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.     

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.     

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.     

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.     

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.     

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 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.     

Comparative assay of Vipera ammodytes antivenom potency

The finding of the most appropriate way to assess precisely the antivenom efficacy represents one of the major issues for antivenom standardization and success increasing of antivenom therapy. The efficacy of experimental Vipera ammodytes antivenom raised in sheep was determined using in vivo mouse lethality test, respectively, L-aminoacid oxidase, total proteinase and phospholipase A₂ antienzymatic effectiveness. The values gained for the antivenom potency depend on the method of measure. So, some of the most toxic venom proteins own phospholipase A₂ activity and provide the highest antivenom potency (lowest effective dose) values by antienzymatic assay method. This value is similar with total antiproteolytic antivenom potency value, but almost three times higher than value obtained by L-aminoacid oxidase (low toxic viper venom protein) antienzymatic assay method.     

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.     

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.     

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.     

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.