallei. The MIC values of AgkTx-II ranged between 85 and 2.76 μM and was most effective against S. aureus, P. vulgaris, P. mirabilis (MIC of 21.25 μM) and B. pseudomallei (MIC of 10.25 μM). This AgkTx-II rapidly killed S. aureus, P. vulgaris and B. pseudomallei in a dose-dependent manner. The effect of the AgkTx-II on bacterial membranes was evaluated by scanning and transmission electron microscopy. AgkTx-II caused morphological alterations apparent on their cellular surfaces, suggesting a killing mechanism based on membrane permeabilization and damage. Cytotoxicity was measured by XTT tetrazolium (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) and lactate dehydrogenase (LDH) assays using U-937 cells (monocytes). The AgkTx-II did not affect cell viability up to 500 μM concentrations but cell death was evident at 1000 μM concentration after 24 and 48 h. Furthermore, the repeated exposure of AgkTx-II (2–14 μM) treated mice showed different tissue alterations, mainly at the brain and kidney; the toxicological potential of AgkTx-II remains to be elucidated. The AgkTx-II exhibits no hemolytic action even at high doses (10–100 μM) in human erythrocytes. However, the AgkTx-II is believed to exert its bactericidal effect by permeabilizing the bacterial membrane by forming pores. In addition, the basic PLA 2 AgkTx-II displays a bactericidal effect, which may be either dependent or independent of catalysis.

Isolation and functional characterization of a new myotoxic acidic phospholipase A 2 from Bothrops pauloensis snake venom

This article reports the purification procedure and the biochemical/functional characterization of Bp-PLA 2, a new myotoxic acidic phospholipase A 2 from Bothrops pauloensis snake venom. It was highly purified through three chromatographic steps (ion-exchange on CM-Sepharose, hydrophobic chromatography on Phenyl-Sepharose and RP-HPLC on a C8 column). Bp-PLA 2 is a single-chain protein of 15.8 kDa and p I 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA 2s from snake venoms. Its specific activity was 585.3 U/mg. It displayed a high indirect hemolytic activity and inhibited platelet aggregation induced by collagen or ADP. It also induced in vivo edema and myotoxicity. Pretreatment of Bp-PLA 2 with BPB reduced the enzymatic activity, the inhibitory action on platelet aggregation and myotoxicity in vitro. Morphological analyses indicated that Bp-PLA 2 induced an intense edema, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection. Acidic myotoxic PLA 2s from Bothrops snake venoms are still not extensively explored and knowledge of their structural and functional features will contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.

C-Terminal Heparin-Binding Peptide of Snake Venom VEGF Specifically Blocks VEGF-Stimulated Endothelial Cell Proliferation

Vascular endothelial growth factor-A165 (VEGF-A165) exhibits diverse biological effects through binding to its receptor KDR (VEGFR-2). Heparin-like molecules are known to modulate their interaction. There have been reports that VEGF-A lacking the C-terminal heparin binding region significantly reduced mitogenic activity. Recently, we found novel heparin-binding VEGFs from snake venoms, designated VEGF-Fs, which specifically recognize kinase domain containing receptor (KDR). The C-terminal heparin-binding region is almost completely absent in VEGF-Fs when compared with other heparin-binding VEGFs, despite their heparin-binding potential. In this congress, we report that the C-terminal heparin-binding region of VEGF-F specifically/preferentially interacts with the VEGF-bondable heparin/heparan sulfate, but not with those associated with bFGF or TFPI. We also present the identification of a VEGF receptor-binding protein from the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys⁴⁹PLA₂, an inactive PLA₂ homologue with strong myoxicity. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. The interaction between KDR-bp and KDR was blocked by VEGF-A165, and KDR-bp specifically inhibited VEGF-A165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. This is the first observation demonstrating that an exogenous factor antagonizes the VEGF receptor, furthermore, it is the first identification of the target molecule of the myotoxic PLA₂ from viper venom.

Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms

Group II phospholipase A(2) (PLA(2)) myotoxins isolated from Viperidae/Crotalidae snake venoms induce a rapid cytolytic effect upon diverse cell types in vitro. Previous studies suggested that this effect could be more pronounced on skeletal muscle myotubes than on other cell types, including undifferentiated myoblasts. This study utilized the murine skeletal muscle C2C12 cell line to investigate whether differentiated myotubes are more susceptible than myoblasts, and if this characteristic is specific for the group II myotoxic PLA(2)s. The release of lactic dehydrogenase was quantified as a measure of cytolysis, 3 h after cell exposure to different group II PLA(2)s purified from Bothrops asper, Atropoides nummifer, Cerrophidion godmani, and Bothriechis schlegelii venoms. In addition, susceptibility to lysis induced by synthetic melittin and group III PLA(2) from bee (Apis mellifera) venom, as well as by anionic, cationic, and neutral detergents, was comparatively evaluated on the two cultures. Myotubes were significantly more susceptible to group II PLA(2) myotoxins, but not to the other agents tested, under the same conditions. Moreover, the increased susceptibility of myotubes over myoblasts was also demonstrated with two cytolytic synthetic peptides, derived from the C-terminal region of Lys49 PLA(2) myotoxins, that reproduce the action of their parent proteins. These results indicate that fusion and differentiation of myoblasts into myotubes induce changes that render these cells more susceptible to the toxic mechanism of group II PLA(2) myotoxins, but not to general perturbations of membrane homeostasis. Such changes are likely to involve myotoxin acceptor site(s), which remain(s) to be identified.

Crystallization and preliminary X-ray diffraction analysis of suramin, a highly charged polysulfonated napthylurea, complexed with a myotoxic PLA 2 from Bothrops asper venom

Suramin is a highly charged polysulfonated napthylurea that interferes in a number of physiologically relevant processes such as myotoxicity, blood coagulation and several kinds of cancers. This synthetic compound was complexed with a myotoxic Lys49 PLA 2 from Bothrops asper venom and crystallized by the hanging-drop vapor diffusion method at 18 °C. The crystals belong to the orthorhombic space group P2 12 12 1, with unit cell parameters a=49.05, b=63.84 and c=85.67 Å. Diffraction data was collected to 1.78 Å.

Bactericidal and neurotoxic activities of two myotoxic phospholipases A 2 from Bothrops neuwiedi pauloensis snake venom

Two basic myotoxic PLA 2s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA 2s showed a M r around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), p I∼7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA 2s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn 2+ ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115–129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA 2s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.

Skeletal muscle degeneration induced by venom phospholipases A 2: insights into the mechanisms of local and systemic myotoxicity

Local and systemic skeletal muscle degeneration is a common consequence of envenomations due to snakebites and mass bee attacks. Phospholipases A 2 (PLA 2) are important myotoxic components in these venoms, inducing a similar pattern of degenerative events in muscle cells. Myotoxic PLA 2s bind to acceptors in the plasma membrane, which might be lipids or proteins and which may differ in their affinity for the PLA 2s. Upon binding, myotoxic PLA 2s disrupt the integrity of the plasma membrane by catalytically dependent or independent mechanisms, provoking a pronounced Ca 2+ influx which, in turn, initiates a complex series of degenerative events associated with hypercontraction, activation of calpains and cytosolic Ca 2+-dependent PLA 2s, and mitochondrial Ca 2+ overload. Cell culture models of cytotoxicity indicate that some myotoxic PLA 2s affect differentiated myotubes in a rather selective fashion, whereas others display a broad cytolytic effect. A model is presented to explain the difference between PLA 2s that induce predominantly local myonecrosis and those inducing both local and systemic myotoxicity. The former bind not only to muscle cells, but also to other cell types, thereby precluding a systemic distribution of these PLA 2s and their action on distant muscles. In contrast, PLA 2s that bind muscle cells in a more selective way are not sequestered by non-specific interactions with other cells and, consequently, are systemically distributed and reach muscle cells in other locations.

