RESUMO
Every year in Mexico, around 300,000 people suffer from accidents related to scorpion stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose venom characterization is described here. From this venom, a total of 114 components were found using chromatographic separation and mass spectrometry analysis. The most abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da respectively, similar to other known K+ and Na+-channel specific scorpion peptides. Using intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these peptides using heterologously expressed human Na+-channels revealed a typical ß-toxin effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3 among other ß-toxins from Centruroides scorpions. Peptide Co52 segregates among peptides of unknown defined functions.
Assuntos
Venenos de Escorpião/química , Escorpiões , Animais , Humanos , Espectrometria de Massas , México , Camundongos , Peptídeos/química , Picadas de EscorpiãoRESUMO
A peptide (Cn29) from the venom of the scorpion Centruroides noxius (about 2% of the soluble venom) was purified and its primary and three-dimensional structures were determined. The peptide contains 27 amino acids with primary sequence: LCLSCRGGDYDCRVKGTCENGKCVCGS. The peptide is tightly packed by three disulfide linkages formed between C2-C23, C5-C18 and C12-C25. Since the native peptide was obtained in limited amounts, the full synthetic peptide was prepared using the standard F-moc-based solid phase synthesis method of Merrifield. The native and synthetic peptides were shown to be identical by sequencing, HPLC separation and mass spectrometry. The solution structure of the peptide solved from NMR data shows that it consists of a well-defined N-terminal region without regular secondary structure extending from Leu 1 to Asp 9, followed by a short helical fragment from Tyr10 to Val14 and two short ß strands (Thr17-Glu19 and Lys22-Val24). The primary and tertiary structures of Cn29 are different from all other scorpion peptides described in the literature. Transcriptome analysis of RNA obtained from C. noxius confirmed the expression of a gene coding for Cn29 in its venom gland. Initial experiments were conducted to identify its possible function: lethality tests in mice and insects as well as ion-channel binding using in vitro electrophysiological assays. None of the physiological or biological tests displayed any activity for this peptide, which at present is considered to be another orphan peptide found in scorpion venoms. The peptide is thus the first example of a novel structural component present in scorpion venoms.
Assuntos
Peptídeos/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Gryllidae , Camundongos , Modelos Moleculares , Peptídeos/isolamento & purificação , Escorpiões , Análise de Sequência de Proteína , Testes de ToxicidadeRESUMO
The number and types of venom components that affect ion-channel function are reviewed. These are the most important venom components responsible for human intoxication, deserving medical attention, often requiring the use of specific anti-venoms. Special emphasis is given to peptides that recognize Na(+)-, K(+)- and Ca(++)-channels of excitable cells. Knowledge generated by direct isolation of peptides from venom and components deduced from cloned genes, whose amino acid sequences are deposited into databanks are nowadays in the order of 1.5 thousands, out of an estimate biodiversity closed to 300,000. Here the diversity of components is briefly reviewed with mention to specific references. Structural characteristic are discussed with examples taken from published work. The principal mechanisms of action of the three different types of peptides are also reviewed. Na(+)-channel specific venom components usually are modifier of the open and closing kinetic mechanisms of the ion-channels, whereas peptides affecting K(+)-channels are normally pore blocking agents. The Ryanodine Ca(++)-channel specific peptides are known for causing sub-conducting stages of the channels conductance and some were shown to be able to internalize penetrating inside the muscle cells.
Assuntos
Canais Iônicos/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Modelos Moleculares , Venenos de Escorpião/classificação , Relação Estrutura-AtividadeRESUMO
Ergtoxin 1 (ErgTx1) is a 42 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius Hoffmann, capable of blocking specifically human potassium channels of the ether-á-go-go-related gene family (hERG). This peptide binds to a partially overlapping site on the channel outer mouth, in which residues of the S5-P linker are critically involved. Here we describe results of site directed mutagenesis of the ErgTx1 gene and its heterologous expression in Escherichia coli. The recombinant products show the fundamental role played by methionine in position 35 (Met35) of the primary structure. Naturally oxidized Met35 decreases by three orders of magnitude the affinity of the peptide for the hERG1 channels. This result is quite relevant, because it shows two possible situations: either Met35 is involved in the proper folding of the molecule or it plays a direct role in the interaction with the channel, i.e., constitutes part of the interacting surfaces. These two situations were evaluated by preparing heterologously expressed ErgTx1 gene and a mutant containing alanine in position 35. Additionally circular dichroism measurements of both native and recombinant peptides were performed. The electrophysiological recordings and the structural values obtained by optical measurements, strongly support the idea that Met35 is indeed a key residue on the interacting surfaces of the toxin with the channels.
