Search

Nucleases, in particular ribo- and deoxyribonucleases, are among the least-studied snake venom enzymes. In the present study we have partially purified different nucleases from Vipera lebetina venom. The DNase activity has been proved by DNA degradation both in solution as well as in-gel (zymogram-method). In DNA-containing SDS-PAGE V. lebetina venom exhibits DNA-degrading activity in bands with molecular masses of ∼120, 30-35 and 22-25 kDa. The 120 kDa band corresponds to phosphodiesterase, a 3', 5'-exonuclease. The endonucleolytic activity of the lower-molecular-mass protein has been confirmed by plasmid degradation and the visualization of the results in agarose gel (with ethidium bromide) electrophoresis. A partial DNA sequence of putative RNase H1 has been determined from the V. lebetina venom gland cDNA library. The translated sequence is similar to the assumed RNase H1 from Crotalus adamanteus (AFJ51163). The RNA/DNA hybrid is hydrolysed by V. lebetina venom and venom fractions. The masses of tryptic peptides from the SDS-PAGE 30-35 kDa band are in concordance with the theoretical peptide masses from the respective translated sequence. For the first time RNase H1-like enzyme activity has been ascertained in snake venom, and sequencing a relevant partial transcript confirmed the identification of this enzyme.

The isolation and characterization of individual snake venom components is important for a deeper understanding of the pathophysiology of envenomations, for improving the therapeutic procedures of patients, and it also opens possibilities for the discovery of novel toxins that might be useful as tools for understanding cellular and molecular processes. This review provides a summary of the different toxins that have been isolated and characterized from the venoms of Vipera lebetina (Macrovipera lebetina) subspecies Macrovipera lebetina cernovi, Macrovipera lebetina lebetina, Macrovipera lebetina obtusa, Macrovipera lebetina transmediterranea, Macrovipera lebetina turanica, the snake species causing the majority of human envenomings in Central Asia (Middle East) and North Africa. The venoms of these snakes contain proteins belonging to different families: Zn2+- metalloproteinases, serine proteinases, L-amino acid oxidase, 5'-nucleotidase, phosphodiesterase, phosphomonoesterase, nucleases, hyaluronidase, phospholipase A2, C-type lectin-like protein, disintegrin, DC-fragment, cystein-rich secretory protein, proteinase inhibitors, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), low molecular weight peptides. Their main biochemical properties, toxic and pharmacological actions have been described. In this review we will provide an overview of the proteins and peptides from the venoms of M. lebetina subspecies, their biochemical, pharmacological and structural features and their role in snake venom toxinology. A lot of contributions have been made for better understandings of these venomous snakes, their venom, and their pharmacological effects. Many of these components are also fascinating models for drug design.

Nerve growth factor (NGF) is a protein which stimulates the differentiation and maintenance of sympathetic and embryonic sensory neurons. Snake venoms are a rich source of NGF. Due to small quantities it is sometimes difficult and laborious to isolate NGF from the venoms. In this study the use of Ni-NTA-agarose for isolation of NGF is studied. Anti-Vipera lebetina NGF antibodies were used for identification of NGF during Ni-NTA-agarose fractionation as well as for cross-reaction studies with 21 snake venoms. All studied venoms contained NGF. The molecular masses of the NGFs from Echis ocellatus, Agkistrodon contortrix contortrix, A. bilineatus, A. blomhoffii, A. saxatilis, Calloselasma rhodostoma, Bothrops jararaca and B. lanceolatus were determined for the first time. Some previous results of the NGF studies are revaluated.

Two novel acidic phospholipase A(2)s (PLA(2)) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA(2)-1 (13,704 Da) and VLPLA(2)-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA(2); its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA(2) were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA(2) were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA(2) possess 615bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA(2)s have significant sequence similarity to many other phospholipase A(2)s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA(2)s grouped with other Asp49 PLA(2)s and they appear to share a close evolutionary relationship with the European vipers.

