The variability of Vipera ammodytes ammodytes venoms from Croatia--biochemical properties and biological activity

Vipera ammodytes ammodytes venom has been used for many years in Croatia for immunization of horses and production of specific therapeutic anti-venoms. The neutralizing effectiveness of anti-venoms is directly dependent on the properties of the snake venom used for immunization. Therefore, appropriate characterization of the whole venom is necessary prior to use in the immunization procedure. In the course of such analyses, the variability in biochemical properties and biological activity was observed in venoms collected from snakes originating from different parts of Croatia. The venom pools also differed with respect to time of snake collection (1992-2003). Analyses of three samples of whole venom pools were carried out revealing differences in lethal activity (LD50), minimum haemorrhagic dose (MHD), minimum necrotizing dose (MND), phospholipase A2 activity and in anticomplementary activity. SDS-PAGE electrophoretic patterns were similar, but not identical, for all tested venom pools with respect to the number of protein bands detected, but intensity of particular components differed. Preliminary immunogenicity testing in terms of determination of specific antibodies revealed similar immunogenicity and high cross-reactivity for three samples tested.     

Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom

The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.     

Ammodytoxin content of Vipera ammodytes ammodytes venom as a prognostic factor for venom immunogenicity

Venoms are complex mixtures of proteins, peptides and other compounds whose biochemical and biological variability has been clearly demonstrated. These molecules have been used as antigens for immunization of anti-venom-producing animals (horses or sheep). Ammodytoxins (Atx) are potently neurotoxic compounds, and the most toxic compounds isolated so far from the Vipera ammodytes ammodytes (Vaa) venom. Recently we have shown that the level of antibodies specific to Vaa venom's most toxic component, ammodytoxin A (AtxA), (anti-AtxA IgG) in Vaa venom immunized rabbit sera highly correlated to the venom toxicity–neutralization potential of these sera. Here we investigated whether Atx content of Vaa venom could influence the outcome of immunization procedure. The novel ELISA was developed for precise determination of Atx content and Atx was quantified in venom samples used for immunization of rabbits. We clearly showed that animals immunized with the venom containing lower amount of Atx produced sera with significantly lower venom toxicity–neutralizing power and, vice versa, animals immunized with venoms containing higher amount of Atx produced sera with higher venom toxicity–neutralizing ability. Thus, the content of Atx in Vaa venom is a relevant parameter of its suitability in the production of highly protective Vaa anti-venom.     

Comparative analysis of the venom proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis

The venom proteomics of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, snakes of public health significance and the most poisonous reptiles in Europe, were analyzed by FPLC, 2-D electrophoresis, sequence analysis, and MS/MS. FPLC analysis showed the presence of l-amino acid oxidase, monomeric and heterodimeric phospholipases A2, C-type lectin protein, and proteinases in the venom of V. a. ammodytes. Representatives of the same protein families were found in the venom of the other subspecies, V. a. meridionalis. N-terminally identical PLA2 neurotoxins were identified in both venoms. Difference in the PLA2 compositions of the venoms was also observed: a monomeric protein with phospholipase A2 activity, identical in the first 20 amino acid residues to the catalitically inactive acidic component of the heterodimeric PLA2 present in both venoms, was found only in that of V. a. meridionalis. Probably, this protein represents an intermediate form of the two components of the heterodimer. 2-D electrophoresis and MS/MS analysis showed that the two venoms shared a number of protein families: monomeric and heterodimeric Group II PLA2s, serine proteinases, Group I, II, and III metalloproteinases, l-amino acid oxidases (LAAOs), cysteine-rich secretory proteins, disintegrins, and growth factors. Totally, 38 venom components of the V. a. ammodytes, belonging to 9 protein families, and 67 components of the V. a. meridionalis venom belonging to 8 protein families were identified. The venom proteome of V. a. ammodytes shows larger diversity of proteins (139) in comparison to that of V. a. meridionalis (104 proteins). Most of the proteins are homologues of known representatives of the respective protein families. The protein compositions explain clinical effects of the V. ammodytes snakebites, such as difficulties in the breathing, paralysis, apoptosis, cloting disorders, hemorrhage, and tissue necrosis. The lists of secreted proteins by the two vipers can be used for further study of structure-function relationships in the toxins and for prediction and treatment of snakebite consequences.     