Assuntos
Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Animais , Células CHO , Dicroísmo Circular , Cricetinae , Cricetulus , Eletrofisiologia , Feminino , Concentração Inibidora 50 , Oócitos/metabolismo , Técnicas de Patch-Clamp , Estrutura Secundária de Proteína , Venenos de Escorpião/genética , Xenopus laevisRESUMO
A new antimicrobial peptide, hadrurin, was isolated from the venom of the Mexican scorpion Hadrurus aztecus, by gel filtration on a Sephadex G-50 column, followed by high performance liquid chromatography. It is a basic peptide composed of 41 amino-acid residues with a molecular mass of 4436 Da, and contains no cysteines. A model of the three-dimensional folding of hadrurin is compatible with that of an amphipatic molecule with two alpha-helical segments. Hadrurin demonstrates antimicrobial activity at low micromolar concentration, inhibiting the growth of bacteria such as: Salmonella thyphi, Klebsiella pneumoniae, Enterococcus cloacae, Pseudomonas aeruginosa, Escherichia coli and Serratia marscences. It also shows cytolytic activity when tested in human erythrocytes. Hadrurin and two analogs (C-terminal amidated, and all D-enantiomer) were chemically synthesized. They were used to study the possible molecular mechanism of action by testing their ability to dissipate the diffusion potential of liposomes of different compositions. The results obtained indicate that there are no specific receptor molecules for the action of hadrurin, and the most probable mechanism is through a membrane destabilization activity. It is surmised that hadrurin is used by the scorpion as both an attack and defense element against its prey and putative invasive microorganisms. It is a unique peptide among all known antimicrobial peptides described, only partially similar to the N-terminal segment of gaegurin 4 and brevinin 2e, isolated from frog skin. It would certainly be a model molecule for studying new antibiotic activities and peptide-lipid interactions.
Assuntos
Antibacterianos/química , Hemólise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Enterococcus/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , México , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Conformação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Salmonella typhi/efeitos dos fármacos , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/farmacologia , Escorpiões , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serratia marcescens/efeitos dos fármacosRESUMO
The Na+-channel-affecting toxin Cn2 represents the major and one of the most toxic components of the venom of the Mexican scorpion Centruroides noxius Hoffmann. A monoclonal antibody BCF2 raised against Cn2 has been shown previously to be able to neutralize the toxic effect of Cn2 and of the whole venom of C. noxius. In the present study the epitope was mapped to a surface region comprising the N- and C-terminal segments of Cn2, using continuous and discontinuous synthetic peptides, designed on the basis of the sequence and a three-dimensional model of Cn2. The study of peptides of varying length resulted in the identification of segments 5-14 and 56-65 containing residues essential for recognition by BCF2. The peptide (abbreviated SP7) with the highest affinity to BCF2 (IC50 = 5.1 microM) was a synthetic heterodimer comprising the amino acid sequence from position 3-15 (amidated) of Cn2, bridged by disulfide to peptide from position 54-66, acetylated and amidated. Similar affinity was found with peptide SP1 [heterodimer comprising residues 1-14 (amidated) of Cn2, bridged with synthetic peptide 52-66 (acetylated)]. SP1 and SP7 were used to induce anti-peptide antibodies in mouse and rabbit. Both peptides were highly immunogenic. The sera obtained were able to recognize Cn2 and to neutralize Cn2 in vitro. The most efficient protection (8.3 microgram Cn2 neutralized per mL of serum) was induced by rabbit anti-SP1 serum.
Assuntos
Venenos de Escorpião/imunologia , Escorpiões/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Mapeamento de Epitopos , Feminino , Humanos , Imunização , Técnicas In Vitro , Dose Letal Mediana , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Conformação Proteica , Coelhos , Venenos de Escorpião/genética , Venenos de Escorpião/toxicidade , Escorpiões/genética , Escorpiões/patogenicidade , Canais de Sódio/efeitos dos fármacosRESUMO
A novel peptide called Noxiustoxin 2 (NTX2) was purified from the venom of the scorpion Centruroides noxius and characterized chemically and functionally. It is composed of 38 amino acid residues linked by three disulfide bridges and its primary structure is 61% identical to that of Noxiustoxin (NTX). It is not toxic to mice (using up to 200 micrograms/20 g mouse weight) and crustaceans (up to 30 micrograms/g of crayfish), but has a paralysing effect on crickets (30 micrograms/g animal). It displaces the binding of [125I]NTX to rat brain synaptosome membranes with a Ki of 0.1 microM, in comparison NTX has a Ki of 100 pM. Similarly, using single Ca2+ activated K+ channels of small conductance obtained from cultured bovine aortic endothelial cells it was shown that NTX2 is over two logarithm units less potent than NTX in producing 50% blockade of the probability of opening the channels. NTX2 is not recognized by a panel of six distinct monoclonal antibodies against NTX, however it is recognized by polyclonal antibodies raised in mouse, with native NTX. Primary structure comparison of both NTX and NTX2 suggests that the N-terminal segments of these peptides are important for channel affinity.
