RESUMO
Crotoxin (Crtx), the main toxin in the venom of Crotalus durissus terrificus snake, is a heterodimer with a basic subunit, CB, and an acidic subunit, CA. CB is a phospholipase A2 that depends on CA to specifically bind to the cell membrane. This toxin acts in the central nervous system (CNS) causing chronic seizure effects and other cytotoxic effects. Here, we report its action on glutamate release in rat cerebral cortex synaptosomes. Aiming at a better understanding of the mechanism of action of Crtx, calcium channel blockers were used and internalization studies were performed in cerebellar granule neurons. Our results show that Crtx induces calcium-dependent glutamate release via N and P/Q calcium channels. In addition, the CB subunit of Crtx is shown to be internalized. This internalization does not depend on the presence of CA subunit neither on the PLA2 activity of CB. A correlation between CB internalization and glutamate release remains to be established.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/metabolismo , Córtex Cerebral/efeitos dos fármacos , Venenos de Crotalídeos/química , Crotalus , Crotoxina/farmacologia , Sinaptossomos/efeitos dos fármacos , Animais , Agonistas dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/química , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/metabolismo , Venenos de Crotalídeos/enzimologia , Crotoxina/antagonistas & inibidores , Crotoxina/metabolismo , Ácido Glutâmico/metabolismo , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Fosfolipases A2 do Grupo II/metabolismo , Fosfolipases A2 do Grupo II/farmacologia , Masculino , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Subunidades Proteicas/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos Wistar , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/metabolismo , Proteínas de Répteis/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single-step affinity chromatography in Sephadex® G-50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α-chain) and two minor components (ß-chain and γ-chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA-like lectin. In comparison with the average molecular mass of α-chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D-glucose, D-mannose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH-sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses.
Assuntos
Canavalia/química , Lectinas de Plantas/isolamento & purificação , Subunidades Proteicas/isolamento & purificação , Sementes/química , Sequência de Aminoácidos , Animais , Artemia/efeitos dos fármacos , Artemia/fisiologia , Cromatografia de Afinidade , Dextranos , Edema/induzido quimicamente , Edema/imunologia , Edema/patologia , Eletroforese em Gel de Poliacrilamida , Fetuínas/química , Hemaglutinação/efeitos dos fármacos , Membro Posterior , Concentração de Íons de Hidrogênio , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Masculino , Dados de Sequência Molecular , Peso Molecular , Monossacarídeos/química , Ovalbumina/química , Lectinas de Plantas/farmacologia , Estabilidade Proteica , Subunidades Proteicas/farmacologia , Ratos , Ratos WistarRESUMO
In this study an in vitro model was developed with the aim of investigating the modulatory effect of cholera toxin (CT) and its counterpart the heat labile toxin of Escherichia coli (LT) on TNF-alpha release induced by murine macrophages and primary human monocytes. Previous studies have demonstrated that the enzymatic activity of CT and LT molecules can inhibit TNF-alpha release by macrophages. The results obtained in this study showed that CT and LT are both, in a dose dependent manner, able either to induce or inhibit TNF-alpha release by murine macrophages and primary human monocytes. The results also showed that recombinant B subunits of CT and LT in the absence of their A subunit induce high levels of TNF-alpha release by macrophages and, in addition, increase the level of TNF-alpha release induced by LPS. The ability of both B subunits (CTB and LTB) in inducing TNF-alpha release by macrophages is not related to the level of LPS contamination, since direct measurements of LPS made in the samples employed in this study showed only traces of LPS (3.4 x 10(-8) EU/ml) which is in our system does not induce TNF-alpha release by macrophages. In contrast to the results obtained with the B subunits, incubation of cells with the A subunit of CT (CTA) inhibit TNF-alpha release induced by native CT, native LT, recombinant LTB and LPS. This inhibitory effect must be related to the activity of the A subunit since viability tests performed in terms of metabolic rate demonstrated that high concentrations of CTA are not toxic to the cells. The data presented herein demonstrate that the A subunits of CT and LT have an inhibitory effect on TNF-alpha release in macrophages, whereas their B subunits have a stimulatory effect on TNF-alpha. The results also suggest that the dose dependent bi-modal effect of native CT and native LT on TNF-alpha release by macrophages is a result of the combined effect of their individual A and B subunits.
Assuntos
Toxinas Bacterianas/farmacologia , Toxina da Cólera/farmacologia , Enterotoxinas/farmacologia , Proteínas de Escherichia coli/farmacologia , Escherichia coli/metabolismo , Macrófagos/efeitos dos fármacos , Subunidades Proteicas/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/farmacologiaRESUMO
Crotoxin (Ctx) is a potent neurotoxin of the venom of Crotalus durissus terrificus (the South American rattlesnake). Ctx is a heterodimer composed of CB, a toxic PLA(2) subunit, and CA, a non-toxic and non-enzymatic subunit, that potentiates the neurotoxicity of CB in vivo. The deleterious action of Ctx upon C. d. terrificus snakes themselves is known to be prevented by a PLA(2) inhibitor (CNF) present in their blood serum. CNF acts by replacing CA in Ctx, thus forming a new stable complex CNF-CB. This complex no longer interacts with the target receptor (TR) to deliver CB to cause its lethal effect. Furthermore, CNF-CB seems to be reminiscent of the interaction Ctx-TR at the pre-synaptic site. In the present work, the binding competition between rat brain synaptosomes (TR) and CNF for Ctx was investigated. Radiolabeled Ctx, made of CA and one isoform of CB (CA-(125)ICB(2)), was used as ligand. The competition by unlabeled Ctx was taken as a reference. The potency of CNF as a competitor was evaluated under different incubation conditions with varying time scale addition of reagents (CA-(125)ICB(2), synaptosomes and CA-CB(2) or CNF). CNF was able to inhibit the binding of the toxin to synaptosomes as well as to partially displace the toxin already bound to its membrane target. The mechanisms of competition involved were discussed and a previous schematic model of interactions between Ctx, TR and CNF was updated.
Assuntos
Crotalus , Crotoxina/antagonistas & inibidores , Glicoproteínas/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Proteínas de Répteis/metabolismo , Sinaptossomos/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacologia , Encéfalo/citologia , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/metabolismo , Crotoxina/química , Crotoxina/metabolismo , Glicoproteínas/química , Glicoproteínas/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Subunidades Proteicas/farmacologia , Ratos , Receptores de Superfície Celular/metabolismo , Proteínas de Répteis/química , Proteínas de Répteis/farmacologia , Sinaptossomos/química , Sinaptossomos/efeitos dos fármacosRESUMO
Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.