RESUMO
Mature Ts1, the main neurotoxin from Tityus serrulatus venom, has its C-terminal Cys amidated, while the isolated isoform of Ts1, named Ts1-G, keeps the non-amidated Gly residue at the C-terminal region, allowing the study of the comparative functional importance of amidation at the C-terminal between these two native toxins. Voltage dependent sodium current measurements showed that the affinity of Ts1-G for sodium channels is smaller than that of the mature Ts1, confirming the important role played by the C-terminal amidation in determining Ts1 activity.
Assuntos
Proteínas de Artrópodes/química , Proteínas de Insetos/química , Neurotoxinas/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/isolamento & purificação , Proteínas de Artrópodes/toxicidade , Glicemia/efeitos dos fármacos , Fracionamento Químico , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/toxicidade , Masculino , Camundongos Endogâmicos , Dados de Sequência Molecular , Neurotoxinas/isolamento & purificação , Neurotoxinas/toxicidade , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/toxicidade , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/toxicidade , Escorpiões , Alinhamento de SequênciaRESUMO
This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.
Assuntos
Venenos de Escorpião/química , Venenos de Escorpião/imunologia , Venenos de Escorpião/intoxicação , Animais , Western Blotting , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Hialuronoglucosaminidase/metabolismo , Soros Imunes/imunologia , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos C57BL , Peru , Fosfolipases A2/metabolismo , Proteólise , Coelhos , Ratos , Ratos Wistar , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, ß-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K(v) channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na(V) channels (Na(V)1.4; Na(V)1.5; Na(V)1.6; Na(V)1.8 and DmNa(V)1) and 12 different subtypes of K(V) channels (K(V)1.1 - K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K(V)1.2 and K(V)1.3 channels with an IC50 value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na(V) channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.
Assuntos
Bloqueadores dos Canais de Potássio/química , Canais de Potássio/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/isolamento & purificação , Venenos de Escorpião/isolamento & purificação , Escorpiões , Análise de Sequência de ProteínaRESUMO
The aim of this work was to evaluate the hematological changes induced by Tityus serrulatus venom (TsV). Blood of Wistar rats was collected 0.5, 2, 6 and 24 h after i.p. injection of TsV (0.5 mg/kg) or saline (controls). Two additional groups were injected with 0.67 mg/kg and 0.25 mg/kg of TsV and the blood was collected after 0.5 and 2 h, respectively. The results showed an increase on hematocrit (Ht), red blood cells (RBC) count, hemoglobin concentration (Hb), albumin and total protein, mainly 2-6 h after envenoming. Increase in serum activities of amylase, creatine kinase and aspartate aminotransferase were also observed, indicating tecidual damages. Hyperglycemia was observed at all times analyzed, as a consequence of catecholamine release. No significant changes were detected in the urea, [Na(+)] and [Ca(2+)], but an increase of [Mg(2+)], [K(+)] and conductivity was observed. TsV induced a reduction of erythrocytes osmotic fragility as consequence of dehydration and increase in plasma electrolytes concentration, as evidenced by its higher conductivity. This study demonstrated that TsV is able to induce severe hematological changes, that appear within the first hours after envenoming, justifying the seeking of medical attention as soon as possible to avoid worsening of clinical symptoms.
Assuntos
Venenos de Escorpião/toxicidade , Escorpiões/química , Albuminas/metabolismo , Animais , Sangue/efeitos dos fármacos , Proteínas Sanguíneas/metabolismo , Contagem de Eritrócitos , Hematócrito , Hemoglobinas/metabolismo , Pressão Osmótica , Ratos , Ratos Wistar , Fatores de Tempo , Testes de ToxicidadeRESUMO
Scorpion stings are a public health problem in Brazil, with most incidents involving the species Tityus serrulatus. Some T. serrulatus toxins may act as immunogens for the production of a specific anti-venom, but many of the component toxins remain poorly characterized. Here, we describe the immunological characteristics of the toxin Ts1 (also known as TsVII and Ts-gamma) and evaluate production of neutralizing antibodies against the crude venom of T. serrulatus. Recombinant Ts1 with one copy (Ts1(1)) or two copies in tandem (Ts1(2)) was expressed in BL21 (DE3) cells. Rabbits and mice were immunized with the recombinant proteins (inclusion bodies) and then tested for production of neutralizing antibodies. Neutralization assays showed that anti-Ts1(1) and anti-Ts1(2) protected animals challenged with T. serrulatus crude venom and native Ts1. Thus, Ts1 could be used in a mixed "cocktail" of immunogens for T. serrulatus anti-venom production.
