RESUMO

The myotoxic mechanism for PLA2-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA2-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA2-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA2-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA2-like toxins in general.

Assuntos

Ácidos Graxos/química , Simulação de Dinâmica Molecular , Fosfolipases A/química , Conformação Proteica , Multimerização Proteica , Animais , Bothrops/metabolismo , Biologia Computacional/métodos , Cristalografia por Raios X , Ácidos Graxos/metabolismo , Ligação de Hidrogênio , Ligantes , Fosfolipases A/metabolismo , Ligação Proteica

RESUMO

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.

Assuntos

Antivenenos/farmacologia , Bothrops/metabolismo , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/metabolismo , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Suramina/farmacologia , Animais , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/toxicidade , Cristalografia por Raios X , Masculino , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfolipases A/química , Fosfolipases A/toxicidade

RESUMO

The proteome of the venom of Micrurus nigrocinctus (Central American coral snake) was analyzed by a "venomics" approach. Nearly 50 venom peaks were resolved by RP-HPLC, revealing a complex protein composition. Comparative analyses of venoms from individual specimens revealed that such complexity is an intrinsic feature of this species, rather than the sum of variable individual patterns of simpler composition. Proteins related to eight distinct families were identified by MS/MS de novo peptide sequencing or N-terminal sequencing: phospholipase A(2) (PLA(2)), three-finger toxin (3FTx), l-amino acid oxidase, C-type lectin/lectin-like, metalloproteinase, serine proteinase, ohanin, and nucleotidase. PLA(2)s and 3FTxs are predominant, representing 48 and 38% of the venom proteins, respectively. Within 3FTxs, several isoforms of short-chain α-neurotoxins as well as muscarinic-like toxins and proteins with similarity to long-chain κ-2 bungarotoxin were identified. PLA(2)s are also highly diverse, and a toxicity screening showed that they mainly exert myotoxicity, although some are lethal and may contribute to the known presynaptic neurotoxicity of this venom. An antivenomic characterization of a therapeutic monospecific M. nigrocinctus equine antivenom revealed differences in immunorecognition of venom proteins that correlate with their molecular mass, with the weakest recognition observed toward 3FTxs.

Assuntos

Antivenenos/análise , Venenos Elapídicos/análise , Elapidae , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão/métodos , Venenos Elapídicos/genética , Venenos Elapídicos/toxicidade , Metaloproteases/química , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Neurotoxinas/análise , Neurotoxinas/genética , Fosfolipases A/química , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos

RESUMO

Phospholipases A(2) (PLA(2)s) are one of the main components of bothropic venoms; in addition to their phospholipid hydrolysis action, they are involved in a wide spectrum of pharmacological activities, including neurotoxicity, myotoxicity and cardiotoxicity. Caffeic acid is an inhibitor that is present in several plants and is employed for the treatment of ophidian envenomations in the folk medicine of many developing countries; as bothropic snake bites are not efficiently neutralized by conventional serum therapy, it may be useful as an antivenom. In this work, the cocrystallization and preliminary X-ray diffraction analysis of the Lys49-PLA(2) piratoxin I from Bothrops pirajai venom in the presence of the inhibitor caffeic acid (CA) are reported. The crystals diffracted X-rays to 1.65 Šresolution and the structure was solved by molecular-replacement techniques. The electron-density map unambiguously indicated the presence of three CA molecules that interact with the C-terminus of the protein. This is the first time a ligand has been observed bound to this region and is in agreement with various experiments previously reported in the literature.

Assuntos

Bothrops/metabolismo , Ácidos Cafeicos/metabolismo , Venenos de Crotalídeos/química , Fosfolipases A2 do Grupo II/química , Animais , Cristalização , Cristalografia por Raios X/métodos , Ligantes , Modelos Moleculares , Fosfolipases A/química , Fosfolipases A/metabolismo , Ligação Proteica

RESUMO

BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+) and in the presence of Mg(2+), Cd(2+) and Mn(2+) it was reduced in presence or absence of Ca(2+). Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.

