Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina

The protein composition of the crude venoms of the three most important vipers of Tunisia was analyzed by RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and in-gel tryptic digestion followed by PMF and CID-MS/MS of selected peptide ions in a quadrupole-linear IT instrument. Our results show that the venom proteomes of Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina are composed of proteins belonging to a few protein families. However, each venom showed distinct degree of protein composition complexity. The three venoms shared a number of protein classes though the relative occurrence of these toxins was different in each snake species. On the other hand, the venoms of the Cerastes species and Macrovipera lebetina each contained unique components. The comparative proteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins, which may contribute to a deeper understanding of the biological effects of the venoms, and may also serve as a starting point for studying structure-function correlations of individual toxins.     

Identification of toxic components from the venom of Cerastes cerastes and Vipera lebetina vipers; purification of phospholipases

Cerastes cerastes and Vipera lebetina venoms have been fractionated and the different components analysed by electrophoresis on polyacrylamid gels. Phospholipases A2 contained in these two venoms have been purified and their electrophoretic properties compared.     

Snake venomics of Macrovipera mauritanica from Morocco, and assessment of the para-specific immunoreactivity of an experimental monospecific and a commercial antivenoms

Proteomic analysis of the venom of the medically relevant snake Macrovipera mauritanica from Morocco revealed a complex proteome composed of at least 45 toxins from 9 protein families targeting the hemostatic system of the prey or victim. The toxin profile of Moroccan M. mauritanica displays great similarity, but also worth noting departures, with the previously reported venom proteome of M. lebetina from Tunisia. Despite fine compositional differences between these Macrovipera taxa, their overall venom phenotypes explain the clinical picture observed in M. mauritanica and M. lebetina envenomings. However, M. mauritanica venom also contains significant amounts of orphan molecules whose presence in the venom seems to be difficult to rationalize in the context of a predator-prey arms race. The paraspecific immunoreactivity of an experimental monospecific (M. mauritanica) antivenom and a commercial bivalent antivenom, anti-C. cerastes and anti-M. lebetina, against the venoms of Moroccan M. mauritanica and Tunisian M. lebetina, was also investigated through an affinity chromatography-based antivenomics approach. Both antivenoms very efficiently immunodepleted homologous venom toxins and displayed a high degree of paraspecificity, suggesting the clinical utility of the two antivenoms for treating bites of both M. mauritanica or M. lebetina.     

Venomics and antivenomics profiles of North African Cerastes cerastes and C. vipera populations reveals a potentially important therapeutic weakness

We report the proteomic analysis of the venom of the medically relevant snake, Cerastes cerastes, from Morocco, and the immunoreactivity profile of an experimental monospecific (CcMo_AV against Moroccan C. cerastes venom) and a commercial (Gamma-VIP against Tunisian C. cerastes and M. lebetina venoms) F(ab')(2) antivenoms towards geographic variants of C. cerastes and C. vipera venoms. The venom of C. cerastes is a low-complexity proteome composed of 25-30 toxins belonging to 6 protein families, mainly targetting the hemostatic system. This toxin arsenal explains the clinical picture observed in C. cerastes envenomings. Despite geographic compositional variation, the monospecific CcMo_AV and the Gamma-VIP divalent antivenom produced at Institut Pasteur de Tunis, showed similar immunocapturing capability towards Moroccan, Tunisian, and Egyptian C. cerastes venom proteins. Proteins partially escaping immunorecognition were all identified as PLA(2) molecules. Antivenomic analysis showed low degree of cross-reactivity of Moroccan CcMo_AV and Tunisian Gamma-VIP antivenoms towards C. vipera venom toxins. This study indicates that a more complete therapeutic cover could be achieved by including C. vipera venom in the formulation of venom immunization mixtures, thereby generating a pan-Cerastes antivenom.     

