Isolation and characterization of an anti‐leishmanial disintegrin from Cerastes cerastes venom

Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy. In this study, we report the characterization of a disintegrin isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Indeed, isolated disintegrin, referred to Disintegrin_Cc, induced 84.75% of parasiticidal activity and deep morphological alterations on the parasites. SDS‐PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. It presents a high percentage of identity with other related snake disintegrins. Predicted 3D structure indicated that this peptide shares partial homology with well‐known active antimicrobial peptides. Disintegrin_Cc inhibited 80% of arachidonic acid‐induced platelet aggregation. The obtained results suggest that the isolated molecule plays a dual role as a disintegrin and as an anti‐leishmanial compound. This component could be useful as a drug in the treatment of leishmaniasis.     

Biochemical and biological characterization of a dermonecrotic metalloproteinase isolated from Cerastes cerastes snake venom

A dermonecrotic metalloproteinase (CcD‐II) was isolated from C. cerastes venom. Venom fractionation was performed using three chromatographic steps (molecular exclusion on Sephadex G‐75, ion‐exchange on DEAE‐Sephadex A‐50, and reversed‐phase high‐performance liquid chromatography on C8 column). CcD‐II presented an apparent molecular mass of 39.9 kDa and displayed a dermonecrotic activity with a minimal necrotic dose of 0.2 mg/kg body weight. CcD‐II showed proteolytic ability on casein chains and on α and β fibrinogen chains that was inhibited by ethylenediamine tetraacetic acid and 1,10‐phenanthroline while remained unaffected by phenylmethylsulphonyl fluoride and heparin. CcD‐II displayed gelatinase activity and degraded extracellular matrix compounds (type‐IV collagen and laminin). These results correlated with histopathological analysis showing a complete disorganization of collagenous skin fibers. These data suggested that CcD‐II belongs to P‐II class of snake venom metalloproteinase. The characterization of venom compounds involved in tissue damage may contribute in the development of new therapeutic strategies in envenomation.     

Purification and properties of the clotting enzyme from limulus lysate

The clotting enzyme from Limulus lysate which is involved in the gelation reaction of lysate with endotoxin has been purified and some of its properties determined. It was isolated from endotoxin-treated lysate and purified by gel filtration, ion exchange chromatography, and disc gel electrophoresis. Reaction of clotting enzyme with lysate clottable protein produces a clot or gel such as occurs with the gelation of lysate by endotoxin. Purified clotting enzyme has an approximate molecular weight of 84,000 (subunit MW 43,000), is isoelectric at pH ca. 5.5, trypsin-like, heat labile and pH sensitive.     

Characterization of phospholipase A2 from the venom of Horned viper (Cerastes cerastes)

Phospholipase A2 has been purified from the venom of Horned viper (Cerastes cerastes) by gel permeation chromatography followed by reverse-phase HPLC. The primary structure was established by sequence analysis of the intact protein and its enzymic peptides. The structure has 120 residues, properties like other group IIB phospholipases, but only 45-55% identity with the enzyme from other viperid species, and large variations even within the species (26% residue differences at known positions in another form).     

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.     

Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom

Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy.     

Purification,Characterization and Antibacterial Activity of l‐amino Acid Oxidase from Cerastes cerastes

Antibiotic resistance presents a real problem in which new antibacterial molecules from natural secretions could be beneficial in the development of new drugs. In this study, Cerastes cerastes venom was investigated for its antibacterial activity against Gram‐positive and Gram‐negative bacteria. The antibacterial activity was evaluated by measuring the halo inhibition and minimum inhibitory concentration (MIC). An l ‐amino acid oxidase (CcLAAO) was purified from this venom using three chromatographic steps; its homogeneity (60 kDa) was confirmed by SDS‐PAGE. LC–MS/MS analysis of CcLAAO showed similarities with other LAAO enzymes from Echis ocellatus and Viridovipera stejnegeri venoms. CcLAAO presents an antibacterial activity against three bacterial strains (Staphylococcus aureus, Methicillin‐resistant S. aureus, and Pseudomonas aeruginosa) with MIC values of 10, 10, and 20 μg/mL, respectively. However, no effect was observed against Escherichia coli and yeast strains. Kinetic parameters of CcLAAO evaluated on l ‐leucine at pH 8.0 and 20°C were K_m = 0.06 mmol and V_max = 164 mmol/min.     