Sequence homology between phospholipase and its inhibitor in snake venom. The primary structure of the inhibitor of vipoxin from the venom of the Bulgarian viper (Vipera ammodytes ammodytes, Serpentes)

We are presenting the first primary structure of a snake venom inhibitor. It was isolated from the neurotoxin vipoxin of the Bulgarian Viper (Vipera ammodytes ammodytes, Serpentes) which represents a complex of a strong toxic basic protein with phospholipase A2 activity (2 isoenzymes) and the nontoxic acidic component functioning as its inhibitor. The sequence was established by automatic degradation in a liquid phase sequenator on the S-carboxymethylated chain and on the peptides obtained by tryptic hydrolysis of the oxidized chain. A limited tryptic digestion of the oxidized chain provided the necessary overlapping peptides. The inhibitor consists of 122 amino-acid residues including 14 cysteine and 10 tyrosine residues and is thus similar to the phospholipases from snake venoms. A comparison of the inhibitor sequence with the primary structure of the phospholipase A2 (CM-II) from the Horned Adder (Bitis nasicornis) venom shows a surprising homology of 52%. The identical amino acids include the cysteine and tyrosine residues and are generally accumulated in the surroundings of cysteine residues. The histidine (pos. 47) in the active center of the phospholipase A2 is substituted by glutamine in the inhibitor, but the tryptophan (pos. 30) which is essential for the enzymatic activity is present. The significant homology between enzyme and inhibitor in the vipoxin complex is believed to originate from a gene duplication. The relatively late development of the reptiles and the snake venom complex explains the highly preserved structure compared to other enzyme-inhibitor systems.     

The role of antibodies specific for toxic sPLA2s and haemorrhagins in neutralizing potential of antisera raised against Vipera ammodytes ammodytes venom

The contribution of antibodies directed against the two main toxic groups of proteins in the Vipera ammodytes ammodytes venom, haemorrhagic metalloproteinases (H) and neurotoxic sPLA2s (Atxs), to the overall protective efficacy of the whole venom antisera was investigated. Using ELISA assays we established a high correlation between the protective efficacy of the whole venom antisera in mice and their anti-Atxs antibody content. As the haemorrhage is the prevailing toxic effect of the venom in human, the lack of correlation also with anti-H IgG content exposed that the mouse model might not be optimal to evaluate the neutralizing potential of the venom-specific antisera for human therapy. We further revealed that Atxs and structurally very similar but non-toxic AtnI2 from the venom are not immuno cross-reactive.     

The C-terminal and beta-wing regions of ammodytoxin A, a neurotoxic phospholipase A2 from Vipera ammodytes ammodytes, are critical for binding to factor Xa and for anticoagulant effect

Ammodytoxin A (AtxA) from the venom of Vipera ammodytes ammodytes belongs to group IIA secreted phospholipase A2 (sPLA2), for which the major pathologic activity is presynaptic neurotoxicity. We show here that this toxin also affects hemostasis because it exhibits strong anticoagulant activity. AtxA binds directly to human coagulation factor Xa (FXa) with Kdapp of 32 nM, thus inhibiting the activity of the prothrombinase complex with an IC50 of 20 nM. To map the FXa-interaction site on AtxA, various mutants of AtxA produced by site-directed mutagenesis and expressed in Escherichia coli were tested in the study. In surface plasmon resonance (SPR) measurements, with FXa covalently attached to the sensor chip, we show that the FXa-binding site on AtxA includes several basic amino acid residues at the C-terminal and beta-wing regions of the molecule. Applying an in vitro biological test for inhibition of prothrombinase activity, we further demonstrate that the same residues are also very important for the anticoagulant activity of AtxA. We conclude that the anticoagulant site of AtxA is located in the C-terminal and beta-wing regions of this phospholipase A2. Synthetic peptides comprising residues of the deduced anticoagulant site of AtxA provide a basis to synthesize novel anticoagulant drugs.     

The first phospholipase inhibitor from the serum of Vipera ammodytes ammodytes

Ammodytoxins are neurotoxic secretory phospholipase A(2) molecules, some of the most toxic components of the long-nosed viper (Vipera ammodytes ammodytes) venom. Envenomation by this and by closely related vipers is quite frequent in southern parts of Europe and serotherapy is used in the most severe cases. Because of occasional complications, alternative medical treatment of envenomation is needed. In the present study, ammodytoxin inhibitor was purified from the serum of V. a. ammodytes using two affinity procedures and a gel exclusion chromatography step. The ammodytoxin inhibitor from V. a. ammodytes serum consists of 23- and 25-kDa glycoproteins that form an oligomer, probably a tetramer, of about 100 kDa. N-terminal sequencing and immunological analysis revealed that both types of subunit are very similar to gamma-type secretory phospholipase A(2) inhibitors. The ammodytoxin inhibitor from V. a. ammodytes serum is a potent inhibitor of phospholipase activity and hence probably also the neurotoxicity of ammodytoxins. Discovery of the novel natural inhibitor of these potent secretory phospholipase A(2) toxins opens up prospects for the development of new types of small peptide inhibitors for use in regulating the physiological and pathological activities of secretory phospholipases A(2).     

The quantities of dried venom collected from specimens of Vipera ammodytes L., 1758 (Reptilia, Viperidae) in captivity in Bulgaria

Some researches have been made to obtain more data about quantities of dried venom collected from Vipera ammodytes L., 1758 in captivity. The minimal quantity of dried venom collected by exemplar is 9.7 mg to 36.4 mg and the maximal quantity is 49.0 mg to 90.3 mg. From 810 exemplars of V. ammodytes of Bulgaria and 9 months of investigations, 10597 samples were made, with a total of 298.164 g of dried venom (average for animal: 28.14 mg).     