We have previously demonstrated that the fibrinolytic enzyme lebetase is synthesized with disintegrin-like domain that is cleaved posttranslationally (Siigur et al., 1996). Now we isolated a heterodimeric disintegrin viplebedin-2 containing this disintegrin-like part from Vipera lebetina venom using size-exclusion chromatography on Sephadex G-100 sf and HPLC on C18 column. The molecular masses of viplebedin-2 and tryptic peptides from both chains of viplebedin-2 were determined by MALDI-TOF mass spectrometry. Using cDNA library of the venom gland of a single V. lebetina turanica snake the viplebedin-2 coding cDNAs were cloned and sequenced. Viplebedin-2 chains are synthesized from two different genes. One chain, containing VGD sequence in disintegrin loop, is synthesized as a disintegrin-like part of the PII-type metalloprotease, lebetase. The other chain, containing MLD sequence in disintegrin loop, is synthesized from the gene without metalloproteinase domain. Two polyadenylation signal sequences have been found in MLD sequence coding chain precursor cDNAs. Viplebedin-2 dose-dependently inhibited adhesion of platelets to immobilized collagen and inhibited collagen-induced platelet aggregation.

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (-20 degrees C, -70 degrees C) and loses its activity by heating at 70 degrees C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC(50) of 0.094 microM and 0.036 microM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.

A variety of factor X activators have been detected in snake venoms. About 10 activators have been isolated from Viperidae, Crotalidae, and Elapidae venoms. Viperidae and Crotalidae venom activators are mainly metalloproteases. Only two factor X activators are characterized from Elapidae venoms, both belonging to serine proteases. Most thoroughly investigated snake venom factor X activators are from Vipera russelli (now renamed Daboia russelli; RVV-X) and Vipera lebetina (now renamed Macrovipera lebetina; VLFXA). VLFXA is cloned and sequenced, and its primary structure is deduced from the cDNA sequence. Both activators consist of a heavy chain and two C-type lectin–like light chains that are held together by disulfide bonds. Primary stuctures of the heavy chain and one light chain are similar for both activators. The primary structure of the second light chain is not detected in RVV-X. Heavy chains of RVV-X and VLFXA contain metalloprotease and disintegrin-like and cysteine-rich domains. All chains of VLF...

In order to better understand the function of fibrinolytic enzyme lebetase isoforms and the synthesis of disintegrins we have isolated a cDNA encoding the most basic isoform (Le-4) from the cDNA library prepared from the poly(A)+ RNA of the venomous gland of an individual Vipera lebetina snake. The truncated 5′-sequence of 1112 basepairs encodes the mature protein with 203 amino acid residues with calculated isoelectric point and size of 5.6 and 22,930 Da, respectively. Multiple comparison of the deduced amino acid sequence of the metalloprotease part of Le-4 is related to short reprolysins, identities were within the range of 60–87%. The two lebetase isoforms are synthesized in different way: Le-4 is synthesized with metalloprotease domain only; Le-3 is synthesized with metalloprotease and disintegrin-like domain and processed posttranslationally. The sequence of the disintegrin-like part of Le-3 is identical to A-chain of the heterodimeric disintegrin VLO5 from Vipera lebetina obtusa venom (Calvete et al., 2003).

Snake venoms contain four classes of metalloproteases that all have a typical zinc-chelating sequence (HEXXHGXXH). N-terminal sequences and internal sequences of different purified metalloproteases were determined using Edman sequencing and LC MS/MS technique. Oligonucleotideswere designed and used as primers for cDNA cloning from Vipera lebetina venom gland cDNA library. We found that isoforms of fibrinolytic enzyme lebetase Le-4 and Le-3 are synthesized in different way: Le-4 is synthesized as P-I type metalloprotease, Le-3 is synthesized with disintegrin-like domain as P-II type protease and processed post-translationally. An endothelial cell apoptosis-inducing heterodimeric glycosylated metalloprotease, V. lebetina apoptosis-inducing protease (VLAIP), belongs to P-III type containing metalloprotease, disintegrin-like and cysteine-rich domains. All these enzymes hydrolyze the Aα-chain and more slowly the Bβ-chain of fibrinogen. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes apoptosis. V. lebetina venom contains also P-IV type-specific coagulant factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein composed of a heavy chain and two C-type lectin-like light chains linked by disulfide bonds. The heavy and light chains of VLFXA are synthesized from different genes.