Assuntos
Antivenenos/biossíntese , Proteínas de Insetos/imunologia , Venenos de Escorpião/antagonistas & inibidores , Animais , Formação de Anticorpos , Antivenenos/imunologia , Antivenenos/isolamento & purificação , Sequência de Bases , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/genética , Dose Letal Mediana , Masculino , Camundongos , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Venenos de Escorpião/química , Venenos de Escorpião/genética , Venenos de Escorpião/imunologia , Venenos de Escorpião/toxicidadeRESUMO
Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A(2) (PLA(2)s). Asp49-PLA(2)s are catalytically active, whereas Lys49-PLA(2)s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA(2) (CB), is a PLA(2)-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA(2)s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA(2)s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Crotoxina/toxicidade , Inibidores Enzimáticos/toxicidade , Músculo Esquelético/efeitos dos fármacos , Fosfolipases A/antagonistas & inibidores , Animais , Dicroísmo Circular/métodos , Creatina Quinase/metabolismo , Crotoxina/análise , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/patologia , Inibidores Enzimáticos/análise , Membro Posterior , Masculino , Camundongos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Fosfolipases A/análise , Fosfolipases A/classificação , Isoformas de Proteínas , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
This paper describes a brief study on the crotoxin mechanism of action, regarding the transport of GABA and L-glutamate in rats cortico-cerebral synaptosomes and in heterologous systems, such as COS-7 cells expressing gabaergic transporters, and C6 glioma cells and Xenopus oocytes expressing glutamatergic transporters. Crotoxin concentrations over 1 µM caused an inhibitory effect of ³H-L-glutamate and ³H-GABA, and reversibly inhibited L-glutamate uptake by C6 glioma cells. When COS-7 cells were assayed, no inhibition of the ³H-GABA transport could be evidenced. Crotoxin kept its inhibitory effect on neurotransmitters uptake even when Ca2+ ions were removed from the medium, therefore, independently of its PLA2 activity. In addition, high concentrations (2 mM) of BPB did not avoid the action of crotoxin on the neurotransmitters uptake. Crotoxin also inhibited ³H-L-glutamate, independently on Na+ channel blockade by TTX. In addition, an evaluation of the lactic dehydrogenase activity indicated that uptake inhibition does not involve a hydrolytic action of crotoxin upon the membrane. We may also suggest that crotoxin acts, at least partially, altering the electrogenic equilibrium, as evidenced by confocal microscopy, when a fluorescent probe was used to verify cell permeability on C6 glioma cells in presence of crotoxin.
RESUMO
This paper describes a brief study on the crotoxin mechanism of action, regarding the transport of GABA and L-glutamate in rats cortico-cerebral synaptosomes and in heterologous systems, such as COS-7 cells expressing gabaergic transporters, and C6 glioma cells and Xenopus oocytes expressing glutamatergic transporters. Crotoxin concentrations over 1 µM caused an inhibitory effect of ³H-L-glutamate and ³H-GABA, and reversibly inhibited L-glutamate uptake by C6 glioma cells. When COS-7 cells were assayed, no inhibition of the ³H-GABA transport could be evidenced. Crotoxin kept its inhibitory effect on neurotransmitters uptake even when Ca2+ ions were removed from the medium, therefore, independently of its PLA2 activity. In addition, high concentrations (2 mM) of BPB did not avoid the action of crotoxin on the neurotransmitters uptake. Crotoxin also inhibited ³H-L-glutamate, independently on Na+ channel blockade by TTX. In addition, an evaluation of the lactic dehydrogenase activity indicated that uptake inhibition does not involve a hydrolytic action of crotoxin upon the membrane. We may also suggest that crotoxin acts, at least partially, altering the electrogenic equilibrium, as evidenced by confocal microscopy, when a fluorescent probe was used to verify cell permeability on C6 glioma cells in presence of crotoxin.
Assuntos
Animais , Masculino , Ratos , GABAérgicos , Crotoxina , Glutamatos , Neurotoxinas , Venenos de Crotalídeos/farmacologia , Sistema NervosoRESUMO
Anti-bothropic complex (ABC) was isolated from the serum of the South American opossum (Didelphis albiventris) by single-step affinity chromatography using a Sepharose-immobilized metalloprotease (BaP1) from Bothrops asper as the binding protein. Biochemical characterization of ABC showed the presence of two glycosylated subunits of 43 and 45 kDa, respectively, with an isoelectric point < 4. The two subunits were separated by ion-exchange HPLC. The N-terminal sequences of both subunits (LKAMDPTPXLWIETESP, where X is Arg-9 and Pro-9, respectively) showed a high degree of identity with other serum inhibitors isolated from different marsupials. Functional studies pointed out that ABC inhibits the hemorrhagic and proteolytic activities on fibrin, fibrinogen, and casein induced by the metalloproteases BaP1 and BaH4 isolated from B. asper venom. In addition to the anti-hemorrhagic and anti-proteolytic activities, ABC also showed anti-myotoxic, anti-lethal, and anti-edematogenic effects against myotoxic phospholipases A(2) isolated from the same venom. Moreover, it had inhibitory effects on the phospholipase A(2) activity of the crude venom as well as the isolated venom phospholipases A(2).
Assuntos
Proteínas Sanguíneas/farmacologia , Proteínas de Transporte/farmacologia , Venenos de Crotalídeos/antagonistas & inibidores , Metaloendopeptidases/antagonistas & inibidores , Neurotoxinas/antagonistas & inibidores , Gambás/sangue , Fosfolipases A/antagonistas & inibidores , Aminoácidos/química , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Bothrops , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Venenos de Crotalídeos/química , Venenos de Crotalídeos/enzimologia , Fosfolipases A2 do Grupo II , Hemorragia/prevenção & controle , Proteínas de Répteis , Análise de Sequência de ProteínaRESUMO
The purification procedure of a hyaluronidase from Tityus serrulatus scorpion venom is described. It involves basically an ion-exchange chromatography on CM-cellulose at pH 7.8 followed by a rechromatography of the active fraction on the same column at pH 4.7. The optima pH and temperature for maximum activity of the isolated enzyme was 6.0 and 40 degrees C, respectively. Its K(M) was 69.7 microg/ml at 37 degrees C and its specific activity was 19,900+/-1,730 turbidity reducing units (TRU)/mg against 845+/-88TRU/mg for the whole desiccated venom, representing a 23- to 24-fold purification range. The hyaluronidase activity of the purified protein (51kDa) was inhibited by some flavonoid compounds. This article also showed that T. serrulatus hyaluronidase affected on the activity of the venom's major toxin, tityustoxin-I (TsTX-I or Ts1), as reflected by alterations in the serum levels of creatine kinase (CK), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) following injection of TsTX-I, in the presence or absence of hyaluronidase.