RESUMO
The isolation and characterization of individual snake venom components is important for a deeper understanding of the pathophysiology of envenomation and for improving the therapeutic procedures of patients. It also opens possibilities for the discovery of novel toxins that might be useful as tools for understanding cellular and molecular processes. The variable venom composition, toxicological and immunological properties of the common vipers (Vipera berus berus) have been reviewed. The combination of venom gland transcriptomics, bottom-up and top-down proteomics enabled comparison of common viper venom proteomes from multiple individuals. V. b. berus venom contains proteins and peptides belonging to 10-15 toxin families: snake venom metalloproteinase, phospholipases A2 (PLA2), snake venom serine proteinase, aspartic protease, L-amino acid oxidase (LAAO), hyaluronidase, 5'-nucleotidase, glutaminyl-peptide cyclotransferase, disintegrin, C-type lectin (snaclec), nerve growth factor, Kunitz type serine protease inhibitor, snake venom vascular endothelial growth factor, cysteine-rich secretory protein, bradykinin potentiating peptide, natriuretic peptides. PLA2 and LAAO from V. b. berus venom produce more pronounced cytotoxic effects in cancer cells than normal cells, via induction of apoptosis, cell cycle arrest and suppression of proliferation. Proteomic data of V. b. berus venoms from different parts of Russia and Slovakian Republic have been compared with analogous data for Vipera nikolskii venom. Proteomic studies demonstrated quantitative differences in the composition of V. b. berus venom from different geographical regions. Differences in the venom composition of V. berus were mainly driven by the age, sex, habitat and diet of the snakes. The venom variability of V. berus results in a loss of antivenom efficacy against snakebites. The effectiveness of antibodies is discussed. This review presents an overview with a special focus on different toxins that have been isolated and characterized from the venoms of V. b. berus. Their main biochemical properties and toxic actions are described.
RESUMO
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.
Assuntos
Venenos de Víboras/química , Venenos de Víboras/farmacologia , Viperidae , Animais , Peptídeos/química , Peptídeos/farmacologia , Proteoma/química , Proteoma/farmacologia , Especificidade da EspécieRESUMO
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 â¼120, 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.
Assuntos
Endonucleases/metabolismo , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Endonucleases/química , Reação em Cadeia da Polimerase , RNA/metabolismo , Homologia de Sequência de Aminoácidos , Venenos de Víboras/isolamento & purificação , ViperidaeRESUMO
5'-Nucleotidase (5'-NT) is widely represented in animal tissues (CD73) as well as in almost all snake venoms. In the present study, a 5'-NT isoform has been isolated from Vipera lebetina venom. The homodimeric isoform consists of monomers with molecular masses of 60 kDa. The enzyme is thermolabile and has pH optimum at 7.5. The 5'-NT activity is inhibited by metal ions Fe(3+), Cu(2+) and Zn(2+), enhanced by Mn(2+) while Mg(2+) and Ca(2+) have no remarkable effect. In addition to 120-kDa protein there are higher molecular forms of 5'-NT present in the V. lebetina venom. The cloning and sequencing of the 5'-NT coding cDNA resulted in 5'-truncated construct. MALDI-TOF and Orbitrap mass-spectrometry of the tryptic peptides confirmed the translated N-terminally truncated protein sequence concordance to the 5'-NT isolated from the venom. The isolated protein strongly inhibited ADP- or collagen-induced platelet aggregation.
Assuntos
5'-Nucleotidase/genética , Modelos Moleculares , Venenos de Víboras/enzimologia , Viperidae/genética , 5'-Nucleotidase/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia em Gel , Cromatografia por Troca Iônica , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Conformação Proteica , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Venenos de Víboras/toxicidade , Viperidae/metabolismoRESUMO
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.
