RESUMO
A novel peptide named Pg8 was purified from the venom of the South African scorpion Parabuthus granulatus and its primary structure was determined. It contains 63 amino acid residues tightly folded by 4 disulfide bridges. The gene coding for this peptide was cloned from a cDNA library. By recursive PCR strategy a hybrid gene was constructed having a factor X recognition site for proteolysis and a modified sequence for preferential codon usage of E. coli. A pQE30 molecular vector already contained a His-tag was used for expression. This construction was expressed in BL21 and Origami strains. The fusion protein from inclusion bodies was separated by HPLC (yield approximately 5mg/L) and properly folded in vitro. Lethality tests showed that the recombinant peptide was toxic and was used to immunize mice. A volume of 0.25ml of the anti-serum produced was capable of protecting up to 3 LD(50) doses of pure toxin Pg8 but also, and more importantly, the entire soluble venom.
Assuntos
Antivenenos/genética , Venenos de Escorpião/genética , Venenos de Escorpião/imunologia , Escorpiões/genética , Sequência de Aminoácidos , Animais , Anticorpos/análise , Antivenenos/imunologia , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Feminino , Genes/genética , Genes/imunologia , Camundongos , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Venenos de Escorpião/enzimologiaRESUMO
The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/fisiologia , Venenos de Escorpião/farmacologia , Espermatozoides/efeitos dos fármacos , Animais , Células Cultivadas , Condutividade Elétrica , Cinética , Masculino , Camundongos , Neurotoxinas/farmacologia , Técnicas de Patch-Clamp , Espermatogônias/efeitos dos fármacos , Espermatogônias/fisiologia , Espermatozoides/fisiologiaRESUMO
This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.