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52–Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.

SummaryAmino acid sequences of two anticoagulant serine fibrinogenases – α- and β-fibrinogenase (VLAF and VLBF) from Vipera lebetina venom have been deduced from the cDNA sequences encoding the enzymes. The mature protein sequences of 234 amino acids (VLAF) and 233 amino acids (VLBF) exhibit significant similarity with other snake venom serine proteinases. Both enzymes contain the catalytic triad His57, Asp102, Ser195, and twelve conserved cysteines forming six disulfide bridges. Unlike typical trypsin-like serine proteinases, they lack the third aspartate, Asp189 which is replaced by Gly189. VLBF is a typical representative of arginine esterases – β-fibrinogenases. α-Fibrinogenase, VLAF, is unique among snake venom serine proteinases with homologous structure. Until now there is no evidence of the anticoagulant serine enzymes degrading fibrinogen α-chain only and lacking esterolytic activity.Parts of this paper were presented at the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.The sequence data of Vipera lebetina mRNA for α- and β-fibrinogenase have been deposited in the GenBank database under accession numbers AF528193 (VLAF) and AF536235 (VLBF).

Nucleases and phosphatases are ubiquitous but mostly marginal components of snake venoms. These proteins have been studied quite extensively but up to now no data regarding their amino acid sequences confirmed at protein level have been published. The present study deals with purification, characterization, and structural properties of a phosphodiesterase from Vipera lebetina venom (VLPDE). The VLPDE with molecular mass of about 120 kDa hydrolyses ADP but not ATP and 5′-AMP. The aggregation of platelets induced by ADP or collagen is dose-dependently inhibited by VLPDE. The cloning and sequencing of the VLPDE-encoding cDNA resulted in 2772-nt sequence with ORF of 2556 nt. The translated sequence comprises 851 amino acids including the 23-amino acid signal peptide. VLPDE is synthesized as a 828-amino acid single-chain protein but subsequently cleaved to form a two-chain protein held together with disulfide bonds. In reducing conditions the enzyme behaves like a heterodimeric protein but, differently from the real heterodimers, it is synthesized as a single-chain protein. VLPDE is the first snake venom phosphodiesterase with established and confirmed primary structure.

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants in the same venom. We showed that V. lebetina venom contains: (1) proteases that degrade fibrinogen, but not fibrin; (2) fibrinolytic enzyme (lebetase); (3) factor X activator (VLFXA); (4) factor V activator (VLFVA). Fibrinolytic enzyme and VLFXA are metalloproteases; the other studied enzymes are serine proteases. α-Fibrinogenase has no homolog among known serine proteases. β-Fibrinogenase is a typical thermostable arginine esterase that hydrolyzes esters and amides of arginine and attacks the β-chain of fibrinogen. Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins. We used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique for the recovery and identification of peptides released by protease hydrolysis and for the detection of human factor X cleavage products after VLFXA hydrolysis. VLFXA cleaves the Arg52-Ile53 bond in the heavy chain of human factor X and the Arg226-Val227 bond in human factor IX precursor; VLFVA cleaves Arg1545-Ser1546 in factor V.

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.

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.

