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 MatrizRESUMO
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-AtividadeRESUMO
A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.
Assuntos
Bothrops/genética , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/isolamento & purificação , Células Endoteliais/metabolismo , Epoprostenol/metabolismo , Fosfolipases A/genética , Fosfolipases A/isolamento & purificação , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/farmacologia , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Epoprostenol/biossíntese , Feminino , Fosfolipases A2 do Grupo II , Fosfolipases A2 do Grupo IV , Humanos , Masculino , Dados de Sequência Molecular , Fosfolipases A/metabolismo , Fosfolipases A/farmacologia , Fosfolipases A2 , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Proteínas de Répteis , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom. Previous work of our group demonstrated that this toxin or its phospholipase A(2) subunit inhibits macrophage spreading and phagocytosis. The phagocytic activity of macrophages is controlled by the rearrangement of actin cytoskeleton and activity of the small Rho GTPases. The effect of crotoxin and its subunit on actin reorganization and tyrosine phosphorylation in rat peritoneal macrophages, during phagocytosis of opsonized zymosan, was presently investigated. The crude venom was used as positive control. In addition, the effect of crotoxin on the activity of Rho and Rac1 small GTPases was examined. Transmission electron studies showed that the venom or crotoxin decreased the extent of spread cells and increased microprojections often extended from macrophage surface. Immunocytochemical assays demosntrated that the venom or toxins increased F-actin content in the cytoplasm of these cells, but induced a marked decrease of phosphotyrosine. These effects were abolished by treatment with zileuton, a 5-lipoxygenase inhibitor. Furthermore, crotoxin decreased membrane-associated RhoA and Rac1 in translocation assays. The present results indicate that the crotalid venom and crotoxin are able to induce cytoskeleton rearrangement in macrophages. This effect is associated with inhibition of tyrosine phosphorylation and of the activity of proteins involved in intracellular signalling pathways important for the complete phagocytic activity of these cells.
Assuntos
Actinas/metabolismo , Crotoxina/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Tirosina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Crotalus/fisiologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/metabolismo , Masculino , Fosfolipases A/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Venenos de Serpentes/farmacologiaRESUMO
The whole venom of Lachesis muta muta is preponderantly neurotoxic but moderately myotoxic on the chick biventer cervicis preparation (BCp). We have now examined these toxic activities of a basic phospholipase A(2), LmTX-I, isolated from the whole venom. LmTX-I caused a significant concentration-dependent neuromuscular blockade in the BCp. The time to produce 50% neuromuscular blockade was 14.7+/-0.75 min (30 microg/ml), 23.6+/-0.9 min (10 microg/ml), 34+/-1.7 min (2.5 microg/ml) and 39.2+/-3.6 min (1 microg/ml), (n=5/concentration; p<0.05). Complete blockade with all tested concentrations was not accompanied by inhibition of the response to ACh. At the highest concentration, LmTX-I (30 microg/ml) significantly reduced contractures elicited by exogenous KCl (20mM), increased the release of creatine kinase (1542.5+/-183.9 IU/L vs 442.7+/-39.8 IU/L for controls after 120 min, p<0.05), and induced the appearance of degenerating muscle fibers ( approximately 15%). Quantification of myonecrosis indicated 14.8+/-0.8 and 2.0+/-0.4%, with 30 and 10 microg/mlvenom concentration, respectively, against 1.07+/-0.4% for control preparations. The findings indicate that the basic PLA(2) present on venom from L. m. muta (LmTX-I) possesses a dominant neurotoxic action on isolated chick nerve-muscle preparations, whereas myotoxicity was mainly observed at the highest concentration used (30 microg/ml). These effects of LmTX-I closely reproduce the effects of the whole venom of L. m. muta in chick neuromuscular preparations.
Assuntos
Venenos de Crotalídeos/enzimologia , Fosfolipases A/isolamento & purificação , Fosfolipases A/farmacologia , Viperidae/fisiologia , Acetilcolina , Animais , Galinhas , Venenos de Crotalídeos/química , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Bloqueadores Neuromusculares/química , Bloqueadores Neuromusculares/isolamento & purificação , Bloqueadores Neuromusculares/farmacologia , Fosfolipases A/química , Fosfolipases A2 , Cloreto de PotássioRESUMO
MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)s or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA(2) homologues and, together with additional strategies, supports the concept of the presence of others "bioactive sites" distinct from the catalytic site in snake venom myotoxic PLA(2)s.
