Differences in pharmacological actions between beta-bungarotoxin and other neurotoxic phospholipases A2 purified from snake venoms

Five highly toxic phospholipases A2 (PLAs) (beta-bungarotoxin, caudoxin, Mojave toxin, notexin and a basic PLA from Naja nigricollis venom) were compared for their pharmacological actions. In the chick biventer cervicis nerve-muscle preparation, all PLA toxins except beta-bungarotoxin (beta-BuTX) inhibited the postsynaptic acetylcholine response and induced contracture of the muscle at a high concentration. Indirect hemolytic activity was found in all PLA toxins and some of the toxins (Naja nigricollis basic PLA and Mojave toxin) even showed a potent direct hemolytic action, while beta-BuTX was devoid of both direct and indirect hemolytic activities on the guinea-pig erythrocytes. All PLA toxins except beta-BuTX caused an increase in muscle tone in the guinea-pig ileum at a concentration as low as 0.05 microgram/ml, and an increase in the contractile force in the guinea-pig atrium at a concentration of 1.0 microgram/ml. In contrast, beta-BuTX had no stimulant effect at concentrations up to 10 micrograms/ml. On the cultured cells, beta-BuTX suppressed the proliferation of neuroblastoma cells, but did not cause lysis of non-neuronal cells of the rat brain. However, beta-BuTX uniquely maintained a high population of viable cells in the neuroblastoma cell cultures. From these results it was concluded that beta-BuTX is the most specific presynaptic neurotoxin among the PLA toxins so far tested.     

Influence of phospholipases A2 from snake venoms on survival and neurite outgrowth in pheochromocytoma cell line PC12

To determine whether the ability to induce neurite outgrowth in rat pheochromocytoma cell line PC12 is characteristic of phospholipases of different types, we have studied the influence of phospholipase A(2) (PLA2) from cobra Naja kaouthia venom and two PLA2s from viper Vipera nikolskii venom on PC12 cells. Phospholipases from the viper venom are heterodimers in which only one of the subunits is enzymatically active, while PLA2 from the cobra venom is a monomer. It was found that all three PLA2s induce neurite outgrowth in PC12. The PLA2 from cobra venom exhibits this effect at higher concentrations as compared to the viper enzymes. We have not observed such an activity for isolated subunits of viper PLA2s, since the enzymatically active subunits have very high cytotoxicity, while the other subunits are not active at all. However, co-incubation of active and inactive subunits before addition to the cells leads to a marked decrease in cytotoxicity and to restoration of the neurite-inducing activity. It has also been shown that all enzymatically active PLA2s are cytotoxic, the PLA2 from cobra venom being the least active. Thus, for the first time we have shown that PLA2s from snake venoms can induce neurite outgrowth in PC12 cells.     

Effect of crotapotin on the biological activity of Asp49 and Lys49 phospholipases A(2) from Bothrops snake venoms

Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A(2) (PLA(2)s). Asp49-PLA(2)s are catalytically active, whereas Lys49-PLA(2)s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA(2) (CB), is a PLA(2)-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA(2)s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA(2)s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction.     

Structure and function comparison of Micropechis ikaheka snake venom phospholipase A2 isoenzymes

Comparison of the crystal structures of three Micropechis ikaheka phospholipase A2 isoenzymes (MiPLA2, MiPLA3 and MiPLA4, which exhibit different levels of pharmacological effects) shows that their C-terminus (residues 110-124) is the most variable. M-Type receptor binding affinity of the isoenzymes has also been investigated and MiPLA4 binds to the rabbit M-type receptor with high affinity. Examination of surface charges of the isoenzymes reveals a trend of increase in positive charges with potency. The isoenzymes are shown to oligomerize in a concentration-dependent manner in a semi-denaturing gel. The C-termini of the medium (MiPLA4) and highly potent (MiPLA2) isoenzyme molecules cluster together, forming a highly exposed area. A BLAST search using the sequence of the most potent MiPLA2 results in high similarity to Staphylococcus aureus clotting factor A and cadherin 11. This might explain the myotoxicity, anticoagulant and hemoglobinuria effects of MiPLA2s.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

Structural and functional comparison of proteolytic enzymes from plant latex and snake venoms