Assuntos

Bothrops , Venenos de Crotalídeos/química , Neurotoxinas/química , Fosfolipases A/química , Sequência de Aminoácidos , Animais , Fracionamento Químico , Galinhas/fisiologia , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/farmacologia , Crotalus/metabolismo , Crotoxina/isolamento & purificação , Crotoxina/farmacologia , Cinética , Masculino , Camundongos , Dados de Sequência Molecular , Bloqueio Neuromuscular , Neurotoxinas/isolamento & purificação , Neurotoxinas/farmacologia , Fosfolipases A/isolamento & purificação , Fosfolipases A/farmacologia , Alinhamento de Sequência , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz

RESUMO

The patterns of myotoxicity induced in mice by crotoxin, crotoxin B and a Lys49 phospholipase A(2) (PLA(2)) homologue were compared. Lys49 PLA(2)-induced local myotoxicity is reflected by creatine kinase (CK) loss in injected gastrocnemius muscle, and by a profile of CK increase in plasma characterized by a rapid increment and drop after intramuscular injection, and by a lack of CK increase in plasma after intravenous injection. In contrast, crotoxin and crotoxin B, which induce local and systemic myotoxicity, provoked a more prolonged increment in plasma CK activity upon intramuscular injection, and induced increments in plasma CK after intravenous injection. The three toxins promoted a similar extent of local myotoxicity, assessed by the loss of CK in injected gastrocnemius. A method for the quantitative assessment of the ability of toxins to induce systemic myotoxicity is proposed, based on the estimation of the ratio between the area under the curve in the plasma CK activity (total myotoxicity) to the loss of CK in injected gastrocnemius (local myotoxicity). The highest ratio corresponded to crotoxin, and the lowest corresponded to Lys49 PLA(2), the former being a systemic myotoxin and the latter a local myotoxin. Neutralization by antivenoms also differed between the toxins: a drastic reduction in plasma CK, with very poor neutralization of local CK loss, was achieved in the case of crotoxin B when antivenom was injected intravenously, whereas no neutralization was achieved in the case of Lys49 PLA(2). When tested in undifferentiated myoblasts in culture, Lys49 PLA(2) induced cytotoxicity, whereas crotoxin and crotoxin B did not, evidencing that the latter are devoid of widespread cytolytic activity. Molecular modeling analysis showed that Lys49 PLA(2) has a conspicuous cationic face, which is likely to interact with diverse membranes. In contrast, crotoxin B, despite its overall basic pI, has a lower density of positively charged residues at this molecular region. It is suggested that Lys49 PLA(2)s homologues interact, through this cationic face, with many different cell types, thus lacking specificity for muscle cells. In contrast, crotoxin B has a more selective interaction with targets in the muscle cell membrane. This selectivity might be the basis for the ability of crotoxin and crotoxin B to induce systemic myotoxicity.

Assuntos

Crotoxina/toxicidade , Músculo Esquelético/efeitos dos fármacos , Fosfolipases A/toxicidade , Animais , Creatina Quinase/sangue , Creatina Quinase/metabolismo , Venenos de Crotalídeos/química , Crotalus/fisiologia , Crotoxina/química , Camundongos , Modelos Moleculares , Necrose/induzido quimicamente , Fosfolipases A/química , Conformação Proteica , Proteínas de Répteis/toxicidade , Fatores de Tempo

RESUMO

PrTX-I, a non-catalytic and myotoxic Lys49-PLA(2) from Bothrops pirajai venom has been crystallized alone and in complex with bromophenacyl bromide (BPB), alpha tocopherol and alpha tocopherol acetate inhibitors. These crystals have shown to diffract X-rays between 2.34 and 1.65 A resolution. All complexes crystals are isomorphous and belong to the space group P2(1) whereas native PrTX-I crystals belong to the P3(1)21.

Assuntos

Brometos/farmacologia , Venenos de Crotalídeos/química , Inibidores Enzimáticos/farmacologia , Lisina/química , Fosfolipases A/química , alfa-Tocoferol/farmacologia , Animais , Bothrops , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica

RESUMO

Zhaoermiatoxin, an Arg49 phospholipase A2 homologue from Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) venom is a novel member of the PLA2-homologue family that possesses an arginine residue at position 49, probably arising from a secondary Lys49-->Arg substitution that does not alter the catalytic inactivity towards phospholipids. Like other Lys49 PLA2 homologues, zhaoermiatoxin induces oedema and strong myonecrosis without detectable PLA2 catalytic activity. A single crystal with maximum dimensions of 0.2 x 0.2 x 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 A using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6(4), with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 A.