The complete amino acid sequence of lebetase is deduced from the nucleotide sequence of a cDNA clone isolated by screening a venomous gland c DNA library of Central Asian Vipera lebetina snake. The cDNA sequence with 2011 basepairs encodes an open reading frame of 478 amino acids which includes an 18 amino acid signal peptide, plus an 175 amino acid segment of zymogen-like propeptide, a mature protein of 204 amino acids, a spacer of 18 amino acids and a disintegrin-like peptide of 63 amino acids. The mature protein lebetase as isolated from the crude venom has the molecular weight of approximately 23.7 kD and, thus, lebetase as well as several other snake venom metalloproteinases is translated as a precursor protein, which may be processed posttranslationally. The lebetase proprotein has a “cysteine switch” motif (PKMCGV) similar to that involved in the activation of matrix metalloproteinase zymogens. The mature protein (residues 223-427) shows the strongest similarity with fibrolase(63% identity), fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. The metalloproteinase domain has a typical zinc-chelating sequence (HEXXHXXGXXH). In the disintegrin-like domain of protein, the RGD sequence is replaced by VGD.

Antibodies to two fractions of fibrinolytic enzyme in Vipera lebetina venom were produced by immunizing mice with chromatographically purified lebetase probes. The antibodies were isolated from mice blood by protein A affinity chromatography. The antibodies to both fractions reacted only with the fibrinolytic enzyme in V. lebetina venom as demonstrated by western immunoblotting. Immunodot assays, ELISA and western immunoblotting revealed that 11 snake venoms including species of Viperidae and Crotalidae but not Elapidae cross-react with lebetase antibodies to varying degrees. The molecular weights of cross-reacting components were detected and compared with the earlier published data.

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin–agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.

Human coagulation factor X is a serine protease zymogen, which circulates in blood as a two-chain molecule. A variety of factor X activators have been detected in snake venoms. About 15 activators have been isolated from Viperidae, Crotalidae and Elapidae venoms. Viperidae and Crotalidae venom activators are mainly metalloproteases. Only two factor X activators are characterized from Elapidae venoms, both belonging to serine proteases. Most thoroughly investigated snake venom factor X activators are from Vipera russellii (now renamed Daboia russellii) - RVV-X, and Vipera lebetina (now renamed Macrovipera lebetina) - VLFXA. RVV-X is a heterotrimeric metalloproteinase with a mammalian ADAM-like heavy chain and two lectin-like light chains. The crystal structure of RVV-X has recently been determined. VLFXA is the first factor X activator that was cloned and sequenced and its primary structure was deduced from the cDNA sequences. Both activators consist of a heavy chain and two C-type lectin–like light chains which are held together by disulfide bonds. Heavy chains of RVV-X and VLFXA contain metalloprotease, disintegrin-like and cysteine-rich domains. All chains of VLFXA and RVV-X are synthesized from different genes. The primary structures of factor X activating snake venom serine proteases are unknown up to now.

Nerve growth factor was isolated from the Vipera lebetina venom by a four-step procedure including gel filtration, ion exchange, heparin and hydrophobic chromatography. The purified protein is a glycosylated non-covalently bound homodimer with monomeric molecular mass of 14,380 Da. The cDNA encoding NGF is cloned and sequenced. The amino acid sequence translated from the cDNA comprises 117 or 119 amino acids depending on the N-terminus (truncated or not). The recombinant NGF (expressed in Escherichia coli) was used to prepare the anti-NGF antiserum. The antiserum interacted with the wild-type NGF and enabled to localize NGF during the purification procedure in parallel with MALDI-TOF analysis of tryptic peptides. The isolated NGF caused neurite outgrowth from PC12 cells in concentrations beginning from 2.5 ng/ml.

An arginine esterase was purified from the venom of Vipera lebetina by gel filtration on Sephadex G-100 and by affinity and DEAE-cellulose chromatography. The enzyme has a mol. wt of 52,500 and pI approximately 3. It is a glycoprotein containing 23% of neutral sugars, and has extremely high thermostability. The esterase activity is inhibited by diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF). The Km and kcat values are for alpha-N-benzoyl-L-arginine ethyl ester (BAEE) 7.7 x 10(-5) M and 43.8 sec-1, for p-tosyl-L-arginine methyl ester (TAME) 3.6 x 10(-4) M and 39.8 sec-1 (pH 8.5, 25 degrees C, and for alpha-N-benzoyl-DL-arginine-4-nitroanilide (BAPNA) 1.8 x 10(-4) M and 0.94 sec-1 (pH 8.3, 25 degrees C), respectively. Lysine esters are not hydrolyzed. The enzyme has weak caseinolytic activity and hydrolyzes glucagon at the sites Lys12-Tyr13, Arg17-Arg18 and Arg18-Ala19. In fibrinogen it cleaves B beta-chain first and later also the A alpha-chain.

