Sequences and structural organization of phospholipase A2 genes from Vipera aspis aspis, V. aspis zinnikeri and Vipera berus berus venom. Identification of the origin of a new viper population based on ammodytin I1 heterogeneity.

We used a PCR-based method to determine the genomic DNA sequences encoding phospholipases A2 (PLA2s) from the venoms of Vipera aspis aspis (V. a. aspis), Vipera aspis zinnikeri (V. a. zinnikeri), Vipera berus berus (V. b. berus) and a neurotoxic V. a. aspis snake (neurotoxic V. a. aspis) from a population responsible for unusual neurotoxic envenomations in south-east France. We sequenced five groups of genes, each corresponding to a different PLA2. The genes encoding the A and B chains of vaspin from the neurotoxic V. a. aspis, PLA2-I from V. a. zinnikeri, and the anticoagulant PLA2 from V. b. berus are described here. Single nucleotide differences leading to amino-acid substitutions were observed both between genes encoding the same PLA2 and between genes encoding different PLA2s. These differences were clustered in exons 3 and 5, potentially altering the biological activities of PLA2. The distribution and characteristics of the PLA2 genes differed according to the species or subspecies. We characterized for the first time genes encoding neurotoxins from the V. a. aspis and V. b. berus snakes of central France. Genes encoding ammodytins I1 and I2, described previously in Vipera ammodytes ammodytes (V. am. ammodytes), were also present in V. a. aspis and V. b. berus. Three different ammodytin I1 gene sequences were characterized: one from V. b. berus, the second from V. a. aspis, V. a. zinnikeri and the neurotoxic V. a. aspis, and the third from the neurotoxic V. a. aspis. This third sequence was identical with the reported sequence of the V. am. ammodytes ammodytin I1 gene. Genes encoding monomeric neurotoxins of V. am. ammodytes venom, ammodytoxins A, B and C, and the Bov-B LINE retroposon, a phylogenetic marker found in V. am. ammodytes genome, were identified in the genome of the neurotoxic V. a. aspis. These results suggest that the population of neurotoxic V. a. aspis snakes from south-east France may have resulted from interbreeding between V. a. aspis and V. am. ammodytes.     

Phospholipases A2 isolated from snake venoms block acetylcholine-elicited currents in identified Lymnaea stagnalis neurons

Phospholipases A₂ (PLA₂s) are the most abundant family of snake venom proteins and play a significant role in prey envenomation. Their content in venoms is rather high. PLA₂s not only have enzyme activity but exhibit other types of biological activities including neurotoxicity. We have earlier shown that a protein bitanarin from the venom of the puff adder Bitis arietans is capable to block the responses of Lymnaea stagnalis neurons to acetylcholine and represents an active PLA₂ at the same time. Further investigation of PLA₂s isolated from the venoms of snakes of two families revealed their capability to interact with nicotinic acetylcholine receptors (nAChRs): PLA₂ from Vipera ursinii (Viperidae family), Naja kaouthia, and Bungarus fasciatus (Elapidae family) suppressed acetylcholine-induced current in identified neurons of L. staganlis. The effect was evident at PLA₂ concentration in the range of tens micromoles. The data obtained suggest the presence in a PLA₂ molecule of a site interacting with nAChR and a possible involvement of nAChR block in toxic action of PLA₂s.     

Molecular phylogeny of Vipera Laurenti, 1768 and the related genera Macrovipera (Reuss, 1927) and Daboia (Gray, 1842), with comments about neurotoxic Vipera aspis aspis populations

We used mtDNA sequences (cytochrome b and NADH dehydrogenase subunit 2) to reconstruct molecular phylogenies of Vipera sensu lato, Vipera sensu stricto, and Vipera aspis. Three major clades were identified within the Vipera s.l. group: (1) the European vipers, (2) the oriental vipers, consisting of Montivipera (Vipera 2) plus Macrovipera lebetina, and (3) a group of Asian and North African vipers consisting of Daboia russelii, V. palaestinae, and Macrovipera mauritanica. We also distinguished three clades within the monophyletic European Vipera group: V. ammodytes, V. aspis, and V. latastei, and Pelias with monophyly of Vipera 1 uncertain. Within V. aspis, the specimens collected in France formed the sister group of an Italian clade. The "neurotoxic" French population of V. aspis, which has a specific venom profile, separated from other French V. aspis early in the history of this group.     

Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

Comparison of Phylogeny,Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A₂ reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.     

