Monoclonal antibody immunoaffinity chromatography of the nerve growth factor from snake venoms

1. Pure monoclonal antibodies to Vipera lebetina venom nerve growth factor have been isolated by affinity chromatography using CNBr-agarose bound antigen. 2. Nerve growth factors from ten snake venoms (Vipera lebetina, Vipera russellii, Vipera berus berus, Vipera ursini, Echis carinatus, Agkistrodon halys, Bungarus caeruleus, Naja naja oxiana, Naja naja, Naja naja atra) were purified using monoclonal antibodies against NGF linked to BrCN-activated agarose.     

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.     

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.     

Evidence for existence of venom 5' nucleotidase in multiple forms through inhibition of concanavalin A

Pharmacologically active 5' nucleotidase is a ubiquitously distributed enzyme in snake venoms. In this study the effect of concanavalin A (Con-A) on different snake venoms 5' nucleotidase activity is tested in order to know the protein nature which will ultimately help in purification of the enzyme with high yield. Con-A inhibited Naja naja, Naja kauthia, Naja melanoleuca, Naja naja sputatrix, Agistrodon halys blomhoffii, Bothrops asper and Oxyranus scutellas venom 5' nucleotidase activity at different concentrations. This indicates the presence of glycopart in the protein, thus glycoprotein in nature. Vipera russellii, Vipera plaestenae, Agistrodon contratrix, Bitis orientis, Echis carinatus and Trimeresures malabaricus was not inhibited by Con-A, indicating absence of glycopart in the protein. This study for the first time shows existence of 5' nucleotidase in multimeric forms.     

Comparative characterisation of Russell's viper (Daboia/Vipera russelli) venoms from different regions of the Indian peninsula.

Russell's viper (Daboia/Vipera russelli) venom from different regions of India was subjected to chromatographic, electrophoretic, biochemical and immunological analysis. The elution profiles from ion-exchange chromatography and protein banding pattern from SDS-PAGE showed a significant variation in the constituents of venoms. The acidic proteins are found to be predominant in the venoms of eastern and western regions while basic proteins are the major contributors of the northern and southern regional venoms. The major variation of phospholipases A(2) in the venom samples of India may be described as: southern regional venom is rich in basic, toxic PLA(2) while this activity showed a dramatic decrease as one moves towards west, north and eastern regions of India. In addition, the caseinolytic, TAME-hydrolytic, anticoagulant, oedema-inducing and haemorrhagic activities of the venoms have also varied from one region to another. The muscle specimens of mice injected with venoms of different regions showed variable change in the muscle fibre damage and cell morphology. The eastern regional venom is most lethal among all the venoms. The lethal potencies for four regional venoms vary as: eastern>western>southern>northern. The polyclonal antibodies prepared against the venom of southern region showed cross-reaction with the venoms of other regions, but the extent of cross-reaction and diffusion patterns are different. However, the polyclonal antibodies prepared against southern regional venom showed no protection against lethal toxicity of other regional venoms.     

Molecular cloning and sequence analysis of a cDNA for factor V activating enzyme.

The complete amino acid sequence of a factor V activator (VLFVA) is deduced from the nucleotide sequence of a cDNA encoding the enzyme. The cDNA was isolated by PCR screening a venomous gland cDNA library of Central Asian Vipera lebetina snake. The full-length cDNA clone, derived from two overlapping fragments, comprises 1563 basepairs which encode an open reading frame of 259 amino acids. The amino acid sequence of VLFVA (235 amino acids) shows significant homology with snake venom and mammalian serine proteinases. It contains 12 half-cysteines which form, by analogy with other serine proteinases, 6 disulfide bridges. VLFVA has the catalytic triad His43-Asp88-Ser182. The amino terminal amino acid valine is preceded by 24 amino acids: a putative signal peptide of 18, mainly hydrophobic, amino acids and an activating peptide of 6, mainly hydrophilic amino acid residues. This is the first cloned factor V activating enzyme from snake venom.     

