Molecular cloning and characterization of Phoneutria nigriventer toxins active on calcium channels

doi:10.1016/S0041-0101(03)00011-4

Toxicon

Volume 41, Issue 7, June 2003, Pages 755-763

https://doi.org/10.1016/S0041-0101(03)00011-4 Get rights and content

Abstract

The aim of the present study was the molecular cloning of toxins active on calcium channels expressed by the spider Phoneutria nigriventer. Clones encoding the toxins Pn3-3A, Pn3-4A, Tx3-5, Pn3-5A, Tx3-6, Pn3-6A and Pn3-6B were identified from a cDNA library derived from the venom gland of this spider, revealing toxins of 49, 76, 45, 39, 55 and 58 amino acids residues, respectively, with polypeptide precursors being composed of three major portions: a signal peptide, a propeptide and finally, the mature toxin. A high degree of homology with the amino acid sequence was found between Pn3-3A and the neurotoxin Tx3-3 (identity of 79%), and between Pn3-4A and the neurotoxin Tx3-4 (identity of 95%). The deduced amino acid sequence for the mature polypeptides Tx3-5 and Tx3-6 confirms the polypeptide sequence previously published for these neurotoxins. In addition, the toxin Pn3-5A showed 58% identity to the Tx3-5 amino acid sequence, and the toxins Pn3-6A and Pn3-6B showed 85 and 33% identity, respectively, to the Tx3-6 amino acid sequence.

Introduction

The importance of calcium as a universal signaling agent in biological processes has been well addressed by several authors (Bootman et al., 2001). Thus, the search for tools that interfere with intracellular calcium concentration can help to understand the function of this ion in the birth, life and death of cells. Several neurotoxins isolated from spiders have been important tools in the investigation of calcium channels, permitting the identification and characterization of different channel subtypes (Mintz et al., 1992, Sutton et al., 1998).

The venom of the spider Phoneutria nigriventer contains a complex mixture of polypeptides active on ionic channels and receptors. This venom was fractioned by Rezende et al. (1991), revealing the presence of four major active fractions named PhTx1, PhTx2, PhTx3 and M. All of these fractions showed neurotoxic effects, except fraction M that was active on smooth muscle isolated from mice. These fractions were further purified and analyzed by Cordeiro et al., 1992, Cordeiro et al., 1993 who described the amino acid sequence of four neurotoxins from fraction PhTx2 and six from fraction PhTx3. Figueiredo et al., 1995, Figueiredo et al., 2001 also reported the isolation of a novel neurotoxic fraction denominated PhTx4 which is active in mice and insects. Some neurotoxic polypeptides isolated from this fraction, named Tx4(6-1) and Tx4(5-5), were shown to have insecticidal activity. The use of molecular biology has improved the knowledge of this rich source of biologically active molecules not only by the characterization of toxins already described but also by the identification of new putative toxins (Diniz et al., 1993, Kalapothakis et al., 1998a, Kalapothakis et al., 1998b, Kushmerick et al., 1999, Penaforte et al., 2000).

The pharmacological action of the toxins isolated from the spider P. nigriventer has been the target of research by our group, and we have demonstrated that these neurotoxins act as potent activators and blockers of ionic channels (Romano-Silva et al., 1993, Araújo et al., 1993, Prado et al., 1996, Guatimosin et al., 1997, Kushmerick et al., 1999, Reis et al., 1999). In the present report we describe the molecular characterization of a group of neurotoxins active on calcium channels isolated from a cDNA library constructed with the venom glands of the spider P. nigriventer.

Section snippets

General molecular biology

Standard recombinant DNA techniques (plasmid purification, phenol extraction, ethanol precipitation, electrophoresis, etc.) were carried out as described by Sambrook et al. (1989).

cDNA library screening

The cDNA library used in the present study has been previously described by Kalapothakis et al. (1998a). The library was constructed using the Uni-Zap™XR vector (Stratagene) with mRNA purified from venom glands of P. nigriventer spiders.

The molecular screening of Pn3-3A was based on the peptide sequence of Tx3-3

Molecular cloning

The venom of the P. nigriventer spider has been demonstrated to be a rich source of neurotoxins active on calcium channels. Rezende et al. (1991) demonstrated that fraction PhTx3 isolated from this venom was lethal (LD₅₀ 137±9 μg/kg) and caused flaccid paralysis (ED₅₀ 0.04 μg/kg) in mice. Gomez et al. (1995) showed that this fraction inhibits the liberation of acetylcholine from the guinea-pig ileum and slices of rat cerebral cortex. The action of this fraction on calcium channels was reported

Acknowledgements

This work was supported by PADCT (Programa de Apoio ao Desenvolvimento Cientı́fico e Tecnológico), FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico) and PRONEX (Programa de Apoio a Núcleos de Excelência).

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The genes encoding for all PhTx3 toxins are identified. It was shown that these toxins are encoded as a precursor peptide composed of a signal peptide, an intervening propeptide, and the mature toxin (Cardoso et al., 2003; Carneiro et al., 2003; Kalapothakis et al., 1998b). Interestingly, certain isoforms identified by molecular cloning have never been purified from the crude venom.
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Molecular cloning and characterization of Phoneutria nigriventer toxins active on calcium channels

Abstract

Introduction

Section snippets

General molecular biology

cDNA library screening

Molecular cloning

Acknowledgements

Semin. Cell Dev. Biol.

FEBS

Toxicon

J. Biol. Chem.

Toxicon

Neurosci. Lett.

Toxicon

Toxicon

Neuropharmacology

Brain Res. Bull.

Toxicon

Neurochem. Int.

J. Biol. Chem.

J. Biol. Chem.

Toxicon

Effects of a toxic fraction PhTx2, from the spider Phoneutria nigriventer on the sodium current

Naunyn-Schmiedeberg's Arch. Pharmacol.