Assuntos
Difosfato de Adenosina/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Estrutura Terciária de Proteína , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Plaquetas/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Diester Fosfórico Hidrolases/genética , Filogenia , Agregação Plaquetária , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
Secretory phospholipasesA(2) (sPLA(2)s) form a large family of structurally related enzymes widespread in nature. Herein, we studied the inhibitory effects of sPLA(2)s from Vipera lebetina (VLPLA(2)), Vipera berus berus (VBBPLA(2)), and Naja naja oxiana (NNOPLA(2)) venoms on (i) human platelets, (ii) four different bacterial strains (gram-negative Escherichia coli and Vibrio fischeri; gram-positive Staphylococcus aureus and Bacillus subtilis) and (iii) five types of cancer cells (PC-3, LNCaP, MCF-7, K-562 and B16-F10) in vitro. sPLA(2)s inhibited collagen-induced platelet aggregation: VBBPLA(2) IC(50) = 0.054, VLPLA(2) IC(50) = 0.072, NNOPLA(2) IC(50) = 0.814 µM. p-Bromophenacylbromide-inhibited sPLA(2) had no inhibitory action on platelets. 36.17 µM VBBPLA(2 )completely inhibited the growth of gram-positive Bacillus subtilis whereas no growth inhibition was observed towards gram-negative Escherichia coli. The inhibitory action of sPLA(2)s (~0.7 µM and ~7 µM) towards cancer cells depended on both venom and cell type. VBBPLA(2 )(7.2 µM) inhibited significantly the viability of K-562 cells and the cell death appeared apoptotic. The sPLA(2)s exhibited no inhibitory effect towards LNCaP cells and some effect (8%-20%) towards other cells. Thus, already sub-µM concentrations of sPLA(2)s inhibited collagen-induced platelet aggregation and from the current suite of studied svPLA(2)s and test cells, VBBPLA(2) was the most growth inhibitory towards Bacillus subtilis and K-562 cells.
Assuntos
Antibacterianos/farmacologia , Citotoxinas/farmacologia , Venenos Elapídicos/enzimologia , Fosfolipases A2/farmacologia , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/química , Citotoxinas/isolamento & purificação , Elapidae , Humanos , Camundongos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Agregação Plaquetária/efeitos dos fármacos , Alinhamento de Sequência , ViperidaeRESUMO
Three cancer cell lines, the human androgen independent prostate cancer PC-3, androgen dependent LNCaP prostate cancer and human chronic myeloid leukaemia cell line K-562, were treated with Sephadex G-100 sf fractions of Vipera lebetina venom and with HUVEC apoptosis inducing heterodimeric metalloproteinase (VLAIP) from the same venom. The venom was separated into nine fractions using size-exclusion chromatography on Sephadex G-100 sf. The effect of V. lebetina venom gel filtration fractions on the viability of studied cancer cells was different: high molecular mass fractions were the most effective on PC-3 cells. The viability of LNCaP cells was inhibited most strongly by the third fraction. The first and the second fractions contain different metalloproteinases including VLAIP that also most effectively reduced the viability of PC-3 cells. VLAIP decreased PC-3 cell viability in a concentration- and time-dependent manner but did not induce apoptosis as shown by DNA fragmentation assay. VLAIP induced changes in cell shape, rounding up and detachment. VLAIP inhibited the PC-3 cell adhesion to extracellular matrix proteins collagen I, fibronectin and vitronectin but not to fibrinogen. VLAIP had no significant effect on the viability of LNCaP and K-562 cells. VLAIP was also capable to inhibit ADP- and collagen-induced platelet aggregation dose-dependently. IC(50) was determined to be 1.89 µM and 0.94 µM, respectively.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Leucemia/tratamento farmacológico , Metaloproteases/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Venenos de Víboras/farmacologia , Animais , Antineoplásicos/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fracionamento Químico , Fragmentação do DNA , DNA de Neoplasias/análise , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Leucemia/patologia , Masculino , Metaloproteases/química , Dados de Sequência Molecular , Neoplasias da Próstata/patologia , Venenos de Víboras/química , Venenos de Víboras/enzimologia , Viperidae/fisiologiaRESUMO
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.