Chemical modifications of phospholipases A 2 from snake venoms: effects on catalytic and pharmacological properties

Phospholipases A 2 (PLA 2s) constitute major components of snake venoms and have been extensively investigated not only because they are very abundant in these venoms but mainly because they display a wide range of biological effects, including neurotoxic, myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anti-coagulant, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoral, anti-malarial and anti-parasitic. Due to this functional diversity, these structurally similar proteins aroused the interest of many researchers as molecular models for study of structure–function relationships. One of the main experimental strategies used for the study of myotoxic PLA 2s is the traditional chemical modification of specific amino acid residues (His, Met, Lys, Tyr, Trp and others) and examination of the consequent effects upon the enzymatic, toxic and pharmacological activities. This line of research has provided useful insights into the structural determinants of the action of these enzymes and, together with additional strategies, supports the concept of the presence of ‘pharmacological sites’ distinct from the catalytic site in snake venom myotoxic PLA 2s.

Inflammatory effects of snake venom myotoxic phospholipases A 2

Snake venom phospholipases A 2 (PLA 2) show a remarkable functional diversity. Among their toxic activities, some display the ability to cause rapid necrosis of skeletal muscle fibers, thus being myotoxic PLA 2s. Besides myotoxicity, these enzymes evoke conspicuous inflammatory and nociceptive events in experimental models. Local inflammation and pain are important characteristics of snakebite envenomations inflicted by viperid and crotalid species, whose venoms are rich sources of myotoxic PLA 2s. Since the discovery that mammalian PLA 2 is a key enzyme in the release of arachidonic acid, the substrate for the synthesis of several lipid inflammatory mediators, much interest has been focused on this enzyme in the context of inflammation. The mechanisms involved in the proinflammatory action of secretory PLA 2s are being actively investigated, and part of the knowledge on secretory PLA 2 effects has been gained by using snake venom PLA 2s as tools, due to their high structural homology with human secretory PLA 2s. The inflammatory events evoked by PLA 2s are primarily associated with enzymatic activity and to the release of arachidonic acid metabolites. However, catalytically inactive Lys49 PLA 2s trigger inflammatory and nociceptive responses comparable to those of their catalytically active counterparts, thereby evidencing that these proteins promote inflammation and pain by mechanisms not related to phospholipid hydrolysis nor to mobilization of arachidonic acid. These studies have provided a boost to the research in this field and various approaches have been used to identify the amino acid residues and the specific sites of interaction of myotoxic PLA 2s with cell membranes potentially involved in the PLA 2-induced inflammatory and nociceptive effects. This work reviews the proinflammatory and nociceptive effects evoked by myotoxic PLA 2s and their mechanisms of action.

Alkylation of myotoxic phospholipases A 2 in Bothrops moojeni venom: a promising approach to an enhanced antivenom production

Bothrops moojeni crude venom (MjCV) and its two major toxins, namely myotoxin I (MjTX-I) and myotoxin II (MjTX-II) were alkylated by p-bromophenacyl bromide (BPB). After alkylation the i.p. LD 50 (mice) of MjCV and MjTX-I/II increased from 6.0 to 15.7 mg/kg and from 8.0 to 45.0 mg/kg, respectively. In addition, doses of 5× LD 50 of alkylated MjTX-I did not cause a single death in mice and no myonecrosis was detected for the alkylated toxins, although both proteins still induced edema. Antibodies to native and modified crude venom or myotoxins cross-reacted with 12 purified class II myotoxic phospholipases A 2 found in snake venoms of the genus Bothrops. Myotoxic PLA 2s from class I and class III were not recognized by the above antibodies. These results suggest that the overall antigenic structure is conserved among class II myotoxic PLA 2s, despite differences in their amino acid sequences. Anti-MjTX-I-BPB and anti-MjTX-II-BPB rabbit serum, obtained against the modified myotoxins, were apparently more efficient than those obtained against the native myotoxins. In neutralization experiments, pre-incubation of crude venom or isolated myotoxins with antibodies raised against the native or modified toxins inhibited their PLA 2 and myotoxic activities. Therefore, alkylation of His48 by BPB strongly reduces the local tissue damage induced by B. moojeni venom or isolated myotoxins while retaining antigenicity, which suggests a promising procedure for an enhanced antiophidian serum production for practical purposes.