Assuntos

Arginina/química , Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Homologia Estrutural de Proteína , Trimeresurus , Substituição de Aminoácidos/genética , Animais , Venenos de Crotalídeos/genética , Cristalização , Masculino , Fosfolipases A/genética , Fosfolipases A2 , Trimeresurus/genética , Difração de Raios X

RESUMO

Some scorpion venom contain heterodimeric phospholipases A2. They were shown to be toxic to insects and to cause edema and/or hemolysis of mammalian erythrocytes. This manuscript describes the results of cDNA cloning of five different heterodimeric phospholipases from the venomous glands of the Mexican scorpion Anuroctonus phaiodactylus. The amino acid sequence deduced from the heterodimeric phospholipases open reading frames corresponds in each case to a different isoform. The nucleotide sequences corresponding to two of these genes were also obtained by directly sequencing genomic DNA. The cDNA isoforms show high similarity with the heterodimeric phospholipase Phaiodactylipin purified from the same scorpion. However, similar phospholipases were also found in scorpions from other species and the sequences available were used to construct a phylogenetic tree. In order to understand better the gene structure and phylogeny of these enzymes we analyzed their sequences and compared them with secretory phospholipases of other sources from groups I, II and III. The genomic DNA sequence of a similar phospholipase from bee venomous glands was also cloned. The information available on a Drosophila phospholipase was included in this analysis. The phospholipases of groups I and II contain a conserved exon-intron structure (four or five exons of the mature segment of the enzyme are separated by three or four introns). Also, the gene structure of the phospholipases from A. phaiodactylus and that of the bee venom, belonging to group III phospholipases, are interrupted by three introns. The mature peptide of the bee enzyme is a single polypeptide chain, coded by four exons, whereas those from the scorpion studied here although having four exons, showed the presence of two different polypeptides in its native state. The mature protein is processed after synthesis, producing the heterodimeric structure: a long and a short-peptide chain, linked by a disulfide bridge. The small subunit is the one coded by the fourth exon. The human phospholipase A2 and that of Drosophila, also classified into the group III phospholipases, have a considerably different exon-intron organization.

Assuntos

Fosfolipases A/genética , Filogenia , Venenos de Escorpião/enzimologia , Escorpiões/enzimologia , Análise de Sequência de DNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Dimerização , Isoenzimas/química , Isoenzimas/genética , Modelos Genéticos , Dados de Sequência Molecular , Fosfolipases A/química , Fosfolipases A2

RESUMO

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 A, and diffracted to 1.75 A resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 A, and diffracted to 2.6 A resolution. The crotapotin crystal diffracted to 2.3 A resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Assuntos

Crotoxina/química , Fosfolipases A/química , Cristalização , Cristalografia por Raios X , Dimerização , Fosfolipases A2 , Conformação Proteica

RESUMO

Scanning alanine mutagenesis has been used to study the structural determinants of several activities of bothropstoxin-I (BthTx-I), a lysine 49 Phospholipases A(2) from the venom of Bothrops jararacussu. A total of 31 mutants were generated in the interfacial recognition site and C-terminal loop regions of the protein. The effects of mutagenesis on the in vivo myotoxic activity, the cytolytic activity against cultured C2C12 myoblasts, the bactericidal activity, and the Ca(2+)-independent membrane damaging activity against liposome membranes were compared. Residues 116-119 and 122-125 in the C-terminal loop region are structural determinants for these activities, indicating that membrane permeabilization by the BthTx-I is an important general property in all the measured effects. The structural determinants of myotoxicity and myoblast membrane permeabilization are highly correlated, demonstrating that cultured C2C12 myoblasts are a good model for the myotoxic effect. However, comparison of the structural determinants for all activities revealed several differences in the structural determinants between the effects against myoblast and bacterial membranes, and further differences when compared to the liposome membrane damaging effect. These membrane dependent effects are interpreted to be the consequence of differences in the activation of the membrane bound form of the protein on biological and artificial membranes.