Sequence homology between phospholipase and its inhibitor in snake venom. The primary structure of phospholipase A2 of vipoxin from the venom of the Bulgarian viper (Vipera ammodytes ammodytes, Serpentes)

The amino-acid sequence of phospholipase A2 from the neurotoxin vipoxin of the Bulgarian Viper (Vipera ammodytes ammodytes, Serpentes) is presented. The enzyme consists of 122 amino-acid residues including 7 disulfide bonds and thus belongs to phospholipases A2 group IIA. The sequence was determined by automatic Edman degradation of the intact chain and of the peptides obtained after tryptic hydrolysis of the oxidized chain. The short cleavage time of 30 min and another limited tryptic digestion of the oxidized and citraconylated chain provided overlapping peptides. Sequencing was done with liquid- and gas-phase sequenators. The complete alignment of all peptides was facilitated by the high degree of homology with known viperid venom phospholipases A2. In common with mammalian phospholipases, the tryptophan residue in position 30 (essential for enzymatic activity) as well as the histidine in position 47 in the active site are present. Vipoxin phospholipase A2 shows 53.3% homology with another phospholipase A2 from Vipera ammodytes ammodytes venom (Ammodytoxin B), whereas 62% homology was found between both subunits of vipoxin phospholipase A2 and its inhibitor. This high degree of identity can be accounted for in terms of a common origin by gene duplication.     

Amino acid and cDNA sequences of a neutral phospholipase A2 from the long-nosed viper (Vipera ammodytes ammodytes) venom.

The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and 100%, respectively.     

Dose dependent effects of standardized nose-horned viper (Vipera ammodytes ammodytes) venom on parameters of cardiac function in isolated rat heart

Direct, dose dependent effects of the nose-horned vipers (Vipera ammodytes ammodytes) venom on various parameters of cardiac action in isolated rat hearts were examined. Biochemical (protein content, SDS polyacrylamide gel electrophoresis) and biological (minimum haemorrhagic and necrotizing dose and lethal dose (LD(50))) characterization of the venom was performed before testing. The hearts were infused with venom doses of 30, 90 and 150 microg/mL for 10 min followed by 30 min of wash out period. Left ventricular pressure, coronary flow, heart rate, atrioventricular conduction, myocardial oxygen consumption, incidence and duration of arrhythmias were measured and relative cardiac efficiency was calculated. Cardiac CPK, LDH, AST and troponin I were measured as biochemical markers of myocardial damage. The venom caused dose dependent electrophysiological instability and depression of contractility and coronary flow. Effects on the heart rate were biphasic; transient increase followed by significant slowing of the frequency. Relative cardiac efficiency decreased as oxygen consumption remained high relative to the heart rate-contractility product, indicating purposeless expenditure of oxygen and energy. Effects by the dose of 30 microg/mL were highly reversible while the dose of 90 mug/mL caused damages that were mostly irreversible. The dose of 150 mug/mL induced irreversible asystolic cardiac arrest.     

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

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

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.     

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

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

Purification and properties of a kininogenin from the venom of Vipera ammodytes ammodytes.

A kininogenin (EC 3.4.21.8) was purified from the venom of Vipera ammodytes ammodytes (European sand viper) by a combination of gel filtration and ion-exchange chromatography. The enzyme is approximately six times more active than bovine trypsin in its ability to release vasoactive peptides from a plasma precursor. The kininogenin is a glycoprotein containing 18-20% by weight of carbohydrate. It showed a mol. wt. of 40500 on gel filtration. Gel electrophoresis of the reduced sample in the presence of sodium dodecyl sulphate and 2-mercaptoethanol revealed the presence of two major components of mol.wt. 34300 and 31300. The heterogeneity, which was also observed on disc electrophoresis, was removed by incubation with neuraminidase. After incubation with neuraminidase the kininogenin retained full enzymic activity and possessed an isoelectric point of pH7.2. The carbohydrate content has been decreased to 10% by weight, and the single component seen on electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol corresponded to a mol.wt. of 29500.     

Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

Enzymatic activity and inhibition of the neurotoxic complex vipoxin from the venom of Vipera ammodytes meridionalis

Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor. Oleyloxyethylphosphocholine, aristolochic acid and vitamin E suppressed the enzymatic activity of vipoxin and its isolated PLA2 subunit. These compounds influence inflammatory processes in which PLA2 is implicated. The peptide Lys-Ala-Ile-Tyr-Ser, which is an integral part of the PLA2 components of the two neurotoxic complexes from V. ammodytes meridionalis and V. russelli formosensis (sequence 70-74) activated vipoxin increasing its PLA2 activity by 23%. This is in contrast to the inhibitory effect of the respective pentapeptides with 70-74 sequences on other group II PLA2s. Surprisingly, the same peptide inhibited 46% of the V. russelli formosensis PLA2 activity. The limited changes in the structure of the two highly homologous neurotoxins lead to considerable differences in their interaction with native peptides.