蛇毒丝氨酸蛋白酶多样性──底物专一性免疫化学及序列比较研究

本文报道烙铁头（Ｔｒｉｍｅｒｅｓｕｒｕｓｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒纤维蛋白原溶酶（ＴＭＶＦｇ），眼镜王蛇（Ｏｐｈｉｏｐｈａｇｕｓｈａｎｎａｈ）蛇毒纤维蛋白原溶酶（ｏｈＳ１），竹叶青（Ｔｒｉｍｅｒｅｓｕｒｕｓｓｔｅｊｎｅｇｅｒｉ）蛇毒专一纤溶酶原激活剂（ｓｖ－ｐＡ）对５种小分子多肽底物的底物专一性，及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用，并和其它蛇毒丝氨酸蛋白酶如矛头蝮（Ｂｏｔｈｒｏｐｓａｔｒｏｘ）蛇毒凝血酶样酶（Ｂａｔｒｏｘｏｂｉｎ）、铜头蝮（Ａｇｋｉｓｔｒｏｄｏｎｃｏｎｔｏｒｔｒｉｘｃｏｎｔｏｒｔｒｉｘ）蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇（Ｖｉｐｅｒａｒｕｓｓｅｌｌｉ）毒第Ⅴ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫交叉反应，并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom ( Apis mellifera ), three snake venoms ( Naja naja sputatrix, Vipera russellii and Crotalus adamanteus ) and the polypeptide melittin was investigated against Escherichia coli . Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom > melittin > N. naja sputatrix venom ≫ V. russellii venom > C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.     

The action of various venoms on Escherichia coli

The antibacterial activity of honeybee venom (Apis mellifera), three snake venoms (Naja naja sputatrix, Vipera russellii and Crotalus adamanteus) and the polypeptide melittin was investigated against Escherichia coli. Minimum inhibitory concentration values, cell lysis and alterations in cell permeability were determined and action against E. coli was in the order: A. mellifera venom greater than melittin greater than N. naja sputatrix venom much greater than V. russellii venom greater than C. adamanteus venom. Cellular damage by A. mellifera and N. naja sputatrix venoms was evident in electron micrographs.     

A comparative study of the biological properties of venoms from snakes of the genus Vipera (true adders)

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.     

Avidity of sera-neutralizing snake venom

Avidity of antivenom sera used for the treatment of snake bites was studied. Sera against the venom of Vipera libetina obtained from producers immunized with crude venoms were more avid than analogous sera obtained to anavenoms. In studying the avidity of polyvalent serum neutralizing the Vipera libetina, echis and cobra venoms showed the serum obtained in immunization with the mixture of crude venoms to be highly avid to all the venoms composing the antigen; besides, it bound the venoms of Vipera libetina and echis more rapidly and more stably than the corresponding monovalent sera.     

Isolation and characterization of nerve growth factor from Vipera berus berus (common viper) venom

Nerve growth factor from Vipera berus berus venom was purified by gel filtration on Sephadex G-100 (superfine), ion-exchange-chromatography on DEAE-Sephadex A-50 and chromatofocusing on PBE 118. The Vipera berus berus venom NGF consists of multiple molecular forms with pls in the interval 9.1-9.7. All isoforms have identical mol. wts approximately 35,000 +/- 3000 (in gel filtration) and 17,000 +/- 2000, 15,000 +/- 2000 (by SDS electrophoresis with beta-mercaptethanol). V. berus berus venom NGF reacted with monoclonal antibodies against Viper lebetina NGF and caused differentiation of pheochromocytoma PC12 cells.     

Venom Toxins: Plausible Evolution from Digestive Enzymes

Some hydrolytic enzymes are common to the pancreas, the mammaliansalivary glands and the snake venom glands. Phospholipase A,which is found in elapid and viperid venoms and in the mammalianpancreas, shows 29 common amino acid residues out of 118–125positions. Presynaptic neurotoxins and other venom toxins areusually composed of 2–3 units or subumts,one of whichis a phospholipase. The Vipera palaestinae two-component toxinretains its lethality when the enzyme is replaced by heterologousvenom phospholipases, but not by the pig pancreatic enzyme.This toxin is neutralized by a factor found in the blood serumof snakes, which binds to the phospholipase and inhibits itsactivity. The blood serum of snakes also neutralizes hemorrhaginsand inhibits the protease activity of the venom. It is hypothesizedthat the developing venom glands first produced enzymes thatwere already secreted by the pancreas and against which inhibitorswere present in the blood. These inhibitors facilitated theevolution of enzyme-based toxins by neutralizing any damagingsubstances that might have escaped from the venom glands.     

Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.     

Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper. A new member of the VEGF family of proteins

During the generation of abundant expressed sequence tags from the Viperidae snake Bothrops insularis venom glands, we identified for the first time a cDNA coding for a putative vascular endothelial growth factor-like (VEGF-like) protein. The deduced primary sequence, after complete sequencing of the longest snake venom VEGF (svVEGF) cDNA, displayed similarity with vertebrate VEGFs and with the hypotensive factor from Vipera aspis venom. Its cDNA was subcloned, expressed in Escherichia coli with a His(6) tag as an insoluble monomer, and purified by Ni(2+)-affinity chromatography after 8 m urea extraction. Antiserum against svVEGF was generated and tested in Western blot against proteins from snake venoms and cellular extracts. The mature svVEGF appears to be ubiquitously distributed throughout snake venoms and was also confirmed by Northern blot studies of other related Viperidae species and by cDNA cloning of svVEGF from Bothrops jararaca pit viper. The produced recombinant protein dimerizes after refolding processes and was biologically characterized, showing ability to increase vascular permeability. These results established that svVEGF is a novel and important active toxin during the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins.     

Analysis of Vipera russelli venom using polyclonal antibodies prepared against its purified toxic phospholipase A2 VRV PL-V.

Vipera russelli venom induces predominantly neurotoxic, myotoxic necrotic and hemorrhagic symptoms in experimental animals and has several hydrolytic enzyme activities. In this study, V. russelli venom is characterized both as a PLA2 and as a toxin. Anti PL-V Ig (antibodies to a toxic phospholipase A2 VRV PL-V of V. russelli venom) nullifies the toxicity of whole V. russelli venom to a great extent. The neurotoxic symptoms vanish completely in the presence of anti PL-V Ig. The cross reacting components of whole V. russelli venom were removed by precipitating them from whole venom by the addition of anti PL-V Ig. The non-cross reacting components present in the supernatant were checked for toxicity. There was a significant reduction in toxicity. The LD50 value of the supernatant had increased from 4.1 mg/kg body weight to 11.7 mg/kg body weight and it showed about 34% of the total venom phospholipase A2 activity. It had edema forming, hemorrhagic and hemolytic activity but failed to induce neurotoxic, anticoagulant and myotoxic effects.     

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.     

Toxinological and immunological studies of capillary electrophoresis fractionated Chrysaora quinquecirrha (Desor) fishing tentacle and Chironex fleckeri Southcott nematocyst venoms

Repeated runs of capillary electrophoresis (CE) were used to study partially-purified jellyfish nematocyst venom protein in concentrations sufficient to perform toxinological assays. Nematocyst venoms from Chironex fleckeri (Cf) and Chysaora quinquecirrha were processed. The CE eluate was divided into quadrants by scanning protein content. The fourth fraction of both jellyfish venoms, contained proteins with the smallest molecular weight components, which were responsible for the highest hemolysins and the humoral and cell-mediated immunological activity. Cytotoxic Cf lethal factor activity against human liver cells was widely dispersed throughout both venoms but more prominent in fraction 4. A V(beta) receptor human T-cell repertoire was not species-specific for either crude or fractionated jellyfish nematocyst venom.     

乌梢蛇血清的抗出血因子:一个有前途的抗蛇毒药物原料

用柱层析和聚丙烯酰胺凝胶盘状电泳法,从乌梢蛇血清中分离纯化了一个抗出血因子。用SDS-聚丙烯酰胺凝胶电泳法测得其分子量大约为65 kD;测定了五种蝮亚科蛇毒(尖吻蝮、竹叶青蛇、原矛头蝮、哈扑和短尾蝮)的最小出血剂量和乌梢蛇血清中抗出血因子对这五种蛇毒的抗出血活性;还测定了七种蛇毒(除上述五种毒蛇外,还包括圆斑蝰和银环蛇)的半数致死量,以及抗出血因子对中毒小鼠的治疗作用。结果显示:从乌梢蛇血清中提纯的抗出血因子的抗蛇毒活性,不仅可以抵抗它的捕食者尖吻蝮的蛇毒,而且还可以抵抗具出血活性的其它蛇毒;但它对不具出血活性的银环蛇毒的致死抑制作用不明显。该抗出血因子不仅在体外实验表现出强的中和出血毒素的活性,而且在体内实验中亦表现出对中毒小鼠良好的治疗作用,因而可能成为新的抗蛇毒药物的有前途的原料。乌梢蛇血清对血循毒的中和能力的获得,可能归因于尖吻蝮与乌梢蛇之间捕食与被捕食相互作用的关系。