Mass spectrometric‐based revision of the structure of a cysteine‐rich peptide toxin with γ‐carboxyglutamic acid, TxVIIA, from the sea snail, Conus textile

Abstract

A mollusk-specific toxin, TxVIIA, having potent paralytic activity was isolated from the venom of sea snail Conus textile (Fainzilber M et al., 1991, Eur JBiochem 202:589-595). The structure reported above was based upon amino acid analysis and the Edman degradation. We have recently reinvestigated this toxin employing some of the most novel techniques in mass spectrometry. We now report a revised structure based primarily on high-energy collision-induced dissociation analysis of the two Asp"-N peptides of the reduced, pyridinylethyl derivative representing the entire sequence using matrix-assisted laser desorption ionization (MALDI) as CGGYSTYCyVDSy CCSDNCVRSYCTLF-NH2 (y, y-carboxyglutamic acid or Gla).The N-terminus of the previous sequence was incorrect, apparently due to a side reaction of reduction and alkylation, which led to the erroneous assignment of Trp for the N-terminal residue.In addition, the last two C-terminal amino acids and the C-terminal amidation had not been detected. Also, a combination of electrospray ionization mass spectrometry and positive and negative ion MALDI mass spectrometry provided information on the molecular weights of the native and derivatized toxin and presence of two Gla residues. Thus, TxVIIA does not have an "unusual" sequence as previously reported, but in fact belongs to the conserved Cys framework for W -and 6-conotoxins. However, the four net negative charges with the cysteine-rich structure of this revised sequence is highly unusual for conopeptides.

A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca 2+ influx

“…Since the discovery of Gla in 1974 and elucidation of its involvement in the Ca 2ϩ -mediated membrane interaction of the vitamin Kdependent blood clotting proteins, it has also been found in two mineralized tissue proteins (25)(26)(27)(28). The discovery of Gla in toxins from invertebrate cone snails (conantokin-G, conantokin-T, conotoxin TxVIIA, and ␥-conotoxin-PnVIIA) suggests a more general role for Gla in biology beyond processes involved in blood coagulation and tissue mineralization (5,11,(29)(30)(31). This is also in accordance with the wide tissue distribution of the vitamin K-dependent carboxylase (32).…”

An Unusual Family of Glycosylated Peptides Isolated from Dendroaspis angusticeps Venom and Characterized by Combination of Collision Induced and Electron Transfer Dissociation

“…More unusual PTMs have also been characterized, especially within the cone snail family. Hydroxylation of prolines [13], bromination of tryptophans [13,14], γ-carboxylation of glutamic acids [15,16], sulphation of tyrosines [17], and Nterminal pyroglutamic acid formations [18] represent the PTMs observed more or less frequently in the toxin world. D-amino acids are also present in toxins [19].…”

Rapid sensitive analysis of cysteine rich peptide venom components

“…The extraordinary complexity of crude venom samples (often containing Ͼ100 components) (4) and the lack of DNA databases for many of the organisms of interest presents a major analytical challenge. Most of the currently known primary structures of peptide toxins have been obtained using Edman sequencing (10), comparison of tandem MS (MS/MS) data with cDNA sequences (11)(12)(13)(14)(15)(16)(17), or a combination of these techniques (18)(19)(20)(21). Although MS reduces the requirement for exhaustive purification compared with Edman sequencing (22,23), it is often difficult to obtain full length de novo sequence exclusively by MS/MS.…”