Assuntos

Alanina/metabolismo , Membrana Celular/metabolismo , Lisina/metabolismo , Membranas Artificiais , Mutagênese , Fosfolipases A/química , Fosfolipases A/metabolismo , Animais , Bothrops/metabolismo , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Creatina Quinase/sangue , Escherichia coli/efeitos dos fármacos , Lipossomos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Proteínas Mutantes/metabolismo , Fosfolipases A/farmacologia , Fosfolipases A2 , Estrutura Secundária de Proteína , Venenos de Serpentes/enzimologia , Venenos de Serpentes/farmacologia , Relação Estrutura-Atividade

RESUMO

BaTX PLA(2), a K49 phospholipase A(2) homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on mu-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The complete amino acid sequence of BaTX PLA(2) contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD(50) of BaTX was 7 mug/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 microM: 40+/-0.4 min and 0.07 microM: 35+/-0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA(2) family, which presents the typical bioactivities described for such proteins.

Assuntos

Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Fosfolipases A/toxicidade , Sequência de Aminoácidos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Técnicas In Vitro , Isoenzimas/química , Dose Letal Mediana , Lisina , Camundongos , Dados de Sequência Molecular , Peso Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Mioblastos Esqueléticos/efeitos dos fármacos , Necrose , Junção Neuromuscular/efeitos dos fármacos , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Conformação Proteica , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo

RESUMO

We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A2 from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios>10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios<8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios>10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant.

Assuntos

Fosfolipases A/química , Dodecilsulfato de Sódio/química , Espectrofotometria Ultravioleta/métodos , Animais , Bothrops , Venenos de Crotalídeos/química , Dimerização , Lisina/química , Modelos Moleculares , Conformação Proteica , Tensoativos/química

RESUMO

A new crotoxin B isoform PLA(2) (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using mu-Bondapack C-18 column analytic. The new crotoxin B isoform PLA(2) (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA(2) (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA(2) (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA(2) (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA(2)s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA(2)s. The new crotoxin B isoform PLA(2) (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA(2) (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA(2) (F6a) was clearly cytotoxic to these cells.

Assuntos

Crotoxina/química , Fosfolipases A/química , Sequência de Aminoácidos , Animais , Galinhas , Crotalus , Masculino , Camundongos , Dados de Sequência Molecular , Neurotoxinas/metabolismo , Nervo Frênico/metabolismo , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz

RESUMO

Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on micro-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry. The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA2, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA2 family, which presents the typical bioactivities described for such proteins.

Assuntos

Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Fosfolipases A/toxicidade , Sequência de Aminoácidos , Animais , Células Cultivadas , Fracionamento Químico , Galinhas , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/toxicidade , Crotalus , Diafragma/efeitos dos fármacos , Ponto Isoelétrico , Isoenzimas , Camundongos , Peso Molecular , Músculo Esquelético/patologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Necrose/patologia , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Nervo Frênico/efeitos dos fármacos , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz

RESUMO

Lys49 phospholipase A2 homologues are highly myotoxic and cause extensive tissue damage but do not display hydrolytic activity towards natural phospholipids. The binding of heparin, heparin derivatives and polyanionic compounds such as suramin result in partial inhibition (up to 60%) of the myotoxic effects due to a change in the overall charge of the interfacial surface. In vivo experiments demonstrate that polyethylene glycol inhibits more than 90% of the myotoxic effects without exhibiting secondary toxic effects. The crystal structure of bothropstoxin-I complexed with polyethylene glycol reveals that this inhibition is due to steric hindrance of the access to the PLA2-active site-like region. These two inhibitory pathways indicate the roles of the overall surface charge and free accessibility to the PLA2-active site-like region in the functioning of Lys49 phospholipases A2 homologues. Molecular dynamics simulations, small angle X-ray scattering and structural analysis indicate that the oligomeric states both in solution and in the crystalline states of Lys49 phospholipases A2 are principally mediated by hydrophobic contacts formed between the interfacial surfaces. These results provide the framework for the potential application of both clinically approved drugs for the treatment of Viperidae snakebites.

Assuntos

Venenos de Crotalídeos/toxicidade , Neurotoxinas/toxicidade , Fosfolipases A/toxicidade , Animais , Sítios de Ligação/efeitos dos fármacos , Bothrops , Creatina Quinase/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Cristalização , Relação Dose-Resposta a Droga , Antagonismo de Drogas , Fosfolipases A2 do Grupo II , Modelos Moleculares , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Necrose/induzido quimicamente , Neurotoxinas/química , Neurotoxinas/metabolismo , Fosfolipases A/química , Fosfolipases A/metabolismo , Fosfolipases A2 , Polietilenoglicóis/farmacologia , Estrutura Secundária de Proteína , Proteínas de Répteis , Espalhamento a Baixo Ângulo , Suramina/farmacologia , Propriedades de Superfície/efeitos dos fármacos , Raios X