Clinical Effects and Antivenom Dosing in Brown Snake (Pseudonaja spp.) Envenoming — Australian Snakebite Project (ASP-14)

Background

Snakebite is a global health issue and treatment with antivenom continues to be problematic. Brown snakes (genus Pseudonaja) are the most medically important group of Australian snakes and there is controversy over the dose of brown snake antivenom. We aimed to investigate the clinical and laboratory features of definite brown snake (Pseudonaja spp.) envenoming, and determine the dose of antivenom required.

Methods and Finding

This was a prospective observational study of definite brown snake envenoming from the Australian Snakebite Project (ASP) based on snake identification or specific enzyme immunoassay for Pseudonaja venom. From January 2004 to January 2012 there were 149 definite brown snake bites [median age 42y (2–81y); 100 males]. Systemic envenoming occurred in 136 (88%) cases. All envenomed patients developed venom induced consumption coagulopathy (VICC), with complete VICC in 109 (80%) and partial VICC in 27 (20%). Systemic symptoms occurred in 61 (45%) and mild neurotoxicity in 2 (1%). Myotoxicity did not occur. Severe envenoming occurred in 51 patients (38%) and was characterised by collapse or hypotension (37), thrombotic microangiopathy (15), major haemorrhage (5), cardiac arrest (7) and death (6). The median peak venom concentration in 118 envenomed patients was 1.6 ng/mL (Range: 0.15–210 ng/mL). The median initial antivenom dose was 2 vials (Range: 1–40) in 128 patients receiving antivenom. There was no difference in INR recovery or clinical outcome between patients receiving one or more than one vial of antivenom. Free venom was not detected in 112/115 patients post-antivenom with only low concentrations (0.4 to 0.9 ng/ml) in three patients.

Conclusions

Envenoming by brown snakes causes VICC and over a third of patients had serious complications including major haemorrhage, collapse and microangiopathy. The results of this study support accumulating evidence that giving more than one vial of antivenom is unnecessary in brown snake envenoming.     

Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes

Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A₂ (PLA₂) isozymeencoding genes, gPLA₂-o1, gPLA₂-o2 and gPLA₂-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA₂-o2) and (gPLA₂-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA₂ isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA₂isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA₂ isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA₂ isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA₂ isozyme genes appears to have been brought about by natural selection for point mutations.     

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 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/Significance

Our 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.     

Crystal structure of phospholipase PA2-Vb,a protease-activated receptor agonist from the Trimeresurus stejnegeri snake venom

Phospholipase A₂ (PLA₂) is an important component in snake venoms. Here, an acidic PLA₂, designated PA2-Vb was isolated from the Trimeresurus stejnegeri snake venom. PA2-Vb acts on a protease-activated receptor (PAR-1) to evoke Ca²⁺ release through the inositol 1,4,5-trisphosphate receptor (IP₃R) and induces mouse aorta contraction. PAR-1, phospholipase C and IP₃R inhibitors suppressed PA2-Vb-induced aorta contraction. The crystal structure reveals that PA2-Vb has the typical fold of most snake venom PLA₂. Several PEG molecules bond to a positively charged pocket. The finding offers a novel pharmacological basis of the structure for investigating the PAR-1 receptor and suggests potential applications for PA2-Vb in the vascular system.     

Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2

Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A₂ inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A₂ (PLA₂s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA₂ contained in this venom. Furthermore, these effects of acidic PLA₂ on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

Characterization of a human coagulation factor Xa-binding site on <Emphasis Type="Italic">Viperidae</Emphasis> snake venom phospholipases A<Subscript>2</Subscript> by affinity binding studies and molecular bioinformatics

Background  

The snake venom group IIA secreted phospholipases A₂ (SVPLA₂), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA₂-inhibitors from snake serum; and (iii) coagulation factors present in human blood.     

Influences of area, isolation and habitat features on distribution of snakes in Mediterranean fragmented woodlands

The effects of isolation-related and vegetational parameters on presence and relative abundance of snakes in patchy forested fragments of Mediterranean central Italy are studied. The most abundant species was Coluber viridiflavus (accounting for 47.7% of the total snake sample observed) followed by Vipera aspis (22%), Elaphe longissima (21.5%), Natrix natrix (7.7%), and Coronella austriaca (1.1%). There was a clear trend for bigger species to be less distributed among the various forest fragments than the smaller species. Presence of Coluber viridiflavus, Coronella austriaca and Natrix natrix was not influenced by woodland area, whereas that of Vipera aspis and Elaphe longissima was positively influenced by woodland area. Woodland isolation parameters did not influence the presence of Coluber viridiflavus, Coronella austriaca and Natrix natrix, but of Vipera aspis and Elaphe longissima. Discriminant stepwise analysis suggested that specific environmental features influenced the occurrence and abundance of the various snake species, Vipera aspis being the taxon more affected by isolation-related parameters. Some conservation implications of our observations are also discussed.     