Evaluation of the effect of gamma rays on the venom of Vipera lebetina by biochemical study

Snake bites represent a serious public health problem in many areas of the world. In Algeria, two widespread snakes are Vipera lebetina and Cerastes cerastes. Vipera lebetina venom causes local hemorrhage and necrosis, and it may lead to permanent limb loss. The principal causes of mortality after snakebites are acute renal failure and hemorrhage, which occur not only locally, at the site of the bite, but also systemically, contributing to the cardiovascular shock characteristic of severe envenomation. Gamma radiation has been shown to be effective for attenuating venom toxicity. Vipera lebetina venom was irradiated with two doses of gamma rays (1 and 2 kGy) from a 60Co source, and the venom's toxic, enzymatic, and structural properties were analyzed. Intraperitoneal injection of the irradiated venoms (100-500 microg/20 g mouse body mass) revealed a significant decrease of the toxicity. Irradiated venoms with 1 and 2 kGy doses were four and nine times less toxic, respectively, than the native venom. A biochemical characterization of in vitro enzymatic activities was performed. Vipera lebetina displayed in vitro caseinolytic, amidolytic, esterasic, coagulant, and phospholipase A2 activities. Caseinolytic, amidolytic, esterasic, and coagulative activities were reduced for the irradiated venoms; only phospholipase A2 activity was abolished in the irradiated venom with a dose of 2 kGy. The native and irradiated venoms were separated by gel filtration and electrophoresis. Chromatographic and electrophoretic profiles were drastically changed as compared with the native venom. Vipera lebetina venom detoxified by gamma rays was used for active immunization, and the presence of antibody in the immune sera was detected by ELISA. The immunogenic properties were preserved and the antisera obtained with the irradiated venoms could cross-react. Antisera were able to neutralize the toxic effect of V. lebetina native venom. These results indicate that irradiation of V. lebetina venom with a dose of 2 kGy can promote a significant detoxification, keeping the immunological properties intact.     

Cerastes cerastes (Egyptian sand viper) venom induced platelet aggregation as compared to other agonists

The Egyptian Sand Viper (Cerastes cerastes) crude venom and subfractions were, for the first time, shown to induce platelet aggregation with agonist activities present in two subfractions. The combined activities of the crude venom components behaved in a unique fashion as compared to the platelet agonists, ADP, collagen and thrombin. The action of the venom was inhibited by conditions that increased cAMP, partially required the formation of thromboxane A2 and was inhibited by the serine protease inhibitor PMSF while being only partially sensitive to leupeptin or soybean trypsin inhibitor. One of the fractionated venom agonists strongly induced serotonin release while the other venom agonist essentially did not. Further characterization of the Cerastes cerastes venom components should broaden our knowledge of the pathology of snake venoms, platelet aggregation and their potential therapeutic value.     

Effect of Some Variables on theIn VivoDetermination of Scorpion and Viper Venom Toxicities

An adequate assessment of scorpion and snake venom LD50 is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy. The LD50 variation of Tunisian scorpion (Androctonus australis garzonii: Aag and Buthus occitanus tunetanus: Bot) venoms with body weight, sex and strain (Swiss or C57BI/6) of mice used, the route of venom injection, the venom-milking procedures (manually or electrically) and the venom batches have been studied over a 7-year period (1990-1996). Aag venom is 3-4 times more toxic than Bot venom. However for both venoms, the LD50 determined in C57BI/6 mice, in small body weight animal or by intraperitoneal route were respectively significantly lower than those determined in Swiss mice, in high body weight or by subcutaneous route. Significant LD50 variations (25-50%) were also seen from one electrically prepared batch to another. A good correlation (r = 0.982) was observed between the concentrations of the crude venom toxic fraction determined by ELISA and LD50 values when assessed in vivo. The LD50 variation of Tunisian viper (Cerastes cerastes: Cc and Vipera lebetina: VI) venoms with the strain (Swiss or BALB/c), sex and body weight of mice used, the season and the year of venom milking were also investigated over a 3-year period (1990-1992). No significant LD50 variations were observed with the mouse strain, the sex or the season of venom milking. However, LD50 varies significantly with the year of the venom collection and the body weight of mice used. Furthermore, SDS-PAGE analysis shows annual variation for VI venom composition where no such variations were observed for Cc venom. These results stress the need either for the standardization of the venom LD50 evaluation or of the venom quality used for the development of an efficient antivenom.     

Immunological study and hemolytic activity of the venom of the horned viper, Cerastes cerastes (Linne, 1758) (Reptilia, Viperidae)

The immunological study of the venom of the Horned Viper, Cerastes cerastes (Linné, 1758) points out eight fractions from which the components have various hemolyticus actions.     

Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system

We report the cloning and sequence analysis of Cerastes vipera and Macrovipera lebetina transmediterranea cDNAs coding for short non-RGD (Arg-Gly-Asp) disintegrins and for dimeric disintegrin subunits. The mRNAs belong to the short-coding class, suggesting that these disintegrin mRNAs may be more widely distributed than previously thought. Our data also argue for a common ancestry of the mRNAs of short disintegrins and those coding for precursors of dimeric disintegrin chains. The Macrovipera lebetina transmediterranea dimeric disintegrin reported to inhibit the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1 was analysed using a proteomic approach and was shown to bear MLD (Met-Leu-Asp) and VGD (Val-Gly-Asp) motifs. The results highlight the fact that disintegrins have evolved a restricted panel of integrin-blocking sequences that segregate with defined branches of the phylogenetic tree of the integrin alpha-chains, providing novel insights into the evolutionary adaptation of the snake venom antagonists to the ligand-binding sites of their target integrin receptors.     

Thrombin-like serine proteases in Cerasted venoms (Cerasted cerastes and Cerastes vipera)

Cerastes cerastes and Cerastes vipera snake venoms are rich of thrombin-like, serine-protease polypeptides. Many proteins have been isolated, purified and characterized from these vipers. These proteins act in various way on blood coagulation pathway and platelet function.     

Snake venomics of the Armenian mountain vipers Macrovipera lebetina obtusa and Vipera raddei

Venoms from the Armenian mountain vipers Macrovipera lebetina obtusa and Vipera raddei were analyzed by RP-HPLC, N-terminal sequencing, MALDI-TOF mass fingerprinting and CID-MS/MS. The venom proteins of M.l. obtusa and V. raddei belong to 9 and 11 families, respectively. The two mountain viper venoms share bradykinin-potentiating/C-natriuretic peptides, and proteins from the dimeric distegrin, DC-fragment, CRISP, PLA(2), serine proteinase, C-type lectin-like, L-amino acid oxidase, and Zn(2+)-dependent metalloproteinase families, albeit each species exhibits distinct relative abundances. M.l. obtusa and V. raddei venoms contain unique components, e.g. the short disintegrin obtustatin in M.l. obtusa, and Kunitz-type serine proteinase inhibitor and VEGF-like molecules in V. raddei. The toxin formulation of M.l. obtusa and V. raddei venoms may be related to their adaptation to rocky mountain ecosystems. On the other hand, the possibility that the VEGF-like proteins from V. raddei underlie the reported potential therapeutic value of V. raddei venom for regenerating damaged peripheral nerves deserves further investigations. Using a similarity coefficient, we estimate that the similarity of venom proteins between M. l. obtusa and M. l. transmediterranea is less than 4%. Although this result would support the classification of M.l. obtusa and M.l. transmediterranea as different species, additional detailed genomic analyses are also required.     

Effects of 60Co gamma radiation on toxicity and hemorrhagic, myonecrotic, and edema-forming activities of Cerastes cerastes venom

Antisera are used as effective antidotes against the local effects of snake bites. To improve antisera production and extend the life of surrogates used to produce antibodies, the chronic effects of venom toxicity must be reduced. The present study evaluated the effectiveness of gamma irradiation to reduce the local effects associated with viperid snake bites by evaluating in NMRI mice the toxicity and edematic, hemorrhagic, and myonecrotic activities of native and irradiated Cerastes cerastes venoms. These results indicated that the toxicity of irradiated venoms (1 and 2 kGy) decreased as compared with that of native venom. The edematic and hemorrhagic activities were also reduced in the detoxified samples, particularly with the 2-kGy radiation dose. Furthermore, the creatine phosphokinase (CPK) activity was significantly increased in the serum and decreased in the myocardium after envenomation with native venom, but no significant enzymatic changes were observed in mice envenomated with irradiated venom. Histopathologic evaluation showed that native venom caused severe degenerative changes in the myocardium. In the case of 2-kGy-irradiated venom, no tissue alterations were observed. These results indicate that irradiation of venom with a 2-kGy dose may offer an effective method for reducing the chronic toxic effects of venom in immunized animals.     