Assuntos
Bothrops , Fosfolipases A/farmacologia , Anidridos Acéticos/química , Acetofenonas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Creatina Quinase/sangue , Brometo de Cianogênio/química , Edema/induzido quimicamente , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Humanos , Células Jurkat , Leishmania/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/farmacologia , Nitrobenzenos/química , Fosfolipases A/química , Fosfolipases A/genética , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/patogenicidade , Análise de Sequência de DNARESUMO
Ophidian accidents caused by the subspecies Crotalus durissus are responsible for high morbity and mortality rates. Acute renal failure is a common complication observed in these accidents. The aim of the present study was to investigate the renal effects promoted by the venom of C. d. collilineatus and its fractions, crotoxin and phospholipase A2. C. d. collilineatus (Cdc; 30 microg mL(-1)), crotoxin (CTX; 10 microg mL(-1)) and phospholipase A2 (PLA2; 10 microg mL(-1)) were tested in isolated rat kidney. The first 30 min of each experiment were used as an internal control and Cdc or its fractions, CTX and PLA2 were added to the system after this period. All experiments lasted 120 min. The venom of Cdc decreased perfusion pressure (PP; control120 = 110.3 +/- 3.69 mmHg; Cdc120 = 96.7+/-8.1 mmHg), renal vascular resistance (RVR; control120 = 6.42+/-0.78 mmHg mL g(-1) min(-1); Cdc120 = 4.8+/-0.56 mmHg/mL g(-1) min(-1)), urinary flow (UF; control120 = 0.19+/-0.03 mL g(-1) min(-1); Cdc120 = 0.12 +/- 0.01 mL g(-1) min(-1)), and glomerular filtration rate (GFR; control120 = 0.79 +/- 0.07 mL g(-1) min(-1); Cdc120 = 0.53 +/- 0.09 mL g(-1) min(-1)), but had no effect on the percent of sodium tubular transport (%TNa+), percent of chloride tubular transport (%TK+) and percent of potassium tubular transport (%TCl-). CTX and PLA2 reduced the GFR, while UF, PP and RVR remained stable during the full 120 min of perfusion. Crotoxin administration also diminished the %TK+ (control120 = 69.94 +/- 6.49; CTX120 = 33.28 +/- 4.78) and %TCl- (control120 = 79.53 +/- 2.67; CTX120 = 64.62 +/- 6.93). PLA2 reduced the %TK+, but exerted no effect on the %TNa+ or on that of TCl-. In conclusion, the C. d. collilineatus venom altered the renal functional parameters evaluated. We suggest that crotoxin and phospholipase A2 were involved in this process, since the renal effects observed would be due to the synergistic action of the components of the venom.
Assuntos
Venenos de Crotalídeos/farmacologia , Crotalus , Rim/efeitos dos fármacos , Animais , Venenos de Crotalídeos/administração & dosagem , Crotoxina/administração & dosagem , Crotoxina/farmacologia , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Rim/fisiologia , Masculino , Fosfolipases A/administração & dosagem , Fosfolipases A/farmacologia , Fosfolipases A2 , Ratos , Ratos Wistar , Resistência Vascular/efeitos dos fármacosRESUMO
The crystal structure of an acidic phospholipase A(2) isolated from Bothrops jararacussu venom (BthA-I) chemically modified with p-bromophenacyl bromide (BPB) has been determined at 1.85 Angstroms resolution. The catalytic, platelet-aggregation inhibition, anticoagulant and hypotensive activities of BthA-I are abolished by ligand binding. Electron-density maps permitted unambiguous identification of inhibitor covalently bound to His48 in the substrate-binding cleft. The BthA-I-BPB complex contains three structural regions that are modified after inhibitor binding: the Ca(2+)-binding loop, beta-wing and C-terminal regions. Comparison of BthA-I-BPB with two other BPB-inhibited PLA(2) structures suggests that in the absence of Na(+) ions at the Ca(2+)-binding loop, this loop and other regions of the PLA(2)s undergo structural changes. The BthA-I-BPB structure reveals a novel oligomeric conformation. This conformation is more energetically and conformationally stable than the native structure and the abolition of pharmacological activities by the ligand may be related to the oligomeric structural changes. A residue of the ;pancreatic' loop (Lys69), which is usually attributed as providing the anticoagulant effect, is in the dimeric interface of BthA-I-BPB, leading to a new hypothesis regarding the abolition of this activity by BPB.
