RESUMO
This work reports the isolation, chemical and functional characterization of two previously unknown peptides purified from the venom of the scorpion Pandinus imperator, denominated Pi5 and Pi6. Pi5 is a classical K+-channel blocking peptide containing 33 amino acid residues with 4 disulfide bonds. It is the first member of a new subfamily, here defined by the systematic number α-KTx 24.1. Pi6 is a peptide of unknown real function, containing only two disulfide bonds and 28 amino acid residues, but showing sequence similarities to the κ-family of K-channel toxins. The systematic number assigned is κ-KTx2.9. The function of both peptides was assayed on Drosophila Shab and Shaker K+-channels, as well as four different subtypes of voltage-dependent K+-channels: hKv1.1, hKv1.2, hKv1.3 and hKv1.4. The electrophysiological assays showed that Pi5 inhibited Shaker B, hKv1.1, hKv1.2 and hKv1.3 channels with Kd = 540 nM, Kd = 92 nM and Kd = 77 nM, respectively, other studied channels were not affected. Of the channels tested only hKv1.2 and hKv1.3 were inhibited at 100 nM concentration of Pi6, the remaining current fractions were 68% and 77%, respectively. Thus, Pi5 and Pi6 are high nanomolar affinity non-selective blockers of hKv1.2 and hKv1.3 channels.
Assuntos
Peptídeos/isolamento & purificação , Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/química , Escorpiões , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Drosophila , Humanos , Leucócitos Mononucleares , Peptídeos/química , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio , Células Sf9 , SpodopteraRESUMO
BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antivenenos/metabolismo , Fragmentos de Imunoglobulinas/imunologia , Mutação/genética , Venenos de Escorpião/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Antivenenos/genética , Antivenenos/imunologia , Evolução Biológica , Clonagem Molecular , Mapeamento de Epitopos , Escherichia coli/metabolismo , Dados de Sequência Molecular , Testes de Neutralização , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Peptídeos/isolamento & purificação , Especificidade por SubstratoRESUMO
Different peptides were purified by chromatographic procedures from the skin-secretory glands of the frog Phyllomedusa distincta. These are the first peptides reported from this frog species. Their primary structure was determined by a combination of automated Edman degradation and mass spectrometry. Peptide Q2 contains 25 amino acid residues, peptide Q1 and L have 28 each, peptide M contains 31, and peptide K has 33 amino acid residues. They all showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria, presenting minimal inhibitory concentrations from 0.6 to 40 microM, when tested against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Peptides K, L, and Q1 were chemically synthesized and shown to be active.
Assuntos
Antibacterianos/isolamento & purificação , Peptídeos , Pele/química , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Anuros , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K+ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K+ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K+ channels with a Kd of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of 125I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels.
Assuntos
Canais de Potássio/metabolismo , Venenos de Escorpião/isolamento & purificação , Escorpiões/química , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , DNA Complementar/genética , Vetores Genéticos/genética , Cinética , Dados de Sequência Molecular , Nucleopoliedrovírus/genética , Canais de Potássio/efeitos dos fármacos , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Superfamília Shaker de Canais de Potássio , Spodoptera/citologia , 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
Seven peptides corresponding to the amino acid sequence of toxin 2 from the scorpion Centruroides noxius were chemically synthesized, purified and assayed in mice for their putative neutralizing properties against scorpion toxins. All the peptides were immunogenic and some produced neutralizing antibodies, as verified by injecting the antisera with toxin into naive animals. However, direct challenge of pre-immunized mice (with the longest synthetic peptides of 27 and 57 amino acid residues) revealed an unexpected sensitization phenomena: the animals did not resist injection of one LD50 of purified toxin 2 (5% survival), but pre-immunization of mice with native toxin protected 100% of the animals. These findings suggest that vaccine preparations with synthetic peptides corresponding to the amino acid sequence of certain toxins should be analyzed cautiously.