Intraspecies variability in Vipera ammodytes ammodytes venom related to its toxicity and immunogenic potential

Vipera ammodytes is the most venomous European snake, whose venom has been used as antigen for immunization of antivenom-producing animals. Same as venom of any other snake, it is a complex mixture of proteins, peptides and other compounds which biochemical and pharmacological variability has been demonstrated at interspecies and intraspecies level. In this work we demonstrated intraspecific variability between 8 venom production batches using both the conventional and the new methodology. Moreover, in contrast to the literature on different venoms' variability, for the first time we were able to select those biochemical differences that are related to and give information on the venom's toxicity and immunogenicity. We have shown that methods quantifying ammodytoxin (the most toxic compound identified so far in the Vipera ammodytes ammodytes venom) content of the venom clearly distinguish between high and low immunogenic venoms.     

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.     

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.     

Amino-acid sequence of ammodytoxin B partially reveals the location of the site of toxicity of ammodytoxins

The complete amino-acid sequence of ammodytoxin B, a presynaptically toxic phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined by manual and automated protein sequencing. Ammodytoxin B (i.v. LD50 = 0.58 mg/kg for white mice) is 30-fold less toxic than ammodytoxin A, the most toxic phospholipase isolated from the same venom. The two proteins (each 122 residues long) differ in only 3 residues located in positions 115, 118 and 119 (numbering according to R. Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634) suggesting that an exposed hydrophobic residue in position 115 and a basic residue in position 118 may be responsible for the increased toxicity of ammodytoxin A and should form at least one part of the site of toxicity in ammodytoxins.     

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 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.     

Crystallization and preliminary X-ray diffraction studies of a toxic phospholipase A2 from the venom of Vipera ammodytes meridionalis complexed to a synthetic inhibitor

A toxic phospholipase A(2) (PLA(2)) is isolated from the neurotoxic complex Vipoxin, the major lethal component of the venom of Vipera ammodytes meridionalis. The enzyme is complexed to the synthetic inhibitor elaidoylamide and crystallized. The crystals belong to the space group P2(1)2(1)2(1), with unit cell dimensions a=46.57 A, b=82.68 A, c=119.47 A and beta=90 degrees. Initial diffraction data to 3.3 A resolution are collected.     

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).     

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.     

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.     

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.     

Relation between the size of the acid gland and the quantity of venom produced in Africanized bee, Apis mellifera L. (Hymenoptera: Apidae), in the region of Dourados, MS, Brazil

Crosses between African and European honeybees in Brazil resulted in a highly defensive hybrid bee. The acid gland is important in the expression of this characteristic, being responsible for venom production. Morphological variations in this gland could influence the quantity of venom. Glandular morphology was analyzed, along with the quantity of venom produced and the bees' genetic characteristics. The gland and the venom reservoir were removed from workers. The gland was placed on a histological frame for measurement and the contents of the reservoir were weighed. The results were submitted to an analysis of regression and submitted to Test Z, to evaluate the differences between the averages. The phenotypes were evaluated according to the standard found in literature. Gland length varied from 7.42 mm to 20.33 mm, the quantity of venom from 0.19 mg to 0.34 mg, and as far as the genetic characteristics are concerned, 63.3% of the colonies had workers with large glands. In 53.3% of the colonies, 90% of individuals had simple glands, suggesting the evolutionary process leading to the loss of branching, since the presence of branching indicates primitiveness. The production of venom is associated with the length of the gland and branching does not influence the quantity of venom. There was no statistical difference between the size of the branched and simple glands or in the quantity of venom produced, therefore the large glands can favor commercial exploration of venom, producing larger quantities.     

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 amino acid region 115-119 of ammodytoxins plays an important role in neurotoxicity

Quadruple (Y115K/I116K/R118M/N119L) and double (Y115K/I116K) mutants of ammodytoxin A, a presynaptically toxic phospholipase A(2) from Vipera ammodytes ammodytes venom, were prepared and characterized. The enzymatic activity of the quadruple mutant on phosphatidylcholine micelles was threefold higher than that of AtxA, presumably due to higher phospholipid-binding affinity, whereas the activity of the double mutant was twofold lower. The substantial decrease by more than two orders of magnitude in the lethal potency of both mutants, together with their decreased binding affinity for neuronal receptors, indicates involvement of the amino acid region 115-119 in neurotoxicity. The similar decrease of toxicity for the two mutants points to the importance of the residues Y115 and I116.     

The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom.

A new myotoxic phospholipase A2 homologue, having a serine residue in position 49 instead of highly conserved aspartic acid, was found in the venom of Vipera ammodytes. The primary structure revealed additional mutations in the positions important for enzymatic activity. Tyr28 is exchanged for a histidine and Gly33 for asparagine. These changes render earlier-reported weak enzymatic activity unlikely. The role of this rather abundant venom fraction is apparently in myotoxicity, which was confirmed in the muscle-cell culture from neonatal rats. The muscle-cell culture proved to be a good tool to investigate the effects of various myotoxins on muscle cells.     

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.     

The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper

A myotoxic, basic phospholipase A2 (pI greater than 9.5) with anticoagulant activity has been purified from the venom of Bothrops asper, and its amino acid sequence determined by automated Edman degradation. It is distinct from the B. asper phospholipase A2 known as myotoxin I [Lomonte, B. and Gutierrez, J. M., 1989, Toxicon 27, 725] but cross-reacts with myotoxin I rabbit antisera, suggesting that the proteins are closely related isoforms. To our knowledge, this is the first myotoxic phospholipase to be sequenced that lacks presynaptic neurotoxicity (iv LD50 approximately equal to 8 micrograms/g in mice). The protein appears to exist as a monomer, contains 122 amino acids, and fits with subgroup IIA of other sequenced phospholipase A2 molecules. Its primary sequence shows greatest identity with ammodytoxin B (67%), a phospholipase A2 presynaptic neurotoxin from Vipera ammodytes ammodytes venom. Hydropathy profiles of B. asper phospholipase and the ammodytoxins also show great similarities. In contrast, even though the amino acid sequence identities between B. asper phospholipase and the basic subunit of crotoxin remain high (64%), their hydropathy profiles differ substantially. Domains and residues that may be responsible for neurotoxicity are discussed.     

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.     

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.     

Serine proteinase inhibitors from Vipera ammodytes venom. Isolation and kinetic studies

Three protein inhibitors of serine proteinases were isolated from the crude venom of the long-nosed viper Vipera ammodytes ammodytes by ion-exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki = 3.4 X 10(-10) and 5.6 X 10(-10) M), while the third one primarily inhibits chymotrypsin (Ki = 4.3 X 10(-9) M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N-terminal group in the former inhibitors is blocked; arginine is the N-terminal amino acid in the latter. Besides trypsin and alpha-chymotrypsin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.