Assuntos
Acetofenonas/química , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/química , Animais , Anticoagulantes/química , Anti-Hipertensivos/química , Sítios de Ligação/efeitos dos fármacos , Bothrops , Cristalização , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Fosfolipases A/farmacologia , Inibidores da Agregação Plaquetária/química , Conformação Proteica , Estrutura Quaternária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Venenos de Víboras/enzimologiaRESUMO
A new PLA2 (F16) was purified from Crotalus durissus terrificus venom by molecular exclusion chromatography followed by analytical reverse phase HPLC. The PLA2 (14.86 kDa by MALDI-TOF mass spectrometry) had an amino acid sequence of SLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGRRRPKDATDRCCFVHDCCYEKVTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNGYMFYPDSRCRGPSETC, and showed highly conserved Ca2+-binding and catalytic sites. F16 showed allosteric behavior with 10 mM Ca2+ and had temperature and pH optima of 25 degrees C and 7.9, respectively. F16 (10 microg/ml) produced neuromuscular blockade in chick biventer cervicis preparations in the absence and presence of crotapotin, indicating that crotapotin was not essential for neuromuscular action in this preparation. In contrast, in mouse phrenic nerve-diaphragm preparations, the neuromuscular blockade produced by the same concentration of toxin was dependent on crotapotin. Pre-incubation with heparin markedly reduced the neurotoxicity of F16. These results show that the biochemical and structural properties of F16 are similar to those of the PLA2 isoforms F15 and F17, but that the neurotoxicity and the requirement for crotapotin to form the crotoxin complex varies according to the neuromuscular preparation.
Assuntos
Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Fosfolipases A/farmacologia , Sequência de Aminoácidos , Animais , Galinhas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Crotalus , Diafragma/efeitos dos fármacos , Masculino , Camundongos , Dados de Sequência Molecular , Junção Neuromuscular/efeitos dos fármacos , Fosfolipases A/isolamento & purificação , Fosfolipases A/metabolismo , Fosfolipases A2 , Nervo Frênico/efeitos dos fármacos , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We have isolated a new phospholipase A2 (MiDCA1) from the venom of the coral snake Micrurus dumerilii carinicauda. This toxin, which had a molecular mass of 15,552Da, shared high sequence homology with the PLA2 toxins MICNI A and B from Micrurus nigrocinctus venom (77.7% and 73.1%, respectively). In chick biventer cervicis preparations, MiDCA1 produced concentration- and time-dependent neuromuscular blockade that reached 100% after 120 min (2.4 microM, n = 6); contractures to exogenously applied carbachol (8 microM) and KCl (13 mM) were still seen after complete blockade. In mouse phrenic-nerve diaphragm preparations, MiDCA1 (2.4 microM; n = 6) caused triphasic changes followed by partial neuromuscular blockade. Intracellular recordings of end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) from mouse diaphragm preparations showed that MiDCA1 increased the quantal content by 386+/-12% after 10 min (n = 14; p<0.05) and caused a triphasic change in the frequency of MEPPs. MiDCA1 also decreased the resting membrane potential, an effect that was prevented by tetrodotoxin and/or low extracellular calcium, but not by d-tubocurarine. The toxin increased the amplitude of mouse sciatic-nerve compound action potentials by 30+/-9% (0.6 microM; p<0.05). Potassium currents elicited in freshly dissociated dorsal root ganglia neurones were blocked by 31+/-1% (n = 4; p<0.05) in the presence of 2.4 microM MiDCA1. These results show that MiDCA1 is a new presynaptic phospholipase A2 that produces neuromuscular blockade in vertebrate nerve-muscle preparations. The triphasic effects seen in mammalian preparations and the facilitatory response were probably caused mainly by the activation of sodium channels, complemented by the blockade of nerve terminal potassium channels. The inability of d-turocurarine to prevent the depolarization by MiDCA1 indicated that cholinergic nicotinic receptors were not involved in this phenomenon.