We have previously demonstrated that the fibrinolytic enzyme lebetase is synthesized with disintegrin-like domain that is cleaved posttranslationally (Siigur et al., 1996). Now we isolated a heterodimeric disintegrin viplebedin-2 containing this disintegrin-like part from Vipera lebetina venom using size-exclusion chromatography on Sephadex G-100 sf and HPLC on C18 column. The molecular masses of viplebedin-2 and tryptic peptides from both chains of viplebedin-2 were determined by MALDI-TOF mass spectrometry. Using cDNA library of the venom gland of a single V. lebetina turanica snake the viplebedin-2 coding cDNAs were cloned and sequenced. Viplebedin-2 chains are synthesized from two different genes. One chain, containing VGD sequence in disintegrin loop, is synthesized as a disintegrin-like part of the PII-type metalloprotease, lebetase. The other chain, containing MLD sequence in disintegrin loop, is synthesized from the gene without metalloproteinase domain. Two polyadenylation signal sequences have been found in MLD sequence coding chain precursor cDNAs. Viplebedin-2 dose-dependently inhibited adhesion of platelets to immobilized collagen and inhibited collagen-induced platelet aggregation.

The l -amino acid oxidase from Vipera lebetina venom was purified to homogeneity using combination of size exclusion, ion exchange and hydrophobic chromatography. The monomeric molecular mass of the homodimeric enzyme is 60.9 kDa. The N-terminal and the tryptic peptides share high homology with other snake venom l -amino acid oxidases. The enzyme displays high specificity towards hydrophobic l -amino acids, the best substrates are l -Met, l -Trp, l -Leu followed by l -His, l -Phe, l -Arg and l -Ile. Six substrates—Gly, l -Ser, l -Thr, l -Pro, l -Cys, l -Asp—were not oxidized. The enzyme has antimicrobial activity inhibiting the growth of both Gram-negative and Gram-positive bacteria. V. lebetina LAAO dose-dependently inhibited platelet aggregation induced by ADP or collagen. In case of ADP-induced aggregation the inhibitory effect was more pronounced on the second wave of aggregation.

An l -amino acid oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venom l -amino acid oxidases. The purified protein catalyzed oxidative desamination of l -amino acids, the most specific substrate is l -Phe. The best substrates among the studied 20 amino acids were: l -Met, l -Leu, l -Phe, l -Ile, l -Arg and l -His. Five amino acids, l -Ser, l -Pro, Gly, l -Thr and l -Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 μM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.

A novel endothelial cell apoptosis inducing metalloprotease (VLAIP) was found in the snake venom of Vipera lebetina. This metalloprotease is a heterodimeric glycoprotein with molecular mass of about 106 kDa. The protease hydrolyzes azocasein, fibrinogen and oxidized insulin B-chain. The enzyme readily hydrolyzes the Aalpha-chain and more slowly Bbeta-chain of fibrinogen. VLAIP does not cleave fibrin. The complete amino acid sequences of the two different monomers of VLAIP are deduced from the nucleotide sequences of cDNAs encoding these proteins. The full-length cDNA sequences of the VLAIP-A and VLAIP-B encode open reading frames of 616 and 614 amino acids that include signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. VLAIP belongs to the metalloprotease/disintegrin family of reprolysins and has high identity with the proteins that induce apoptosis of endothelial cells. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes cell death. We demonstrated that VLAIP inhibits endothelial cell adhesion to extracellular matrix proteins: fibrinogen, fibronectin, vitronectin, collagen I, and collagen IV. The induction of apoptosis by VLAIP was shown by means of a typical DNA fragmentation pattern of apoptotic cells as well as by monitoring phosphatidylserine externalization using annexin V-FITC staining and flow cytometric analysis.

