Distinct primary structures of the major peptide toxins from the venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium channel¹

Abstract

Six peptide toxins (Magi 1^6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx-II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na + channel modi¢er N N-atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125 I-labelled peptide toxins clearly demonstrated the speci¢c binding a⁄nity of Magi 1^5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhK KIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K i s of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K i = 267 nM) and site 4 in rat brain synaptosomes (K i = 1.2 nM), whereas it showed no a⁄nities for either mammal binding site 3 or insect binding site 4. Magi 5 is the ¢rst spider toxin with binding a⁄nity to site 4 of a mammalian sodium channel. ß

Solution Structure and Alanine Scan of a Spider Toxin That Affects the Activation of Mammalian Voltage-gated Sodium Channels

“…Peptide Synthesis and Characterization-Magi 5 was synthesized with its C terminus as the free carboxyl, as observed for the native toxin by mass spectrometry (12). The identity between the synthetic and native polypeptides was verified by MALDI-TOF mass spectrometry and capillary zone electrophoresis.…”

“…Hydrogen bonds between the amide of Glu 14 and the oxygen of Asn 11 , the amide of Cys 16 and the oxygen of Lys 3 , the amide of Met 27 and the oxygen of Ala 20 were present in 14, 19, and 20 structures, respectively. The Glu 14 to Asn 11 hydrogen bond, together with the and angles for Lys 12 and Lys 13 (Ϫ67°/Ϫ32°and Ϫ82°/5°, respectively), indicate that the chain reversal around residues 11-14 is a type I ␤-turn. The chain reversal around residues 15-18 does not fit the description of any of the classical turn types, possibly because of distortions associated with the disulfide links to Cys 15 and Cys 16 .…”

“…The six free cysteine residues were allowed to oxidize in air for 24 h at room temperature in 0.5 M aqueous ammonium acetate solution (pH 8.0) containing 1 mM reduced glutathione, 0.1 mM oxidized glutathione. The folded synthetic toxins were purified on the same semi-preparative C 8 reversed phase HPLC column using a 40-min linear gradient from 20 to 60% aqueous acetonitrile, 0.1% trifluoroacetic acid (2 ml/min), followed by cation exchange chromatography as previously described for the native toxins (12). The structural identity between synthetic and native Magi 5 was verified by capillary zone electrophoresis and cation exchange HPLC coelution experiments, circular dichroism, and MALDI-TOF mass spectrometry (see supplemental materials).…”

“…Recently, it was found that this receptor site 4 in mammals was also recognized by a spider toxin, Magi 5, from the hexathelid spider Macrothele gigas (Iriomote, Japan) that binds specifically to the receptor site 4 in mammals (12). Magi 5 is also found in the venom of the hexathelid spider Macrothele raveni (Guangxi, China), where it was named raventoxin III (21).…”

“…It is lethal to mice upon intracranial injection and competes with the scorpion ␤-toxin Css IV with a K i of 1.2 nM. Magi 5 and Css IV bind to the same receptor on the sodium channel from rat brain synaptosomes (12).…”