Assuntos
Venenos Elapídicos/enzimologia , Elapidae , Fosfolipases A/química , Fosfolipases A/farmacologia , Sequência de Aminoácidos , Animais , Galinhas , Diafragma/inervação , Venenos Elapídicos/farmacologia , Masculino , Camundongos , Dados de Sequência Molecular , Fosfolipases A/toxicidade , Fosfolipases A2 , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA2 isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to study the effect of flavonoids on PLA2. We observed that a treatment of PLA2 with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA2 possess a second pharmacological site which does not affect or depend on the enzymatic activity.
Assuntos
Crotalus , Flavonoides/farmacologia , Fosfolipases A/química , Fosfolipases A/metabolismo , Venenos de Serpentes/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Galinhas , Dicroísmo Circular , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/tratamento farmacológico , Flavonoides/química , Bactérias Gram-Positivas/efeitos dos fármacos , Masculino , Fosfolipases A/farmacologia , Fosfolipases A2 , Ratos , Ratos Wistar , Xanthomonas/efeitos dos fármacosRESUMO
The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A(2), and myotoxin II (MT-II), a catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen peroxide (H(2)O(2)) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse peritoneal macrophages at concentrations higher than 25 microg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcgamma, complement, mannose and beta-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and beta-glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H(2)O(2) by thioglycollate-elicited macrophages, MT-III being the most potent stimulator. MT-II induced the release of H(2)O(2) only at a concentration of 3.2 microg/ml (130% increment) while MT-III induced this effect at all concentrations tested (0.5-2.5 microg/ml; average of 206% increment). It is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages up regulating phagocytosis, mainly via mannose and beta-glucan receptors, and the respiratory burst.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fosfolipases A/farmacologia , Animais , Candida albicans , Proteínas do Sistema Complemento/fisiologia , Fosfolipases A2 do Grupo II , Peróxido de Hidrogênio/metabolismo , Fragmentos Fc das Imunoglobulinas/fisiologia , Técnicas In Vitro , Indicadores e Reagentes , Macrófagos/metabolismo , Masculino , Manose/fisiologia , Camundongos , Fagocitose/efeitos dos fármacos , Receptores Imunológicos/fisiologia , Proteínas de Répteis , Ovinos , ZimosanRESUMO
The ability of PLA2 and crotapotin, isolated from Crotalus durissus collilineatus rattlesnake venom, to stimulate insulin secretion from isolated rat islets was examined. PLA2 and crotapotin stimulated insulin secretion at 2.8 mmol/L glucose, whereas at a high glucose concentrations (16.7 mmol/L) only PLA2 stimulated secretion. Nifedipine (10 micromol/L) did not alter the ability of PLA2 to increase insulin secretion stimulated by a depolarizing concentration of K+ (30 mmol/L). PLA2 did not affect 14CO2 production but significantly increased the efflux of arachidonic acid from isolated islets. These results indicate that PLA2-stimulated secretion is not dependent on an additional influx of Ca2+ through L-type Ca(2+)-channels but rather is associated with arachidonic acid formation in pancreatic islets.