Amino acid sequences of two anticoagulant serine fibrinogenases - alpha- and beta-fibrinogenase (VLAF and VLBF) from Vipera lebetina venom have been deduced from the cDNA sequences encoding the enzymes. The mature protein sequences of 234 amino acids (VLAF) and 233 amino acids (VLBF) exhibit significant similarity with other snake venom serine proteinases. Both enzymes contain the catalytic triad His57, Asp102, Ser195, and twelve conserved cysteines forming six disulfide bridges. Unlike typical trypsin-like serine proteinases, they lack the third aspartate, Asp189 which is replaced by Gly189. VLBF is a typical representative of arginine esterases - beta-fibrinogenases. alpha-Fibrinogenase, VLAF, is unique among snake venom serine proteinases with homologous structure. Until now there is no evidence of the anticoagulant serine enzymes degrading fibrinogen alpha-chain only and lacking esterolytic activity.

Proteases play crucial role starting from fertilization until to cell death. Our studies of the two Viperidae venoms (Levantine viperVipera lebetina, Common viperVipera berus) have demonstrated the existence of biomedically important proteases, both coagulants and anticoagulants that may be useful as diagnostic tools or potential therapeutics. We showed that venoms of both snakes contain: (i) metalloproteases and serine proteases that degrade fibrinogen, but not fibrin; (ii) factor X activators (VLFXA, VBFXAE); (iii) bradykinin-releasing serine proteases. AdditionallyVipera lebetinasnake venom contains thrombolytic fibrin degrading metalloenzyme (lebetase), HUVEC cell apoptosis inducing metalloprotease (VLAIP), factor V activator (VLFVA), thermostable β-fibrinogenase and α-fibrinogenase which has no homolog among known serine proteases. We examined the activity of snake venom proteases against bradykinin, substance P, insulin B-chain and 6–10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α2-macroglobulin bait region and pregnancy zone protein (PZP). We used MALDI TOF mass spectrometry technique for the discovery and identification of peptides released by protease hydrolysis. The sensitive and quick MALDI-TOF mass spectrometry methodology allows us to obtain the primary information about the substrate specificity of different proteases against various peptides and proteins.

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6–9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly – factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly – factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that VLFXA cleaves the Arg52-Ile53 bond in the heavy chain of human factor X and the Arg226-Val227 bond in human factor IX precursor. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.

Two glycosylated serine fibrinogenases isolated from Vipera lebetina venom have homologous N-terminal sequences and antigenic determinants but can be clearly differentiated according to substrate specificity, glycosylation levels, molecular mass and fibrinogen degradation. alpha-Fibrinogenase has no homolog among known serine proteinases. It has N-terminal similarity with snake venom arginine esterases but does not hydrolyze the esters of arginine, lysine and tyrosine. The enzyme has strong proteolytic activity and degrades alpha-chain of fibrinogen altering its clottability by thrombin. beta-Fibrinogenase is a typical arginine esterase which hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen.

Four clones encoding homologous protein(ase)s were isolated from the Vipera lebetina (snake) venom gland cDNA library. One of them represented DNA encoding factor V activating enzyme ( Siigur et al., 1999 ), the other is homologous to VLFVA but has two principal discrepancies in the translated protein sequence in comparison with snake venom serine proteinase structures: in the active site triad Ser195 is replaced by Asn195 and His57 by Arg57. The third and the fourth clone represent combinations of the first two clones. The possibilities of generation of such clones via trans-splicing of the primary gene transcript, by exon shuffling or by unequal crossing-over on the genome level are discussed.