Infections and deaths of horned vipers, Cerastes cerastes (L., 1758) and lebetin vipers, Vipera lebetina (L., 1758) caused by Pseudomonas aeruginosa (Schr?eter, 1885)

The authors present some data about the buccal and intestinal microflora of Ophidian Reptiles schooling in the vivaria of the Institute Pasteur of Tunis. They describe two cases of infestation and dead, chiefly symptomatology, of a horned Viper, Cerastes cerastes (L. 1758) and a lebetin Viper, Vipera lebetina (L., 1758) by Pseudomonas aeruginosa Schr?eter, 1885.     

Morphological Changes of Proteolipid Giant Unilamellar Vesicles Affected by Macrovipera lebetina obtusa Venom Visualized with Fluorescence Microscope

As a rule, zootoxins are complex and biologically active, and therefore the greater part of zootoxins is subjected to biotransformation and interacts with biological membranes. In this case, the interaction of different venom components with the membranes is not always the same. The present study shows how the giant unilamellar vesicles (GUV) from bovine brain proteolipids interact with Macrovipera lebetina obtusa venom. GUV (mean diameter 30 μm) were formed by the electroformation method. We used 8-anilino-1-naphthalenesulfonic acid and pyrene as fluorescence probes, which allowed us to quantify the fluidity changes in the membrane by measuring the fluorescence intensity.     

Short term effects of animal venoms on the mitotic index of the duodenal mucosa of albino rats.

Short term administration of the venoms of the snakes Naja haje, Naja nigricollis, and Cerastes vipera and of the scorpion Leiurus quinquestriatus on the mitotic index of the duodenal mucosal cells of the white rat, Rattus rattus, has been studied. All the venoms increased the number of dividing cells of the duodenal mucosa significantly. Naja haje crude venom was fractionated into three fractions. Fraction I had no effect on the mitotic index whereas fractions II and III increased it significantly. Treatment of rats with Naja haje venom fractions II and III after blocking the histamine or the serotonin receptors did not affect the stimulatory action of the two venom fractions on the mitotic index, which it increased significantly. It was suggested that the venoms of Naja haje, Naja nigricollis, Cerastes vipera, and Leiurus quinquestriatus and Naja haje venom fractions possessed a mitogenic activity. Fraction II of Naja haje venom acted through both the muscarinic and adrenergic receptors while fraction III acted on the adrenergic ones.     

Molecular events associated with Macrovipera lebetina obtusa and Montivipera raddei venom intoxication and condition of biomembranes

Studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including BLMs, SUVs and GUVs. The present study was undertaken to elucidate how the plastic properties (namely, its microviscosity, thickness, permeability) of model membranes from native lipids of different tissues of rats change in the course of Macrovipera lebetina obtusa (MLO), Montivipera raddei (MR) and Naja kaouthia (NK) venoms processing. The presence of viper venom in organism leads to increasing of the electrical resistance of BLMs from liver and muscle lipids approximately on a sequence, while the BLMs from brain lipids have not shown noticeable differences of plastic properties compared to the control. Giant unilamellar vesicles (GUVs) with a mean diameter of 30μm have a minimum curvature and mimic cell membranes in this respect. Snake venom was added to the sample chamber before the vesicles were formed. The membrane fluorescence probes, ANS and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. Fluorescent spectra were acquired on a Varian fluorometer instrument. ANS and pyrene allow us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. The presence of viper venom in GUVs media reveals a noticeable decreasing of membrane fluidity compared to the control, while the binding of fluorophores with GUVs modified by venom leads to the appearance of channel activity. These studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom.     

Macrovipera lebetina obtusa Snake Venom as a Modulator of Antitumor Effect in S-180 Sarcoma Mouse Model

Molecular Biology - Macrovipera lebetina obtusa (MLO) is a venomous snake endemic to Middle East. Here we describe the therapeutic potential of the MLO snake venom. In S-180 sarcoma-bearing mouse...     

Cross-reactivities of polyclonal antibodies against factor V activating enzyme, a serine proteinase from Vipera lebetina (snake) venom

Antibodies to the factor V activating enzyme from Vipera lebetina venom were produced by immunizing a rabbit with chromatographically purified factor V activating enzyme probes. The antibodies cross-reacted with different protein fractions in 23 snake venoms (ten viperid, eight crotalid, and five elapid venoms) as demonstrated by western immunoblotting. In the venom of Vipera russelli the antibodies recognized only one protein band which probably belonged to factor V activating enzyme.