Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins

Ronald C Hogg,Rustam Kh Ziganshin,Daniel Bertrand,Vladislav G Starkov,Olga V Vorontsova,Yana V Makarova,Marina V Serebryakova,Audrey Benoit,Tatyana V Andreeva,Igor E Kasheverov,Victor I Tsetlin,Yuri N Utkin,Alexey V Osipov

doi:10.1074/jbc.m802085200

Abstract

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Highlights

Three-fingered toxins (TFTs)2 are the main components of the Elapidae snake venoms
In this paper we report identification in the Naja kaouthia cobra venom of a new class of naturally occurring TFTs, which represent disulfide-bound heterodimers of ␣-cobratoxin (␣-CT), a long-chain ␣-neurotoxin, with cytotoxins, as well as a homodimer of ␣-CT
Materials—The N. kaouthia Thailand cobra venom was obtained from living adult cobras kept in captivity as described earlier [12]. ␣-CT and cytotoxins (CX) 1, 2, and 3 were purified from this venom according to Kukhtina et al [13]

Summary

EXPERIMENTAL PROCEDURES

Materials—The N. kaouthia Thailand cobra venom was obtained from living adult cobras kept in captivity as described earlier [12]. ␣-CT and cytotoxins (CX) 1, 2, and 3 were purified from this venom according to Kukhtina et al [13]. 1:1 and 2:1 of DTT to protein molar ratios were employed in a 0.2 M Tris-HCl buffer, pH 8.5, followed by the addition of excess of 4-vinylpyridine and incubation under the nitrogen for 45 min. Receptor Binding Studies—For competition binding assays, suspensions of nAChR-rich membranes from T. californica ray electric organ (1.2 nM ␣-bungarotoxin binding sites suspended in 20 mM Tris-HCl buffer, pH 8.0, containing 1 mg/ml bovine serum albumin), human ␣7 nAChR-transfected GH4C1 cells (1.2 nM ␣-bungarotoxin binding sites prepared in PBS with 0.1% Tween 20 (PBS-T) buffer), or solutions of expressed AChBP from L. stagnalis (6.4 nM in PBS-T buffer) were incubated for 90 min with various amounts of the toxins followed by an additional 90-min incubation with 0.2 nM [125I]␣-bungarotoxin. Excitation wavelengths were 280 or 292 nm, and emission was registered in the range from 300 to 450 nm

RESULTS

Mass of additional components observed at the elevated laser energy

DISCUSSION