Search

Background:Conus amadis is a carnivorous snail found abundantly in coastal waters of India. Despite its abundance in southern coastal waters of India and the fact that most of the conotoxin act in neuronal system, research work on Conus amadis venom was not much focused. So we have made a brief study on the venom complex of Conus amadis to identify the library of novel conotoxins and to screen the natural venom for neurological function.Objective:De novo sequencing of novel conopeptides from the venom cocktail of Conus amadis and to screen its natural venom for the presence of biological activities in zebrafish model.Methods:Proteome based MALDI-TOF and LC-MS-MS analysis for identification of novel conotoxins and subsequent sequencing. Due to the complex disulfide rich nature of the venom peptides, the study also involves global chemical modification experiments of the venom extract to unambiguously determine the sequence of novel conotoxins. Biological function analysis of natural venom was tested in zebrafish model to ascertain anti-epileptic properties.Results:In this study, we have identified 19 novel conotoxins containing 1, 2 & 3 disulfides, belonging to different classes. Among them, 2 novel contryphans, 3 T-superfamily conotoxins, 2 A-superfamily conotoxins and 2 Mini M-Superfamily conotoxins were sequenced to its amino acid level from the fragmented spectrum of singly and doubly charged parent ions using de novo sequencing strategies. ama1054, a contryphan peptide toxin, possesses post translationally modified bromo tryptophan at its seventh position. Except ama1251, all the sequenced peptide toxins possess modified C-terminal amidation. Crude venom exhibited anticonvulsant properties in pentylenetetrazole-induced seizure in zebrafish larvae, which suggested anti-epileptic property of the venom cocktail. Acetylcholinesterase activity was also identified in the venom complex.Conclusion:Based on the preliminary evidence, if this study is extended further through bioassay guided purification, could possibly yield peptide toxins with anticonvulsant and other neurologically active molecules.

Marine cone snails are predatory gastropods characterized by a well-developed venom apparatus and highly evolved hunting strategies that utilize toxins to paralyze prey and defend against predators. The venom of each species of cone snail has a large number of pharmacologically active peptides known as conopeptides or conotoxins that are usually unique in each species. Nevertheless, venoms of only very few species have been characterized so far by transcriptomic approaches. In this study, we used transcriptome sequencing technologies and mass spectrometric methods to describe the diversity of venom components expressed by a worm-hunting species, Conus bayani. A total of 82 conotoxin sequences were retrieved from transcriptomic data that contain 54 validated conotoxin sequences clustered into 21 gene superfamilies including divergent gene family, 17 sequences clustered to 6 different conotoxin classes, and 11 conotoxins classified as unassigned gene family. Seven new conotoxin sequences showed unusual cysteine patterns. We were also able to identify 19 peptide sequences using mass spectrometry that completely overlapped with the conotoxin sequences obtained from transcriptome analysis. Importantly, herein we document the presence of 16 proteins that include five post-translational modifying enzymes obtained from transcriptomic data. Our results revealed diverse and novel conopeptides of an unexplored species that could be used extensively in biomedical research due to their therapeutic potentials.

Background Conotoxins are neuro-pharmacologically active cysteine rich peptides isolated from the venom complex of marine cone snails. These are usually made of even number of cysteines. Method In this study we characterised six novel conotoxin sequences from the venom of Conus leopardus collected from the Andaman Sea, namely Le907 (C-C), Le868 (C-C), Le933 (-C-CC), Le949 (-C-CC), Le1988 (C-C-CC-C) and Le1642 (CC-C-C) using de novo mass spectrometrybased sequencing methods. Astonishingly 3 of these peptides possess novel arrangements of cysteine residues with odd number of cysteines (-C-CC; C-C-CC-C), namely Le933, Le949 and Le1988. Further, a post-translational variant of peptide Le933 was identified and experimentally determined to contain hydroxyproline. Results The unusual cysteine arrangements observed suggests novel class of conotoxins. These results expand our understanding of the diversity of odd cysteine arrangements in conotoxins.

The marine snail Conus araneosus has unusual significance due to its confined distribution to coastal regions of southeast India and Sri Lanka. Due to its relative scarceness, this species has been poorly studied. In this work, we characterized the venom of C. araneosus to identify new venom peptides. We identified 14 novel compounds. We determined amino acid sequences from chemically-modified and unmodified crude venom using liquid chromatography-electrospray ionization mass spectrometry and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Ten sequences showed six Cys residues arranged in a pattern that is most commonly associated with the M-superfamily of conotoxins. Four other sequences had four Cys residues in a pattern that is most commonly associated with the T-superfamily of conotoxins. The post-translationally modified residue (pyroglutamate) was determined at the N-terminus of two sequences, ar3h and ar3i respectively. In addition, two sequences, ar3g and ar3h were C-terminally amidated. At a dose of 2 nmol, peptide ar3j elicited sleep when injected intraperitoneally into mice. To our knowledge, this is the first report of a peptide from a molluscivorous cone snail with sleep-inducing effects in mice. The novel peptides characterized herein extend the repertoire of unique peptides derived from cone snails and may add value to the therapeutic promise of conotoxins. (C) 2015 Elsevier Ltd. All rights reserved.

The occurrence of contryphans, a class of single-disulfide-bond-containing peptides, is demonstrated by the analysis of the venom of nine species of cone snails. Ten full gene sequences and two partial gene sequences coding for contryphan precursor proteins have been identified by next-generation sequencing and compared with available sequences. The occurrence of mature peptides in isolated venom has been demonstrated by LC–ESI–MS/MS analysis. De novo sequencing of reduced, alkylated contryphans from C. frigidus and C. araneosus provides evidence of sequence variation and post-translational modification, notably gamma carboxylation of glutamic acid. The characterization of Fr965 (C. frigidus) provides a rare example of a sequence lacking Pro at position 5 in the disulfide loop. The widespread occurrence of contryphan genes and mature peptides in the venom of diverse cone snails is suggestive of their potential biological significance.