Metalloproteinase with factor X activating and fibrinogenolytic activities from Vipera berus berus venom

We have previously shown that Vipera berus berus venom contains several factor X activating enzymes. In the present study we have investigated one of them. The enzyme was separated from venom by gel filtration on Sephadex G-100 superfine and chromatography on agarose HPS-7 and phenyl-agarose. The enzyme is a glycosylated metalloproteinase containing hexoses, hexosamines and neuraminic acid. The purified factor X activating enzyme consists of two equal chains (59 kDa). The specificity studies have shown that enzyme is nonspecific factor X activating proteinase hydrolysing also proteins such as azocasein, gelatin and fibrinogen. The enzyme hydrolyses oxidized insulin B-chain at the positions Ala¹⁴–Leu¹⁵ and Tyr¹⁶–Leu¹⁷ but it is inactive on fibrin, plasminogen and prothrombin. We used 8–10 amino acid residues containing peptides, which reproduce the sequence around the cleavage sites in factor X, factor IX and fibrinogen, as potential substrates for enzyme. Cleavage products of peptide hydrolysis were determined by MALDI-TOF MS. The peptide Asn–Asn–Leu–Thr–Arg–Ile–Val–Gly–Gly—factor X fragment was cleaved by enzyme at positions Leu³–Thr⁴ and Arg⁵–Ile⁶. The fibrinogen peptide fragment Glu–Tyr–His–Thr–Glu–Lys–Leu–Val–Thr–Ser was hydrolysed at position Lys⁶–Leu⁷.     

Purification and characterization of a prophenoloxidase activating enzyme from crayfish blood cells

A proteinase was purified from crayfish haemocytes by affinity chromatography on heparin-sepharose and phenyl-sepharose, followed by DEAE-cellulose ion-exchange chromatography. This proteinase could mediate the conversion of prophenoloxidase (proPO) to its active form, phenoloxidase (PO), and its was therefore designated a prophenoloxidase activating enzyme, ppA.The purified ppA had a molecular mass of about 36,000 Da. Since ppA was a proteinase able to cleave chromogenic peptide substrates of trypsin, and serine proteinase inhibitors were strongly inhibitory towards ppA activity, the enzyme appeared to be a serine type proteinase. It exhibited maximal enzyme activity at neutral and slightly alkaline pH, and was sensitive to heat inactivation at 58°C.     

Purification and properties of the thrombin-like enzyme from the venom of Lachesis muta muta

1. The coagulating enzyme of the Lachesis muta muta venom was purified to homogeneity by a combination of a gel filtration in Sephadex G-100 and affinity chromatography on agarose-agmatine resin. 2. Several forms of the enzyme were prepared by isoelectric focusing with pIs ranging from 3.1 to 5.0; the asialoenzyme focused as a narrow band at pH 8.7. SDS-PAGE analysis of the purified enzyme revealed a single broad band with apparent Mr of 41-47 kDa. 3. The enzyme cleaves only fibrinopeptide A from fibrinogen; it does not activate factor XIII and is devoid of kallikrein-like activity. 4. Kinetic properties of the enzyme were determined for representative synthetic chromogenic substrates and inhibitors.     

Model of a specific interaction: Salt-bridges form between prothrombin and its activating enzyme blood clotting factor Xa

In order for intricate biochemical pathways to function properly in the heterogeneous environment of a cell or organism, high specificity of enzymesubstrate reactions is essential. The molecular nature of this specificity is examined for the case of the specific activation of prothrombin by factor X_a of the blood coagulation system using model-building methods.The structure of blood clotting factor X_a was modeled by homology to the known structures of the mammalian serine proteases. The sequence about the cleaved peptide bond in prothrombin was placed into the active site of factor X_a in a conformation similar to that of bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor, when bound to trypsin, and to that of the tripeptide chloromethyl ketones, when bound to γ-chymotrypsin and Streptomyces griseus protease B.The model of the complex between prothrombin and factor X_a shows the expected salt-bridge between the Arg preceding the cleaved peptide bond and the primary specificity residue Asp189 of factor X_a. Two other salt-bridges appear to be formed: between a Glu three residues before the cleaved bond in prothrombin and Arg143 of factor X_a, and between a second Glu three residues after the cleaved bond and Lys62 in factor X_a. Several hydrogen bonds and hydrophobic interactions also occur in the complex. A stereogeometric requirement for a Gly two residues before the cleaved bond is also imposed by the factor X_a structure.Examination of the known serine protease sequences and model building suggest that the two new salt-bridges are unique to the prothrombin-factor X_a complex. Therefore, these charge interactions, and the requirement for a Gly, are likely to be at least partially responsible for the high specificity of the activation of prothrombin by factor X_a.     