Assuntos
Venenos de Crotalídeos/química , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Fosfolipases A/farmacologia , Animais , Ácido Araquidônico/metabolismo , Venenos de Crotalídeos/farmacologia , Crotoxina/farmacologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Ratos , Ratos WistarRESUMO
The venoms of Micrurus lemniscatus carvalhoi, Micrurus frontalis frontalis, Micrurus surinamensis surinamensis and Micrurus nigrocinctus nigrocinctus were assayed for biological activities. Although showing similar liposome disrupting and myotoxic activities, M. frontalis frontalis and M. nigrocinctus nigrocinctus displayed higher anticoagulant and phospholipase A2 (PLA2) activities. The latter induced a higher edema response within 30 min. Both venoms were the most toxic as well. In the isolated chick biventer cervicis preparation, M. lemniscatus carvalhoi venom blocked the indirectly elicited twitch-tension response (85+/-0.6% inhibition after a 15 min incubation at 5 microg of venom/mL) and the response to acetylcholine (ACh; 55 or 110 microM), without affecting the response to KCl (13.4 mM). In mouse phrenic nerve-diaphragm preparation, the venom (5 microg/mL) produced a complete inhibition of the indirectly elicited contractile response after 50 min incubation and did not affect the contractions elicited by direct stimulation. M. lemniscatus carvalhoi inhibited 3H-L-glutamate uptake in brain synaptosomes in a Ca2+-, but not time, dependent manner. The replacement of Ca2+ by Sr2+ and ethylene glycol-bis(beta-aminoethyl ether) (EGTA), or alkylation of the venom with p-bromophenacyl bromide (BPB), inhibited 3H-L-glutamate uptake. M. lemniscatus carvalhoi venom cross-reacted with postsynaptic alpha-neurotoxins short-chain (antineurotoxin-II) and long-chain (antibungarotoxin) antibodies. It also cross-reacted with antimyotoxic PLA2 antibodies from M. nigrocinctus nigrocinctus (antinigroxin). Our results point to the need of catalytic activity for these venoms to exert their neurotoxic activity efficiently and to their components as attractive tools for the study of molecular targets on cell membranes.
Assuntos
Venenos Elapídicos/enzimologia , Venenos Elapídicos/farmacologia , Elapidae/fisiologia , Animais , Anticorpos/imunologia , Bioensaio , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Venenos Elapídicos/imunologia , Ácido Glutâmico/metabolismo , Lipossomos/metabolismo , Camundongos , Músculos/efeitos dos fármacos , Neurotoxinas/imunologia , Neurotoxinas/farmacologia , Fosfolipases A/metabolismo , Fosfolipases A/farmacologia , Fosfolipases A2 , Nervo Frênico/efeitos dos fármacosRESUMO
Snakebites are a relevant public health problem in Central and South America. Snake bite envenomations cause intense pain, not relieved by anti-venom. The fangs of many species are short, causing subcutaneous injection. Fangs of larger species inflict subcutaneous or intramuscular envenomation. To understand pain induced by subcutaneous venom, this study examined spinal mechanisms involved in pain-enhancing effects of subcutaneous Lys49 and Asp49 secretory phospholipase-A(2) (sPLA2), two components of Bothrops asper snake venom showing highly different enzymatic activities. Unilateral intraplantar sPLA2-Lys49 (catalytically inactive) or sPLA2-Asp49 (catalytically active) into rat hindpaws each induced mechanical hyperalgesia (Randall-Selitto test), whereas only catalytically active sPLA2-Asp49 caused mechanical allodynia (von Frey test). Effects induced by both sPLA2s were inhibited by intrathecal fluorocitrate, a reversible glial metabolic inhibitor. In support, immunohistochemical analysis revealed activation of dorsal horn astrocytes and microglia after intraplantar injection of either sPLA2. Spinal proinflammatory cytokines, nitric oxide, and prostanoids each appear to be involved in the pain-enhancing effects of these sPLA2s. Blockade of interleukin-1 (IL1) inhibited hyperalgesia induced by both sPLA2s, while leaving allodynia unaffected. Blockade of tumor necrosis factor reduced responses to sPLA2-Asp49. An inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7-NI), inhibited hyperalgesia induced by both sPLA2s, without interfering with allodynia induced by sPLA2-Asp49. On the other hand, L-N(6)-(1-iminoethyl)lysine (L-NI), an inhibitor of the inducible nitric oxide synthase, did not alter any sPLA2-induced effect. Lastly, celecoxib, an inhibitor of cyclooxygenase-2, attenuated sPLA2 actions. These data provide the first evidence of spinal mediators involved in pain facilitation induced by subcutaneous venoms.