Assuntos
Fatores de Crescimento Neural/química , Proteínas de Répteis/química , Venenos de Serpentes/química , Animais , Antivenenos/química , Reações Cruzadas , Fatores de Crescimento Neural/isolamento & purificação , Proteínas de Répteis/isolamento & purificaçãoRESUMO
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 Bß-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.
Assuntos
Angiotensinas/metabolismo , Quimotripsina/química , Serina Proteases/química , Serina Proteases/metabolismo , Tirosina , Venenos de Víboras/enzimologia , Viperidae , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Estabilidade Enzimática , Hidrólise , Dados de Sequência Molecular , Alinhamento de Sequência , Serina Proteases/genética , Especificidade por SubstratoRESUMO
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.
Assuntos
Fatores de Crescimento Neural/isolamento & purificação , Venenos de Víboras/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Cromatografia em Gel , Primers do DNA , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Glicosilação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/toxicidade , Células PC12 , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidade , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Fosfolipases A2/química , Venenos de Víboras/enzimologia , Viperidae/metabolismo , Acetofenonas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Colágeno/farmacologia , DNA Complementar/química , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/farmacologia , Filogenia , Agregação Plaquetária/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Venenos de Víboras/química , Viperidae/genéticaRESUMO
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.
Assuntos
Desintegrinas/metabolismo , Viperidae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Complementar/metabolismo , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/isolamento & purificação , Humanos , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Venenos de Víboras/metabolismo , Venenos de Víboras/farmacologiaRESUMO
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.
Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Aminoácido Oxirredutases/farmacologia , Venenos Elapídicos/enzimologia , Elapidae , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacos , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
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 60.9kDa. 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.
Assuntos
Bactérias/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , L-Aminoácido Oxidase/isolamento & purificação , L-Aminoácido Oxidase/toxicidade , Venenos de Víboras/enzimologia , Viperidae , Sequência de Aminoácidos , Animais , Bactérias/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , L-Aminoácido Oxidase/química , Dados de Sequência Molecular , Peso Molecular , Agregação Plaquetária/efeitos dos fármacos , Alinhamento de Sequência , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Especificidade por SubstratoRESUMO
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 microM. 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.
Assuntos
Apoptose/efeitos dos fármacos , L-Aminoácido Oxidase/isolamento & purificação , L-Aminoácido Oxidase/metabolismo , Inibidores da Agregação Plaquetária/isolamento & purificação , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Fragmentação do DNA/efeitos dos fármacos , Células HeLa , Humanos , Cinética , Dados de Sequência Molecular , Peso Molecular , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
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).
Assuntos
DNA Complementar/biossíntese , Metaloendopeptidases/biossíntese , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Desintegrinas/química , Focalização Isoelétrica , Isoenzimas/biossíntese , Isoenzimas/genética , Metaloendopeptidases/genética , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , ViperidaeRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Metaloproteases/química , Venenos de Víboras/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Adesão Celular/efeitos dos fármacos , Células Endoteliais/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Humanos , Metaloproteases/metabolismo , Metaloproteases/farmacologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Venenos de Víboras/enzimologiaRESUMO
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.
Assuntos
Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Venenos de Víboras/biossíntese , Venenos de Víboras/genética , Viperidae/genética , Viperidae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Fator X/química , Fator X/metabolismo , Humanos , Técnicas In Vitro , Metaloendopeptidases/química , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Venenos de Víboras/químicaRESUMO
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 Ala(14)-Leu(15) and Tyr(16)-Leu(17) 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 Leu(3)-Thr(4) and Arg(5)-Ile(6). The fibrinogen peptide fragment Glu-Tyr-His-Thr-Glu-Lys-Leu-Val-Thr-Ser was hydrolysed at position Lys(6)-Leu(7).
Assuntos
Fator X/metabolismo , Fibrinogênio/metabolismo , Metaloproteases/metabolismo , Venenos de Víboras/enzimologia , Viperidae/metabolismo , Animais , Metaloproteases/isolamento & purificação , Peptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
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.