Utkin_2012_J.Biol.Chem_287_27079

Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a beta-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with alpha-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 +/- 0.03 mum) and with lower efficiency to human alpha7 nAChR (IC(50) 22 +/- 2 mum). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (alpha1beta1epsilondelta) than the fetal form (alpha1beta1gammadelta), EC(50) being 0.44 +/- 0.1 mum and 1.56 +/- 0.37 mum, respectively. The peptide had no effect on GABA(A) (alpha1beta3gamma2 or alpha2beta3gamma2) receptors at a concentration up to 100 mum or on 5-HT(3) receptors at a concentration up to 10 mum. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.