Assuntos
Venenos de Crotalídeos/farmacologia , Hiperalgesia/fisiopatologia , Limiar da Dor/fisiologia , Fosfolipases A/farmacologia , Medula Espinal/fisiologia , Animais , Anticorpos/farmacologia , Biomarcadores , Citratos/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Citocinas/metabolismo , Fosfolipases A2 do Grupo II , Injeções Subcutâneas , Interleucina-1/imunologia , Interleucina-1/metabolismo , Masculino , Neuroglia/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Prostaglandinas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas de Répteis , Medula Espinal/citologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III--AY145836) is related to an acidic PLA2 sharing similarity, within the range 55-81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Bothrops/genética , Venenos de Crotalídeos/enzimologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/farmacologia , Fosfolipases A/genética , Fosfolipases A/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antineoplásicos/química , Sequência de Bases , Bioensaio , Linhagem Celular Tumoral , Clonagem Molecular , DNA Complementar/genética , Precursores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Fosfolipases A2 do Grupo II , Humanos , Camundongos , Dados de Sequência Molecular , Fosfolipases A/química , Fosfolipases A2 , Filogenia , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas de Répteis , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/efeitos dos fármacosRESUMO
BthTx-I (bothropstoxin-I) is a myotoxic Lys49-PLA2 (phospholipase A2 with Lys49) isolated from Bothrops jararacussu venom, which damages liposome membranes by a Ca2+-independent mechanism. The highly conserved Phe5/Ala102/Phe106 motif in the hydrophobic substrate-binding site of the Asp49-PLA2s is substituted by Leu5/Val102/Leu106 in the Lys49-PLA2s. The Leu5/Val102/Leu106 triad in BthTx-I was sequentially mutated via all single- and double-mutant combinations to the Phe5/Ala102/Phe106 mutant. All mutants were expressed as inclusion bodies in Escherichia coli, and the thermal stability (Tm), together with the myotoxic and Ca2+-independent membrane-damaging activities of the recombinant proteins, were evaluated. The far-UV CD profiles of the native, wild-type recombinant and the L106F (Leu106-->Phe) and L5F/F102A/L106F mutant proteins were identical. The L5F, V102A, L5F/V102A and V102A/L106F mutants showed distorted far-UV CD profiles; however, only the L5F and L5F/V102A mutants showed significant decreases in Tm. Alterations in the far-UV CD spectra correlated with decreased myotoxicity and protein-induced release of a liposome-entrapped marker. However, the V102A/L106F and L5F/V102A/L106F mutants, which presented high myotoxic activities, showed significantly reduced membrane-damaging activity. This demonstrates that the topology of the substrate-binding region of BthTx-I has a direct effect on the Ca2+-independent membrane damage, and implies that substrate binding retains an important role in this process.
Assuntos
Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Lisina/química , Fosfolipases A/química , Animais , Sítios de Ligação , Bothrops/metabolismo , Dicroísmo Circular/métodos , Venenos de Crotalídeos/química , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/farmacologia , Cristalografia por Raios X/métodos , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Lipossomos/química , Lipossomos/metabolismo , Lisina/genética , Lisina/farmacologia , Mutagênese Sítio-Dirigida/genética , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/farmacologia , Fosfolipases A/genética , Fosfolipases A/farmacologia , Fosfolipases A2 , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas de Répteis , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Venenos de Serpentes/farmacologia , Especificidade por SubstratoRESUMO
Snakebites constitute a serious public health problem in Central and South America, where species of the lancehead pit vipers (genus Bothrops) cause the majority of accidents. Bothrops envenomations are very painful, and this effect is not neutralized by antivenom treatment. Two variants of secretory phospholipases A2 (sPLA2), corresponding to Asp49 and Lys49 PLA2s, have been isolated from Bothrops asper venom. These sPLA2s induce hyperalgesia in rats following subcutaneous injection. However, venom in natural Bothrops bites is frequently delivered intramuscularly, thereby potentially reaching peripheral nerve bundles. Thus, the present series of experiments tested whether these sPLA2s could exert pain-enhancing effects following administration around healthy sciatic nerve. Both were found to produce mechanical allodynia ipsilateral to the injection site; no thermal hyperalgesia was observed. As no prior study has examined potential spinal mechanisms underlying sPLA2 actions, a series of anatomical and pharmacological studies were performed. These demonstrated that both sPLA2s produce activation of dorsal horn astrocytes and microglia that is more prominent ipsilateral to the site of injection. As proinflammatory cytokines and nitric oxide have each been previously implicated in spinally mediated pain facilitation, the effect of pharmacological blockade of these substances was tested. The results demonstrate that mechanical allodynia induced by both sPLA2s is blocked by interleukin-1 receptor antagonist, anti-rat interleukin-6 neutralizing antibody, the anti-inflammatory cytokine interleukin-10, and a nitric oxide synthesis inhibitor (L-NAME). As a variety of immune cells also produce and release sPLA2s during inflammatory states, the data may have general implications for the understanding of inflammatory pain.