Molecular cloning and expression of a functional snake venom serine proteinase,with platelet aggregating activity,from the Cerastes cerastes viper

The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets. Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes. Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor. The recombinant protein was designated rCC-PPP (for C. cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin. The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases. The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities. Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM). It is able to clot purified fibrinogen and to hydrolyze alpha-chains. Thus, rCC-PPP could be therefore considered a cerastocytin isoform. By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys(42). This implies that rCC-PPP lacks the conserved Cys(42)-Cys(58) disulfide bridge. A structural analysis performed by molecular modeling indicated that the segment of residues Tyr(67)-Arg(80) of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation. Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe(90)-Val(99)), Tyr(172), and Trp(215) residues, which might be involved in the fibrinogen clotting activity of rCC-PPP.     

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 microg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-L-Arg-pNA by leuc was competitively inhibited by benzamidine (Ki=1.61+/-0.25 mM).     

Molecular modeling,biochemical characterization,and pharmacological properties of Cc3‐SPase: A platelet‐aggregating thrombin‐like enzyme purified from Cerastes cerastes venom

Cc₃‐SPase (30 kDa‐proteinase; pI 5.98) was isolated from Cerastes cerastes venom. Its sequence of 271 residues yielded from LC‐MALDI‐TOF showed high degrees of homology when aligned with other proteinases. Cc₃‐SPase cleaved natural and synthetic proteins such as casein and fibrinogen leaving fibrin clots unaffected. Cc₃‐SPase was fully abolished by ion chelators, whereas aprotinin, antithrombin III (Sigma Aldrich, Saint‐Louis, Missouri, USA), and heparin were ineffective. Affinity of Cc₃‐SPase to benzamidine indicated the presence of an aspartate residue in the catalytic site as confirmed by three‐dimensional structure consisting of 14 β‐strands and four α‐helices. Molecular mechanisms revealed that Cc₃‐SPase is capable of promoting dysfunctional platelet aggregation via two signaling pathways mediated by the G‐coupled protein receptors and αIIbβ3 integrin. Cc₃‐SPase is involved in both extrinsic/intrinsic coagulation pathways in deficient plasmas by replacing defective/lacking factors FII, FVII, and FVIII but not FX. Cc₃‐SPase could substitute missing factors in blood diseases related to plasma factor deficiencies.     

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.     

The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor.

The yeast proteinase that causes activation of the chitin synthetase zymogen has been purified by a procedure that includes affinity chromatography on an agarose column to which the proteinaceous inhibitor of the enzyme had been covalently attached. The purified enzyme yielded a single band upon disc gel electrophoresis at pH 4.5 in the presence of urea. At the same pH, but without urea, a faint band was detected in coincidence with enzymatic activity, whereas at pH 9.5, either in the absence or in the presence of sodium dodecyl sulfate, no protein zone could be seen. From sedimentation and gel filtration data, a molecular weight of 44,000 was estimated. The proteinase was active within a wide range of pH values, with an optimum between pH 6.5 AND 7. Titraton of the activity with the protein inhibitor from yeast required 1 mol of inhibitor/mol of enzyme. A similar result was obtained with phenylmethylsulfonyl fluoride, an indication that 1 serine residue is required for enzymatic activity. The enzyme exhibited hydrolytic activity with several proteins and esterolytic activity with many synthetic substrates, including benzoylarginine ethyl ester and acetyltyrosine ethyl ester.A comparison of the properties of the enzyme with those of known yeast proteinases led to the conclusion that the chitin synthestase activating factor is identical with the enzyme previously designated as proteinase B (EC 3.4.22.9). This is the first time that a homogeneous preparation of proteinase B has been obtained and characterized.