Snake venoms are known to contain a number of factors affecting blood coagulation. These venoms have been found to have potent effects on coagulation through both pro-and anticoagulant mechanisms (reviews: Kornalik, 1985, 1990; Seegers and Ouyang, 1979; Tu, 1977; Stocker, 1990a, b; Markland, 1991; Siigur and Siigur, 1992; Ouyang et al., 1992.

A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-Vgamma (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine ester substrates, such as benzoylarginine ethyl ester (BAEE), tosylarginine methyl ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70 degrees C does not alter the arginine esterase activity of the enzyme.

Lebetase, the fibrinolytic enzyme isolated from Vipera lebetina (Levantine viper) snake venom is a metalloenzyme that contains one mole of Zn2+ and one mole of Ca2+ per mole of protein. Lebetase is inhibited by dithiothreitol, suggesting that disulfide bonds are necessary for holding the structure. Vipera lebetina venom contains several isoforms of lebetase in the interval of pI 4.6–5.4. Two lebetase fractions I (pI of the main component 5.0) and II (pI of the main component 5.3) degrade fibrin and fibrinogen by hydrolysis of the alpha and beta chains. The molecular weights of the cleavage products produced by the two different lebetase fractions are identical. The metal ions, Cd2+, Cu2+, Co2+, inhibit fibrinolytic and caseinolytic activity of lebetase I and II. Using mass spectrometry we characterized differences in molecular masses of lebetase I and II (22719 Da and 22912 Da). Vipera lebetina venom from a single snake contains mainly one form of lebetase. Lebetase I is more stable at low pH than lebetase II. The lebetases I and II inhibit platelet aggregation induced by ADP in a dose-dependent manner.

An anticoagulant proteinase, lebetase, was isolated from the venom of Vipera lebetina by gel filtration and anion-exchange chromatography. The proteinase consists of a single polypeptide chain with a molecular weight of 23,700. Amino acid analysis indicates that lebetase contains 214 residues. It consists of several isoforms in the pH range 4.6-5.4, all having fibrinolytic activity. Lebetase hydrolyzes casein, fibrinogen and fibrin, and oxidized insulin B-chain in the positions Ala14-Leu15 and Tyr16-Leu17. Its fibrinolytic activity is inhibited by EDTA and lost upon heating at 70 degrees C for 10 min. The enzyme readily hydrolyzes the A alpha-chain and more slowly the B beta-chain of fibrinogen. In fibrin, the same chains are attacked. Thus, the enzyme is an A alpha, B beta-fibrinogenase. The fibrinolytic activity of lebetase is direct, without any plasminogen activation. E280 1% is 13.0 for lebetase. The enzyme showed low hemorrhagic activity.

M. Samel , E. Siigur and J. Siigur . Purification and characterization of two arginine ester hydrolases from Vipera berus berus (common viper) venom Toxicon 25, 379 – 388, 1987.—Two arginine ester hydrolases, designated EI and EII, consist of multiple molecular forms with pI values in the range 4.0–4.6 for EI and 3.3–3.9 for EII. Isoforms had identical molecular weights: 38,500 for EI and 41,000 for EII (SDS electrophoresis). The N -terminal amino acid for both enzymes was valine and their amino acid contents were very similar, with both containing carbohydrate. After treatment of EI and EII with neuraminidase both enzymes migrated identically in the electrofocusing system. Neither esterase hydrolyzed casein, α- N -benzoyl- dl -arginine- p -nitroanilide (BAPNA), yet both hydrolyzed α- N -benzoyl- l -arginine methylester (BAEE), p -tosyl- l -arginine methylester (TAME) and Pro-Phe-Arg-MCA. The esterase activities of the two enzymes were inhibited by organophosphorus inhibitors and benzamidine. The K m value for EI with BAEE was 3.3 × 10 −5 M, with TAME 3.0 × 10 −5 M, and for EII 2.7 × 10 −5 M (BAEE) and 5.9 × 10 −5 M (TAME). EII possessed kinin-releasing activity, as shown by the twitch response of an isolated rat uterus. The physiological role of EI is unknown. Neither esterase has thrombin-like or fibrinolytic activitives.

