Venom

The venom of cone snails is a potent cocktail of peptides, proteins, and other small molecules. Several of the peptides (conopeptides and conotoxins) target ion channels and receptors and have proven useful as biochemical probes or pharmaceutical leads. In this study, the venom of a fish-hunting cone snail, Conus purpurascens was analyzed for intraspecific variability; α-conotoxins from the venom were isolated by high performance liquid chromatography, identified by mass spectrometry and nuclear magnetic resonance, and tested in a electrophysiological assay in Drosophila melanogaster; the effects of diet change on venom composition was investigated. It has been determined that each specimen of C. purpurascens expresses a distinct venom, resulting in the expression of more than 5,000 unique conopeptides across the species. α- conotoxin PIA was shown to inhibit the Dα7 nicotinic acetylcholine receptor.

Cone snails are venomous marine predators whose venom is a complex mixture of modified peptides (conopeptides). Conopeptides have direct specificity towards voltage- and ligand-gated ion channels and G-protein coupled receptors. More specifically, alpha conotoxins target nicotinic acetylcholine receptors (nAChR) and are of great interest as probes for different nAChR subtypes involved in a broad range of neurological function. Typically, the amount of peptide provided directly from the cone snails (from either dissected or “milked” venom) is minimal, thus hindering the wide use of bioassay-guided approaches for compound discovery. Biochemical-based approaches for discovery by means of identification and characterization of venom components can be used due to their compatibility with the small quantities of cone snail venom available; however, no direct assessment of the bioactivity can be gleaned from these approaches. Therefore, newly discovered conotoxins must be acquired synthetically, which can be difficult due to their complicated folding motifs.
The ability to test small quantities of peptide for bioactivity during the purification process can lead to the discovery of novel components using more direct approaches. Presented here is the description of use of an effective method of bioassay-guided fractionation for the discovery of novel alpha conotoxins as well as further biological characterization of other known alpha conotoxins. This method requires minimal amounts of sample and evaluates, via in vivo electrophysiological measurements, the effect of conotoxins on the functional outputs of a well-characterized neuronal circuit in Drosophila melanogaster known as the giant fiber system. Our approach uses reversed-phase HPLC fractions from venom dissected from the ducts of Conus brunneus in addition to synthetic alpha conotoxins. Fractions were individually tested for activity, re-fractionated, and re-tested to narrow down the compound responsible for activity. A novel alpha conotoxin, bru1b, was discovered via the aforementioned approach. It has been fully characterized in the giant fiber system through the use of mutant flies, as well as tested in Xenopus oocytes expressing nicotinic acetylcholine channels and against the acetylcholine binding protein. Other well-known alpha conotoxins have also been characterized in the giant fiber system.

Cone snails are predatory marine mollusks found in the genus Conus that use a complex cocktail of peptides to capture prey and deter predators. Most of the venom components selectively target ion channels or receptors, making them invaluable tools in neurophysiological studies. In this study, the venom of Conus brunneus, a common Panamic vermivorous cone snail species, was characterized by the use of high performance liquid chromatography, nuclear magnetic resonance, mass spectrometry, and automated Edman degradation sequencing. Three novel peptide sequences were reported: two peptides were members of the M-superfamily and one peptide was classified as an alpha-conotoxin. The disulfide connectivity of a previously isolated P-conotoxin was also determined. These peptides comprise a partial peptide library of Conus brunneus and may prove useful in numerous structural and neurological studies.

Cone snails (genus conus) are marine gastropods of tropical waters. They capture their prey by envenomation and for this; they have evolved a highly efficient and diverse venom cocktail of proteins and peptides. This has elicited extreme interest in venom composition and their molecular targets. In this study, the venom of Conus jaspedius, a small Atlantic cone snail was extracted, venom components isolated and analyzed, by a combination of Size exclusion and High performance liquid chromatography, NMR, Mass spectrometry and Edman sequencing. Here, four novel conopeptide sequences are reported, namely; an alpha conotoxin, a member of the A and O superfamilies and a new family.

Cone snails are predatory marine mollusks that utilize their peptide rich venom to capture prey, deter competitors and defend themselves. Each of the 1000 known species expresses over 100 conotoxins with little overlap between species. Most of these conotoxins selectively target a specific neuronal ion-channel or receptor. Because of their unprecedented diversity and specificity, they hold enormous potential as neuropharmacological agents, and as neuroscience research tools. In this study, the venom of a common shallow water cone snail that thrives in the Indo-Pacific to the Panamic region, Conus tessulatus , was analyzed; conopeptide components of the venom were isolated and investigated by high performance liquid chromatography, nuclear magnetic resonance, mass spectrometry, and automated Edman degradation sequencing. Five new peptide sequences are herein reported, among which there are three members of the M superfamily, one alpha conotoxin, and a conophan. The novel peptides comprise a partial peptide library of this particular cone.