Viperidae snake venoms block nicotinic acetylcholine receptors and voltage-gated Ca2+ channels in identified neurons of fresh-water snail Lymnaea stagnalis

Snake venoms contain a vast array of toxic polypeptide components interacting with a variety of cell targets. Thus, Elapidae snake venoms contain α-neurotoxins with very high affinity for nicotinic acetylcholine receptors (nAChRs) and a few toxins able to suppress the activity of Ca²⁺ and K⁺ channels. Experimental evidence for the presence of nAChR antagonists and voltage-gated ionic channel blockers in venoms of Viperidae snakes is very scarce. In this study, effects of crude venoms of seven snake species (Vipera nikolskii, Echis multisquamatus, Gloydius saxatilis, Bitis arietans, Vipera renardi, Vipera lebetina, and Naja kaouthia) on nAChRs and voltage-gated Ca²⁺ channels were studied for the first time. The experiments were carried out on isolated identified neurons of the fresh-water mollusc Lymnaea stagnalis using voltage-clamp and intracellular perfusion techniques. All Viperidae snake venoms under study blocked nAChRs and voltage-gated Ca²⁺ channels. The potency of these venoms against nAChRs was significantly lower in comparison with N. kaouthia venom which is rich of α-neurotoxins; however, the extent of Ca²⁺ channel block by venoms of Viperidae snakes and N. kaouthia was similar. The data obtained suggest that Viperidae snake venoms tested in this study contain peptides with affinity both for nAChRs and for voltage-gated Ca²⁺ channels.     

Purification and Functional Characterisation of Rhiminopeptidase A,a Novel Aminopeptidase from the Venom of Bitis gabonica rhinoceros

Background

Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros.

Methodology and Principal Findings

The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped β domain, a mixed α and β catalytic domain, a β-sandwich domain and a C-terminal α helical domain.

Conclusions

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.     

Mutagenesis analyses explore residues responsible for the neurotoxic and anticoagulant activities of Trimucrotoxin, a pit-viper venom Asn6-phospholipase A2

Trimucrotoxin (TmCT) is an Asn⁶-containing phospholipase A₂ (PLA₂) from Protobothrops mucrosquamatus (pit-viper) venom. In an attempt to characterize the amino acid residues responsible for the neurotoxic and anticoagulant activities of TmCT, the recombinant fusion proteins of TmCT wild type and mutants were expressed in Escherichia coli. Correct refolding and processing of 37 TmCT mutants were confirmed by their HPLC retention times, circular dichroism spectra, and masses obtained from ESI-MS spectrometry. Each mutant was assayed by pH-stat titration using zwitterionic as well as anionic micelle substrates, and the neurotoxicity was evaluated by using the contractile responses of chick biventer cervicis muscles. The results demonstrated that the residues Asn¹, Asn⁶, Lys⁷, Ile¹¹, Met¹², Gly⁵³, Thr⁷⁹, His¹⁰⁸ and Met¹¹⁸ are important to TmCT neurotoxicity. Through various tests, we also confirmed that enzymatic activity, as opposed to binding to Factor Xa, was a necessary part of TmCT’s anticoagulant effect. In addition, pulldown assays of the WT and selected mutants revealed that TmCT’s in vitro binding to crotoxin acidic subunit may involve a broad surface area. We conclude that the hot spot mutations at specific positions 53, 79, 108, and 118 during the pit-viper Asn⁶-PLA₂ evolution regulate their neurotoxicities, and that many of the neurotoxic site residues and the anticoagulant mechanism of TmCT are different from those of ammodytoxin A (a true-viper venom neurotoxic PLA₂).     

Inhibition of Nicotinic Acetylcholine Receptors,a Novel Facet in the Pleiotropic Activities of Snake Venom Phospholipases A2

Phospholipases A₂ represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A₂, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A₂ from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A₂ content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A₂, which add a new target to the numerous activities of these enzymes.