A novel arrangement of Cys residues in a paralytic peptide of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting snail from the Gulf of Mexico

Abstract

The present study details the purification, the amino acid sequence determination, and a preliminary characterization of the biological effects in mice of a new conotoxin from the venom of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting cone snail collected in the western Gulf of Mexico (Mexico). The 23-amino acid peptide, called as25a, is characterized by the sequence pattern CX1CX2CX8CX1CCX5, which is, for conotoxins, a new arrangement of six cysteines (framework XXV) that form three disulfide bridges. The primary structure (CKCPSCNFNDVTENCKCCIFRQP*; *, amidated C-terminus; calculated monoisotopic mass, 2644.09Da) was established by automated Edman degradation after reduction and alkylation, and MALDI-TOF and ESI mass spectrometry (monoisotopic mass, 2644.12/2644.08Da). Upon intracranial injection in mice, the purified peptide provokes paralysis of the hind limbs and death with a dose of 240 pmol (~0.635 μg, ~24.9 ng/g). In addition, a post-translational variant of this peptide (as25b) was identified and determined to contain two hydroxyproline residues. These peptides may represent a novel conotoxin gene superfamily.

Fig. 1

Purification of peptides as24a and as24b. Panel A) Fractionation of the venom extract (~0.3 mg total protein) on an analytical C18 column eluted with an isocratic step at 5% solution B for 10 min followed by a linear gradient from 5-55% solution B in 100 min, at 1 ml/min. HPLC solutions were: 0.1% (v/v) TFA in water (solution A), and 0.085% (v/v) TFA in 90% aqueous ACN (solution B). The absorbance was monitored at 220 nm. Panel B) The peak highlighted by an arrow in panel A was further purified on the same column, using an isocratic step at 20% solution B for 20 min, and a linear gradient from 20-35% solution B in 60 min, at 1 ml/min. Panel C) The peak shown in panel B was further purified on an analytical C8 column using an isocratic step at 20% solution B for 10 min, and a linear gradient from 20-35% solution B in 60 min, at 1 ml/min. Panel D) The peak highlighted by an arrow and an asterisk in panel A was further purified on an analytical C8 column using an isocratic step at 20% solution B for 10 min, and a linear gradient from 20-35% solution B in 60 min, at 1 ml/min.

Fig. 2

Determination of the molecular mass of conotoxin as24a. Panel A) The MALDI-TOF monoisotopic m/z signal at 2,645.12 ([M+H]⁺) corresponds to a monoisotopic mass of 2,644.12 Da. Panel B) The full scan mass spectrum obtained in an ESI-Orbitrap shows m/z signals corresponding to the triply ([M+3H]³⁺, 882.69) and doubly charged ([M+2H]²⁺, 1323.54) ions. The isotopic resolution (R=60,000) of the doubly charged ion (inset) indicates a monoisotopic mass of 2,644.08 Da. Panel C) HCD fragmentation spectrum showing the product ions derived from the triply charged precursor ion at m/z 882.69. The values of the main y ion series clearly demonstrate that the C-terminal of peptide as24a is amidated.

Fig. 3

Panel A) Amino acid sequence of peptides as24a (upper line) and as24b (lower line) from C. cancellatus. Cys residues are displayed in bold font and underlined. For post-translational modifications, * and O represent, respectively, the amidation of the C-terminal residue, and 4-hydroxyproline residues, whereas ? indicates that the amidation of the C-terminus was not directly confirmed. Panel B) ClustalW2 alignment of peptide as24a (bold type) and 11 conotoxins similar to it: Cp3-R01 (C. capitaneus), Co3-R02, Co3-H03 and Co3-H01 (C. coronatus), Vx3-R01 (C. vexillum), Eb3-H03 (C. ebraeus), Bt3-I04 and Bt3-I03 (C. betulinus), S3-I05 (C. striatus), Mi3-D01 (C. miles), and Ca-AFI99276 (C. caracteristicus). Amidations of the C-termini of as24a and Co3-H01 are not displayed. Identical residues and conserved substitutions with respect to toxin as24a are highlighted, respectively, in black and grey backgrounds. For all the sequences, asterisks and dots indicate, respectively, identical residues and semi-conserved substitutions. Figures from left to right are, respectively, number of residues in each peptide, and percent identity. v = vermivorous; p = piscivorous; IP = Indopacific; EP = eastern Pacific.