Abstract
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 β-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 α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT(3) receptors at a concentration up to 10 μm. 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.
Highlights
Venoms from rare snake species may contain toxins of new structural or/and pharmacological types
In this paper we describe the determination of its amino acid sequence and biological activity of azemiopsin; like waglerins, azemiopsin selectively blocks muscle-type nicotinic acetylcholine receptor (nAChR) but has a markedly different primary structure and does not contain disulfide bridges
Receptor Binding Studies—For competition binding assays, suspensions of nAChR-rich membranes from T. californica ray electric organ (1.25 nM ␣-bungarotoxin binding sites suspended in 20 mM Tris-HCl buffer, pH 8.0, containing 1 mg/ml bovine serum albumin (BSA)), human ␣7 nAChR-transfected GH4C1 cells (0.4 nM ␣-bungarotoxin binding sites suspended in 20 mM Tris-HCl buffer, pH 8.0, containing 1 mg/ml BSA), or solutions of expressed acetylcholine-binding proteins (AChBP) from L. stagnalis and A. californica (2.4 and 150 nM in phosphate-buffered saline containing 0.7 mg/ml BSA and 0.05% Tween 20 (PBS-T)) were incubated for 90 min with various amounts of the toxins, followed by an additional 5-min incubation with 0.1– 0.2 nM 125I-labeled ␣-bungarotoxin
Summary
Venoms from rare snake species may contain toxins of new structural or/and pharmacological types. Results: Amino acid sequence of the new polypeptide azemiopsin isolated from Azemiops feae viper venom was established, and its biological activity was determined. Conclusion: Azemiopsin is the first natural toxin that blocks nicotinic acetylcholine receptors and does not contain disulfide bridges. Waglerins are polypeptides, comprising 22–24 amino acid residues, isolated from the venom of the Asian pit viper Tropidechis waglerii. These polypeptides are selective blockers of muscle-type nicotinic acetylcholine receptor (nAChR)3 [11]. In this paper we describe the determination of its amino acid sequence and biological activity of azemiopsin; like waglerins, azemiopsin selectively blocks muscle-type nAChR but has a markedly different primary structure and does not contain disulfide bridges
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