This work describes classification, functions, location, inhibition, activation, and therapeutic applications of proteases from snake venoms and vegetables. Snake venoms and vegetables can present toxins that unchain necrosis or proteolysis due to the direct cytotoxic action of venom proteases. These proteases are potential tools in the development of drugs for the prevention and treatment of several illnesses. We report herein mainly fibrinogenolytic metallo proteases and serine proteases (“thrombin-like”). These enzymes are extensively used in the treatment and prevention of thrombotic disorders, since they serve as defibrinogenating agents. The therapeutic uses of fibrin(ogen)olytic metallo proteases hold promise for clinical application due to potential in reversing the effects of thrombosis; this has been shown to be an alternative approach to the prevention and treatment of cardiovascular disorders, which are among the most prominent causes of mortality around the world. Plant proteases can be utilized for many cellular and molecular activities, in antibacterial and anticancer therapies, and in the treatment of snakebites, inhibiting snake venom activities such as blood-clotting, defibrinogenation, and fibrin(ogen)olytic and hemorrhagic actions. These toxins also display potential for clinical use in the treatment of hemostatic disorders.     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Characterization of phospholipases A2 of Naja melanoleuca snake venom modified at the N-terminal region

In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the epsilon-amidinated derivatives without reaction of the alpha-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity. Starting from AMPA, chemical modification reactions were carried out at the alpha-amino function. This group was blocked with a tert-butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Asn1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine residue. Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme.     

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.     

Myotoxic phospholipases A(2) in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu

Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA(2) homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA(2) homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins.     

Characterization of the snake venoms from seven Brazilian species of Bothrops by FPLC anion-exchange chromatography.

1. The elution profiles and the caseinolytic, myotoxic, coagulant and hemorrhagic activities of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column were analyzed. 2. Each venom separated into 16-20 peaks, with good reproducibility and the activities were concentrated in virtually discrete regions of the chromatogram. 3. There is a considerable overlap of active proteins in the different species venoms and our results indicate that a venom pool with the species B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox venoms would contain the major active proteins determined in the seven species.     

Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A2

The crude aqueous extract from the leaves of Casearia sylvestris, a plant found in Brazilian open pastures, was assayed for its ability to inhibit phospholipase A2 (PLA2) activity and some biological activities of bee and several snake venoms, and of a number of isolated PLA2s. The extract induced partial inhibition of the PLA2 activity of venoms containing class I, II and III PLA2s. When tested against the purified toxins, it showed the highest efficacy against class II PLA2s from viperid venoms, being relatively ineffective against the class I PLA2 pseudexin. In addition, C. sylvestris extract significantly inhibited the myotoxic activity of four Bothrops crude venoms and nine purified myotoxic PLA2s, including Lys-49 and Asp-49 variants. The extract was able to inhibit the anticoagulant activity of several isolated PLA2s, with the exception of pseudexin. Moreover, it partially reduced the edema-inducing activity of B. moojeni and B. jararacussu venoms, as well as of myotoxins MjTX-II and BthTX-I. The extract also prolonged the survival time of mice injected with lethal doses of several snake venoms and neutralized the lethal effect induced by several purified PLA2 myotoxins. It is concluded that C. sylvestris constitutes a rich source of PLA2 inhibitors.     

Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages

The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A₂ (PLA₂), and BaltTX-II, an Asp49 catalytically active PLA₂ isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA₂ BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA₂, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA₂s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.     

Isolation and characterization of three Ca2+-dependent beta-galactoside-specific lectins from snake venoms.

Three lactose-inhibited lectins from the venoms of the snakes Agkistrodon contortrix contortrix (southern copperhead), Ancistrodon piscivorous leukostoma (western cottonmouth moccasin) and Crotalus atrox (western diamondback rattlesnake) have been isolated and newly characterized. The three lectins are similar to thrombolectin, a lectin isolated from the venom of Bothrops atrox (fer-de-lance) (Gartner, Stocker & Williams, 1980), with regard to sugar specificity, Mr, Ca2+ requirements and sensitivity to reducing agents. Each lectin is a dimer (Mr 28 000) consisting of monomers (Mr 14 000) indistinguishable on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Haemagglutination activity is dependent on the presence of Ca2+ and is inhibited by reducing agents. The lectins are not identical and can be distinguished on the basis of relative affinities for inhibiting sugars, isoelectric points and immunoprecipitation assays using anti-(cottonmouth lectin) serum.