Structural, enzymatic and biological properties of new PLA 2 isoform from Crotalus durissus terrificus venom

We isolated a new PLA 2 from the Crotalus durissus terrificus venom that designated F15, which showed allosteric behavior with a V max of 8.5 nmol/min/mg and a K m of 38.5 mM. The incubated heparin salt of this isolated F15 act a positive allosteric effector by increasing the V max to 10.2 nmol/min/mg, with decreasing the V max value to 20.5 mM. The crotapotin, on the other hand acts as a negative allosteric effector by increasing the V max values to 58.4 mM. F15 also showed high calcium dependence for its catalysis similar to that found for other PLA 2 enzymes isolated from these snake venoms. The replacement of calcium by other divalent ions such Mg 2+, Mn 2+, Cd 2+, Sn 2+ and Cu 2+ resulted in lower enzymatic activity. The optimum pH and temperature for the enzyme was 8.5 and 18 °C, respectively. F15 alone showed moderate neurotoxic activity in isolated mouse phrenic nerve diaphragm in comparison to other strong myotoxic PLA 2 such as bothropstoxin-I (BThtx-I), but this activity was highly neurotoxic in a chick biventrer cervis preparation, whereas BthTx-I did not reveal this high neurotoxicity. This new protein showed a high bactericidal effect against both Gram-negative and Gram-positive bacterial strains. F15 contained 122 amino acid residues, with a primary structure of: HLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGQRRPKDATDRCCFVHDCCYGKLTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPKSRCRRPSETC. Its molecular mass and isoeletric point were 14.5 kDa and 8.85, both estimated by two dimensional electrophoresis. The amino acid sequence of the F15 revealed high sequence homology with F16 and F17. F15 and the other PLA 2s revealed highly conserved amino acid sequences principally for calcium binding loop and active site helix. F15 also showed a high homology with the lysine-rich region of myotoxic PLA 2.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A 2 from Bothrops jararacussu snake venom

An acidic (pI∼4.5) phospholipase A 2 (BthA-I-PLA 2) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an ∼13.7 kDa single chain Asp49 PLA 2 with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 1 S LWQFGKMINYVM–GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC 51. Crystals of this acidic protein diffracted beyond 2.0 Å resolution. These crystals are monoclinic and have unit cell dimensions of a=33.9, b=63.8, c=49.1 Å, and β=104.0°. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3–4 times more active than BthTX-II, a basic D49 myotoxic PLA 2 from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA 2 caused a hypotensive response in the rat and inhibited platelet aggregation. Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA 2, while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA 2 homologues and that either of these two activities may exist alone. BthA-I-PLA 2, in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies.

Cloning and cDNA sequence analysis of Lys 49 and Asp 49 basic phospholipase A 2 myotoxin isoforms from Bothrops asper

Snake venom myotoxic phospholipases A 2 contribute to much of the tissue damage observed during envenomation by Bothrops asper, the major cause of snake bites in Central America. Several myotoxic PLA 2s have been identified, but their mechanism of myotoxicity is still unclear. To aid in the molecular characterization of these venom toxins, the complete open reading frames encoding two Lys 49 and one Asp 49 basic PLA 2 myotoxins from the Central American snake B. asper (terciopelo) were obtained by cDNA cloning from venom gland poly-adenylated RNA. The amino acid sequence deduced from the myotoxins II and III open reading frames corresponded in each case to one of the reported amino acid sequence isoforms. The sequence of a new myotoxin IV-like sequence (MT-IVa) contains conservative Val→Leu 18 and Ala→Val 23 substitutions when compared with the reported N-terminus of the native myotoxin IV, suggesting minor isoform variations among specimens of a single species. Sequence alignment studies indicated significant (>75% sequence identity) identities with other crotalid venom Lys 49 PLA 2s, particularly bothropstoxin I/Ia isoforms of B. jararacussu and myotoxin II of B. asper.