High-throughput identification of novel conotoxins from the Chinese tubular cone snail (Conus betulinus) by multi-transcriptome sequencing.

BACKGROUND: The venom of predatory marine cone snails mainly contains a diverse array of unique bioactive peptides commonly referred to as conopeptides or conotoxins. These peptides have proven to be valuable pharmacological probes and potential drugs because of their high specificity and affinity to important ion channels, receptors and transporters of the nervous system. Most previous studies have focused specifically on the conopeptides from piscivorous and molluscivorous cone snails, but little attention has been devoted to the dominant vermivorous species. RESULTS: The vermivorous Chinese tubular cone snail, Conus betulinus, is the dominant Conus species inhabiting the South China Sea. The transcriptomes of venom ducts and venom bulbs from a variety of specimens of this species were sequenced using both next-generation sequencing and traditional Sanger sequencing technologies, resulting in the identification of a total of 215 distinct conopeptides. Among these, 183 were novel conopeptides, including nine new superfamilies. It appeared that most of the identified conopeptides were synthesized in the venom duct, while a handful of conopeptides were identified only in the venom bulb and at very low levels. CONCLUSIONS: We identified 215 unique putative conopeptide transcripts from the combination of five transcriptomes and one EST sequencing dataset. Variation in conopeptides from different specimens of C. betulinus was observed, which suggested the presence of intraspecific variability in toxin production at the genetic level. These novel conopeptides provide a potentially fertile resource for the development of new pharmaceuticals, and a pathway for the discovery of new conotoxins.

Two novel O-superfamily conotoxins from Conus vexillum.

O-superfamily conotoxins include several families that have diverse pharmacological activity on Na+, K+ or Ca2+ channels. These superfamily toxins have been mainly found in fish-hunting and mollusk-hunting Conus species. Here, we reported two novel O-superfamily conotoxins, vx6a and vx6b, purified from a worm-hunting cone snail, Conus vexillum. Though their cysteine framework and signal peptides share high similarity with those of other members of O-superfamily, the mature vx6a and vx6b both have a low sequence homology with others. To test the biological function of vx6a, the toxin was chemically synthesized and then tested on the locust dorsal unpaired median (DUM) neuron system which containing various ion channels. Although no any activity on ion channels was found on the DUM neuron system, vx6a could clearly elicit a series of symptoms in mouse via intracranial injection, such as quivering, climbing, scratching, barrel rolling and paralysis of limbs at different dose.

A novel M-superfamily conotoxin with a unique motif from Conus vexillum.

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds for the purpose of feeding and defence, each evolved to act in a highly specific manner on different parts of the nervous system. Here, we report the peptide purification, molecular cloning, chemical synthesis, and functional characterization of a structurally unique toxin isolated from the venom of Conus vexillum. The novel peptide, designated Vx2, was composed of 21 amino acid residues cross-linked by 3 disulfide bonds (WIDPSHYCCCGGGCTDDCVNC). Intriguingly, its mature peptide sequence shows low level of similarity with other identified conotoxins, and its unique motif (-CCCGGGC-) was not reported in other Conus peptides. However, its signal peptide sequence shares high similarity with those of the M-superfamily conotoxins. Hence, Vx2 could be classified into a new family of the M-superfamily.

[Determination of primary structure of a novel peptide from mistletoe and its antitumor activity].

AIM: To study the antitumor peptide components in the stems and leaves of mistletoe (Viscum coloratum (Kom.) Nakai), the primary structure of the novel peptide was elucidated. METHODS: Cation exchange, gel filtration and HPLC were employed for isolation and purification. Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry was used to determine the mass. The complete amino acid sequence of the novel peptide was obtained by Edman degradation combined with enzyme digestion. The antitumor activity of the peptide in vitro was studied with MTT method. RESULTS: The primary stucture of the peptide named as viscotoxin B2 is KSCCKNTTGRNIYNTCRFAGGSRERCAKLSGCKIISASTCPSDYPK. The IC50 value of viscotoxin B2 on the Rat Osteoblast-like Sarcoma 17/2.8 cells in vitro is 1.6 mg x L(-1). CONCLUSION: Viscotoxin B2 in V. coloratum, which has high similarity with viscotoxins from V. album, showed antitumor activity.

A novel conotoxin from Conus betulinus, kappa-BtX, unique in cysteine pattern and in function as a specific BK channel modulator.

A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.