1. 1. Nerve growth factor from Vipera lebetina venom was purified by gel filtration and ion exchange chromatography steps. 2. 2. The NGF preparation obtained is a glycoprotein with weak arginine esterase activity. It hydrolyzes benzoylarginine ethyl ester (BAEE). 3. 3. Vipera lebetina NGF consists of multiple forms of protein with pI in the interval 9–10.5. All isoforms have identical molecular weights of 32,500.

A proteinase from the venom of Vipera lebetima was purified by chromatography on Sephadex G-100 and CM-cellulose. The purified proteinase was homogeneous on SDS-polyacrylamide gel electrophoresis and consisted of a single chain with molecular weight of 37 000±1500. The isoelectric point of the proteinase was over 10. The enzyme was active on casein but not on esters and amides of arginine. It split the oxidized insulin B-chain at the peptide bonds of Tyr 16 -Leu 17 , Phe 24 -Phe 25 and Phe 25 -Tyr 26 , and glucagon at the bonds Tyr 10 -Ser 11 , Leu 14 -Asp 15 and Leu 26 -Met 27 . The enzyme was inhibited by DFP and PMSF, and partially by soybean trypsin inhibitor, but not with EDTA.

By means of Sephadex G-100 column chromatography, Vipera berus berus venom has been separated into ten fractions which have been tested for the following enzyme activities: phosphomonoesterase, phosphodiesterase, 5′-nucleotidase, protease, arginine ester hydrolase, hyaluronidase, ribonuclease and phospholipase. Isoelectric focusing over a pH range of 3–10 revealed fractions with the following isoelectric points: arginine ester hydrolase, 4·0 and 4·5; phosphodiesterase, 4·0 and 6·3; proteases, 4·5 and 6·1; 5′-nucleotidase, 5·6; phospholipase, 5·1; 6·7 and 9·3. Phosphomonoesterase and hyaluronidase lost their activities during the electrofocusing procedure. Organophosphorus inhibitors and ethylenediaminetetra-acetic acid have been used to classify the proteases and the arginine ester hydrolase. The protease with the higher mol. wt and with a pI of 6·1 was a metalloenzyme while the protease with the lower mol. wt and with a pI of 4·5 as well as arginine ester hydrolase were serine esterases.

Our studies of Levantine viper venom have demonstrated that the venom is a rich source of biomedically important proteins and peptides. The venom contains metalloproteases: thrombolytic, fibrin-degrading lebetase, an endothelial cell apoptosis inducing metalloprotease (VLAIP), factor X activator (VLFXA); serine proteases: factor V activator, bradykinin-releasing serine proteases, β-fibrinogenase, α-fibrinogenase and chymotrypsin-like protease and different other enzymes such as phosphodiesterase, 5`-nucleotidase, ribonuclease, phospholipase A2s and L-amino acid oxidase. Among nonenzymatic components venom contains: nerve growth factor, vascular endothelial growth factor, disintegrins, C-type lectins. Here we report the isolation and characterization of proteins and peptides from Vipera lebetina venom using size exclusion, ion exchange, hydrophobic interaction and affinity chromatography, HPLC, UPLC and MALDI-TOF MS methods. N-terminal sequences and internal sequences of tryptic peptides of different proteins were determined using Edman sequencing and LC-ESI-MS/MS techniques. On the basis of fragmental sequences of proteins the oligonucleotides were designed and used as primers for cDNA cloning. Using cDNA library of the venom gland of a single snake the cDNAs coding proteins were cloned and sequenced. Protein sequences were deduced from cDNA sequences. The substrate specificity of venom proteases against insulin B-chain, bradykinin, substance P, and 6-10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α2-macroglobulin bait region and pregnancy zone protein were studied using MALDI-TOF mass spectrometry technique.

1. 1. Pure monoclonal antibodies to Vipera lebetina venom nerve growth factor have been isolated by affinity chromatography using CNBr-agarose bound antigen. 2. 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.