Conus dalli is a cone snail species that preys upon mollusks (molluscivorous) and it belongs to the same clade as the better studied Conus textile. They have different biogeographical distribution: C. dalli is restricted to the Panamic area; whereas C. textile is a widespread species found from the Red Sea to Hawaii. The venom of C. textile is an extremely complex mixture of conopeptides characterized for their very high content of modified amino acids; particularly, for their high content of gamma-carboxy glutamate (Gla). Therefore, it is expected that the venom of C. dalli is equally complex and it might provide us with a library of novel conopeptides. We have collected 6 specimens of C. dalli from the Pacific coast of Panama. Their venom ducts were dissected and 40 mg of crude venom were extracted. Venom was separated using SE-HPLC and RP-HPLC and several single-component fractions with unique molecular weights have been found. 1D and 2D NMR methods in conjunction with mass spectrometry techniques have been applied to the main components of the venom. Three novel conopeptides have been isolated and characterized; dal_C1011h, dal_C0910, and dal_C0805g. dal_C1011h is a 27-residue hydrophobic conotoxin that belongs to O-superfamily, dal_C0910 is a 16-residue conotoxin that belongs to M-superfamily, and dal_C0805g is a 12-residue linear conopeptide the belong to the Conorfamide family. The details on the characterization of these conopeptides along with a comparison with previous data obtained from C. textile are presented.

Cone snails are marine gastropods belonging to the genus Conus that inhabit in tropical habitats throughout the world. They are predators that paralyze their prey by injection of venom, containing a complex mixture of conopeptides. The venom of cone snails has been found to be a valuable source of specific drugs for disorders ranging from stroke to chronic pain. For this work, the venom of Conus nux, a Panamic cone snail species, was extracted and analyzed. Components of the venom were isolated using Size Exclusion Chromatography (SEC) and Reversed Phase High Performance Liquid Chromatography techniques. A novel conopeptide sequence, determined by Edman Degradation is reported. The arrangement of the cysteines residues within this sequence suggest that it member of the M-superfamily of conotoxins.

Cone snails are carnivorous marine mollusks, utilizing their neuropeptide-rich venom for prey capture. The venom of Conus brunneus, a wide-spread Eastern Pacific vermivore, has not been extensively studied. In the current work, peptides from the dissected venom were characterized and tested using preliminary bioassays. Six peptides (A-F) were isolated and tested. Three peptide identities were determined by comparison with previously reported data: bru9a (A), bru3a (F), and an a-conotoxin (E). Preliminary screening in a stroke-related model of induced glutamate excitotoxicity in primary neuronal cells and PC12 cell cultures indicated potential neuroprotective activity of peptide fractions A, D, and F. Further testing is necessary to determine and verify structure, activity, target, and mechanism of action of the promising peptides from C. brunneus, which may prove effective neuropharmacological agents to treat stroke.

Cone snails are predatory marine gastropods that use venom for means of predation and defense. This venom is a complex mixture of conopeptides that selectivity binds to ion channels and receptors, giving them a wide range of potential pharmaceutical applications. Conus brunneus is a wide spread Eastern Pacific cone snail species that preys upon worms (vermivorous). Vermivorous cone snails have developed very specific biochemical strategies for the immobilization of their prey and their venom has not been extensively studied to date. The main objective of this dissertation is the characterization of novel conopeptides isolated from Conus brunneus. Chapter 1 is an introduction and background on cone snails and conopeptides. Chapter 2 details the isolation and characterization of a novel P-superfamily conotoxin. Chapter 3 presents the 3D solution structure of the novel P-superfamily conotoxin. Chapter 4 details the isolation and characterization of two novel M-superfamily conotoxins. Chapter 5 covers the use of nano-NMR to characterize a novel P-superfamily conotoxin using nanomole quantities of sample. Chapter 6 is a reprint of a paper published in the Journal of the American Chemical Society in which we combined and implemented techniques developed in the previous chapters to report the presence of D-(Sd(B-Hydroxyvaline in a polypeptide chain. This dissertation contains the first reported work of a P-superfamily structure obtained directly from the crude venom therefore accurately representing native post-translational modifications. In this paper, crude cone snail venom was characterized by: high performance liquid chromatography, nuclear magnetic resonance spectroscopy, nanonuclear magnetic resonance spectroscopy, mass spectrometry, amino acid analysis, Edman degradation sequencing, and preliminary bioassays.

Cone snails are predatory marine animals that rely on their venom components to immobilize and capture their prey. According to the type of prey preference, cone snails can be divided into three groups: vermivorous, molluscivorous and piscivorous. Conus ermineus had been identified as the only piscivorous snail of the Atlantic Ocean. Cone snail venom is a complex and rich sources of natural toxins. The majority of the components of the venom are peptidic in nature, and they act over different ionic channels and membrane receptors. Initial studies using mixture of venom collected from dissected venom ducts concluded that the venom from the same species do not exhibit unusual peptide polymorphism [Olivera, Hillyard, et al., 1995] and that the only major difference between individuals of the same species are different concentrations of the venom components [Vianna, et al., 2005]. For this study, peptides in the injected venom were collected from individual snails and characterized usin g analytical RP-HPLC for a maximum of three years. The different fractions collected were processed through capillary HPLC coupled with Q-TOF ESI-MS, and compared with analytical RP-HPLC fractions processed with MALDI-TOF MS. This study demonstrates that there is an animal-to-animal variation in the peptide components of the injected venom. The injected venom remains relatively constant over time for specific specimens in captivity. Finally, there are some peptides that had been found in all specimens both by MALDI-TOF MS and by ESI-MS. In this study, these peptides are called "molecular fingerprint" peptides. Based on matches of their derived masses to those predicted by published cDNA sequences, nine novel peptides were putatively identified. This study establishes that variations due to enzymatic posttranslational modification are omitted when we consider only information extrapolated from cDNA.