RESUMO
Almost all marine snails within superfamily Conoidea produce venoms containing numerous neuroactive peptides. Most toxins characterized from members of this superfamily are produced by species belonging to family Conidae. These toxins (conotoxins) affect diverse membrane proteins, such as voltage- and ligand-gated ion channels, including nicotinic acetylcholine receptors (nAChRs). Family Turridae has been considerably less studied than their Conidae counterpart and, therefore, turrid toxins (turritoxins) have just been barely described. Consequently, in this work the most prominent chromatographic (RP-HPLC) fractions from the East Pacific species Polystira nobilis venom duct extract were isolated. The biological activity of six selected fractions was assayed on human (h) α7 AChRs expressed in Xenopus laevis oocytes. One of these fractions, F21, inhibited the acetylcholine-elicited response by 62 ± 12%. Therefore, this fraction was further purified and the F21-2 peptide was obtained. This peptide (at 5.6 µM) strongly and irreversibly inhibited the acetylcholine-induced response on hα7 and hα3ß2 nAChRs, by 55 ± 4 and 91 ± 1%, respectively. Electrospray mass spectrometry indicates that the average molecular mass of this toxin is 12 358.80 Da. The affinity for hα3ß2 nAChRs is high (IC50 of 566.2 nM). A partial sequence without cysteines was obtained by automated Edman degradation: WFRSFKSYYGHHGSVYRPNEPNFRSFAS ; blastp search revealed that this sequence has low similarity to some non-Cys-containing turripeptides. This is the first report of a turritoxin from a species of the American Pacific and the second description of a turripeptide inhibiting nAChRs.
Assuntos
Conotoxinas/farmacologia , Venenos de Moluscos , Receptores Nicotínicos/efeitos dos fármacos , Animais , Humanos , Venenos de Moluscos/química , Venenos de Moluscos/isolamento & purificação , Venenos de Moluscos/metabolismo , Venenos de Moluscos/toxicidade , Oócitos , Proteínas Recombinantes/farmacologia , Caramujos/metabolismo , Xenopus laevisAssuntos
Animais , Neurotoxinas/efeitos adversos , Neurotoxinas/metabolismo , Neurotoxinas/intoxicação , Saúde Pública/educação , Saúde Pública , Saúde Pública/tendências , Saxitoxina/efeitos adversos , Saxitoxina/metabolismo , Saxitoxina/intoxicação , Brasil , Crustáceos/metabolismo , Venenos de Peixe/efeitos adversos , Venenos de Peixe/metabolismo , Venenos de Moluscos/efeitos adversos , Venenos de Moluscos/metabolismoRESUMO
The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1-19) and Pro (aa 20-53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.