Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

Abstract

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3′,5′-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum–Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C.

The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5 Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (Benzinger et al., 1998), having 61.7% β-sheet and no α-helix.

Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD₅₀ of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5±160.5%. The increase in contractile amplitude reached a plateau value within 3–5 min after addition of this toxin.

Introduction

Sea anemone is a kind of primitive organism belonging to Anthozoa of Coelenterata. They have numerous tentacles which containing specialized sting cells or cnidocytes. These venom apparata are used in the capture of prey as well as for defense against predators. Therefore, sea anemone contains a variety of biologically active compounds, which may serve as a defense system during evolution, including some potent toxins. Polypeptide neurotoxins with molecular weights of approximately 3 and 5 kDa are the most thoroughly characterized toxins (Kem, 1988, Norton, 1991). These toxins can slow down the inactivation process of voltage dependent sodium channels in excitable tissues. Another group of toxins with the molecular weights in the range of 15–20 kDa are cytolysins which form pores in various lipid membranes (Kem and Dunn, 1988, Jiang et al., 2002).

Sea anemone neurotoxins consist of three classes of polypeptides, two classes of molecules containing 46–49 amino acid residues and one of shorter polypeptides containing 27–31 residues (Norton, 1991). Sea anemone neurotoxins act by binding to the specific receptor sites on the voltage-gated sodium channel of excitable tissue. They can prolong action potentials due to their action on slowing down the inactivation of the Na⁺ channel. The toxins can cause intracellular Na⁺ and Ca²⁺ increase, resulting in the enhancement of heart contraction (Alsen et al., 1976, Beress et al., 1975). However, its activities are not associated with any significant effects on heart rate or blood pressure in vivo (Gross et al., 1985). On the other hand, the toxins have been shown to increase the action potential duration of cardiocytes and similar to class III antiarrhythmic effects (Platou et al., 1986). Thus the toxins may serve as a valuable lead in the development of new therapeutic agent for the treatment of heart failure (Norton, 1991).

Neurotoxins Ap-A and Ap-B share a similar three-dimensional structure consisting of four short strands of antiparallel beta-sheet connected by three loops. The structure of Ap-A differs from that of Ap-B at seven amino acid positions (Monks et al., 1995, Pallaghy et al., 1995). Some residues important for Ap-B activity have been identified from site-directed mutagenesis studies. Trp-33 contributes significantly to apparent affinity (Dias-Kadambi et al., 1996), and the residues at positions 12 and 49 are critical for both high binding affinity and isoform discrimination (Khera et al., 1995). Lys-37 that is conserved among many sea anemone toxins may play a role in channel interaction (Khera and Blumenthal, 1996, Benzinger et al., 1998).

Anthopleura sp. is a representative type of sea anemone in South China Sea (Pei and Huang, 1994). Based on the potential value of the neurotoxins, it is very interesting to screen new neurotoxins from the sea anemone Anthopleura sp. Here we report the cloning of Hk2a cDNA sequence and its functional analysis via an efficient recombinant expression in E. coli.