RESUMO

The effects of two cationic synthetic peptides, derived from the C-terminal region of Lys49 phospholipase A2 homologues from snake venoms, upon various murine tumor cell lines (B16 melanoma, EMT6 mammary carcinoma, S-180 sarcoma, P3X myeloma, tEnd endothelial cells) were evaluated. The peptides are 13-mers derived from Agkistrodon piscivorus piscivorus Lys49 PLA2 (p-AppK: KKYKAYFKLKCKK) and Bothrops asper Lys49 myotoxin II (pEM-2[D]: KKWRWWLKALAKK), respectively, in the latter case with slight modifications and with all-D amino acids. All tumor cells tested were susceptible to the lytic action of the peptides. The susceptibility of tumor cell lines was not higher than that of C2C12 skeletal muscle myoblasts, utilized as a non-transformed cell line control. However, in a murine model of subcutaneous solid tumor growth of EMT6 mammary carcinoma, the intraperitoneal administration of pEM-2[D] caused a tumor mass reduction of 36% (p<0.05), which was of similar magnitude to that achieved by the administration of paclitaxel, an antitumor drug in clinical use. Thus, the C-terminal peptides of Lys49 phospholipase A2 homologues present antitumor effects that might be of interest in developing therapeutic strategies against cancer.

Assuntos

Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Venenos de Crotalídeos/química , Fosfolipases A2 do Grupo II , Injeções Intraperitoneais , Neoplasias Mamárias Experimentais/tratamento farmacológico , Membranas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fosfolipases A/química , Fosfolipases A2 , Proteínas de Répteis , Células Tumorais Cultivadas

RESUMO

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.

Assuntos

Bothrops/metabolismo , Fosfolipases A/metabolismo , Venenos de Serpentes/enzimologia , Sequência de Aminoácidos , Animais , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Carcinoma de Ehrlich/induzido quimicamente , Carcinoma de Ehrlich/enzimologia , Carcinoma de Ehrlich/patologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Ensaio de Imunoadsorção Enzimática , Hemoglobinas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Agregação Plaquetária/efeitos dos fármacos , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/farmacologia

RESUMO

A new phospholipase A(2) (PLA(2))-inhibitory protein was isolated from the plasma of Atropoides nummifer, a crotaline snake from Central America. This inhibitor was named AnMIP, given its ability to neutralize the activity of basic PLA(2) myotoxins of its own and related venoms. The cDNA of AnMIP was cloned and sequenced, showing that it belongs to the alpha group of phospholipase A(2) inhibitors (PLIs). AnMIP appears as a homotrimer in the native state, held together by non-covalent forces, with a subunit molecular mass of 22,247-22,301 and an isoelectric point of 4.1-4.7. This trimeric structure is the first observed in a PLIalpha from American crotaline snakes, previously reported only in Asian species. Sequencing, mass spectrometry, and analytical isoelectrofocusing indicated the existence of isoforms, as reported for other PLIalphas isolated from snake plasma. The inhibitory profile of AnMIP showed specificity towards group II PLA(2)s, either belonging to the catalytically-active (D49) or -inactive (K49) subtypes, exemplified in this study by Bothrops asper myotoxin I and A. nummifer myotoxin II, respectively. By phylogenetic analysis it was shown that AnMIP is closely related to CgMIP-II, previously isolated from the plasma of Cerrophidion godmani, showing 93% amino acid sequence identity.

Assuntos

Proteínas Sanguíneas/genética , Venenos de Crotalídeos/antagonistas & inibidores , Isoformas de Proteínas/genética , Viperidae/genética , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Clonagem Molecular , Fosfolipases A2 do Grupo II , Dados de Sequência Molecular , Fosfolipases A/química , Fosfolipases A/genética , Fosfolipases A2 , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Estrutura Quaternária de Proteína , Proteínas de Répteis , Homologia de Sequência de Aminoácidos

RESUMO

Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (æBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10æg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0 percent (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71 percent homology with bothropstoxin-II and 54 percent homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92 percent homology with Basp-III and 62 percent homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2.

Assuntos

Animais , Bothrops/metabolismo , Venenos de Crotalídeos , Fosfolipases A/química , Neurotoxinas/intoxicação