Assuntos
Venenos de Crotalídeos/farmacologia , Citocinas/metabolismo , Neuroglia/fisiologia , Óxido Nítrico/metabolismo , Fosfolipases A/farmacologia , Ciática , Animais , Anticorpos/farmacologia , Biomarcadores , Inibidores Enzimáticos/farmacologia , Temperatura Alta , Hiperalgesia/induzido quimicamente , Hiperalgesia/imunologia , Hiperalgesia/metabolismo , Interleucina-10/farmacologia , Interleucina-6/imunologia , Vértebras Lombares , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Pressão , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-1/antagonistas & inibidores , Ciática/induzido quimicamente , Ciática/imunologia , Ciática/metabolismo , Medula Espinal/citologiaRESUMO
Phaiodactylipin was purified from the venom of the scorpion Anuroctonus phaiodactylus. It is the first protein to be purified from a scorpion of the family Iuridae and has a molecular mass of 19 172 atomic mass units. The mature protein is composed of two subunits, the large one consisting of 108 amino acid residues, whereas the small subunit has only 18 residues, and the structure is stabilized by five disulfide bridges. The heterodimer is expressed from a single message containing 769 base pairs and a signal peptide with 16 and/or 25 amino acid residues. During maturation an internal hexapeptide is excised. There are three putative sites of N-glycosylation, one of which is situated in the small subunit region. The carbohydrate composition of this site was determined by mass spectrometry analysis and was found to contain three hexoses, two N-acetyl-hexoses and two deoxyhexoses. The protein has a calcium dependent phospholipase A(2) type of activity. It is lethal to arthropods (insects and isopods), but not toxic to mammals, using doses up to 20 microg per 20 g mouse body weight. For crickets, a dose of 5 microg per animal is lethal; however, when injected into mice it is capable of causing only muscular inflammation, without rupture of the basal membrane of cells. It has a direct hemolytic effect in human erythrocytes and retards the coagulation time of blood. It is an unusual phospholipase A(2), with only 36% and 50% amino acid sequence identities to the closest known phospholipases, imperatoxin I and phospholipin, respectively. Identities with bee and Heloderma venom phospholipase are only in the order of 28%.
Assuntos
Fosfolipases A/química , Fosfolipases A/farmacologia , Venenos de Escorpião/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/química , Cálcio/metabolismo , Carboidratos/análise , Carboidratos/química , DNA Complementar/genética , Dimerização , Eritrócitos/efeitos dos fármacos , Glicosilação , Hemólise/efeitos dos fármacos , Membro Posterior , Humanos , Inflamação/induzido quimicamente , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfolipases A/genética , Fosfolipases A/metabolismo , Fosfolipídeos/metabolismo , Filogenia , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
ACL myotoxin (ACLMT) is a Lys49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus. The aim of this work was to study the effect of ACLMT on water transport in the toad bladder. Water flow through the membrane was measured gravimetrically in bag preparations of the bladder. ACLMT (20 nM) increased the baseline water flow and partially inhibited arginine-vasopressin (AVP), 8-chlorophenylthio-cAMP (8-CPT-cAMP) and forskolin-stimulated water flow. The effect of ACLMT on baseline or AVP-stimulated water flow was prevented by lanthanum (0.1 mM) indicating that the effect of ACLMT on water transport may be mediated through an increase in intracellular calcium. The effect of ACLMT on baseline water flow was also prevented by nifedipine (0.1 mM) indicating the participation of exogenous calcium in this effect. Carbachol (0.1 mM) has been shown to enhance baseline water flow while inhibiting AVP-stimulated water flow. The effects of ACLMT and carbachol on baseline water flow and AVP-stimulated water flow were not additive, suggesting that both agents alter water transport by a similar mechanism. Indomethacin (10 microM) reduced the effect of ACLMT on forskolin-stimulated water flow, suggesting an increase in prostaglandin biosynthesis. These results suggest that the effects of ACLMT on water transport may be mediated by increasing intracellular calcium and stimulation prostaglandin biosynthesis.