Assuntos
Bloqueadores dos Canais de Potássio , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Células Cultivadas , Crustáceos/efeitos dos fármacos , Gryllidae/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Ratos , Venenos de Escorpião/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
A novel peptide was purified and characterized from the venom of the scorpion Pandinus imperator. Analysis of its primary structure reveals that it belongs to a new structural class of K+-channel blocking peptide, composed of only 35 amino acids, but cross-linked by four disulphide bridges. It is 40, 43 and 46% identical to noxiustoxin, margatoxin and toxin 1 of Centruroides limpidus respectively. However, it is less similar (26 to 37% identity) to toxins from scorpions of the geni Leiurus, Androctonus and Buthus. The disulphide pairing was determined by sequencing heterodimers produced by mild enzymic hydrolysis. They are formed between Cys-4-Cys-25, Cys-10-Cys-30, Cys-14-Cys-32 and Cys-20-Cys-35. Three-dimensional modelling, using the parameters determined for charybdotoxin, showed that is it possible to accommodate the four disulphide bridges in the same general structure of the other K+-channel blocking peptides. The new peptide (Pil) blocks Shaker B K+ channels reversibly. It also displaces the binding of a known K+-channel blocker, [125I]noxiustoxin, from rat brain synaptosomal membranes with an IC50 of about 10 nM.
Assuntos
Bloqueadores dos Canais de Potássio , Canais de Potássio , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Cisteína/química , Dissulfetos/química , Técnicas In Vitro , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/toxicidade , Ratos , Venenos de Escorpião/genética , Venenos de Escorpião/metabolismo , Homologia de Sequência de Aminoácidos , Superfamília Shaker de Canais de Potássio , Spodoptera , Sinaptossomos/metabolismoRESUMO
Binding of [125I]-Noxiustoxin to rat brain synaptosome membranes and displacement with synthetic peptides corresponding to the amino acid sequence of Noxiustoxin, show that the N-terminal segment of this toxin is implicated in the recognition of brain K(+)-channels. Cleavage of NTX with cyanogen bromide, endopeptidase V8 from Staphylococcus aureus and endopeptidase Lys-C support these findings. On the contrary, a synthetic C-terminal tetradecapeptide of charybodotoxin (ChTX), shows that in this K(+)-channel blocker, the C-terminal region, rather than the N-terminal is capable of displacing 125[I]-NTX binding to brain membranes.
Assuntos
Charibdotoxina/química , Bloqueadores dos Canais de Potássio , Venenos de Escorpião/química , Sinaptossomos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Ensaio Radioligante , Ratos , Venenos de Escorpião/metabolismo , Venenos de Escorpião/farmacologia , Relação Estrutura-Atividade , Sinaptossomos/metabolismoRESUMO
The soluble venom of the scorpion of Colima, Mexico, was fractionated by Sephadex G-50 gel filtration followed by separation of the toxic fraction (number II) with carboxymethyl-cellulose columns in 20 mM ammonium acetate buffer, pH 4.7. Of 24 fractions five were toxic to mice and were further separated in 50 mM phosphate buffer, pH 6.0 using the same ion exchange resin. Final separation included chromatography of the toxic subfractions in the same resin, but in 75 mM ammonium acetate buffer, pH 5.0. By this procedure at least five distinct toxins were obtained in pure form according to gel electrophoresis analysis. Amino acid composition of toxin 1 (component II.20.3.4) and toxic component II.22.5 is included. The N-terminal amino acid sequence of component II.22.5 was shown to be: Lys-Glu-Gly-Tyr-Ile-Val-Asn-Tyr-His-Thr-Gly-Cys-Lys-Tyr-Thr-Cys-Ala-Lys- Leu-Gly - Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-.