Comparative Venomics Reveals the Complex Prey Capture Strategy of the Piscivorous Cone Snail Conus catus.

Venomous marine cone snails produce a unique and remarkably diverse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.

Peptide toxin glacontryphan-M is present in the wings of the butterfly Hebomoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae).

Protein profiling has revealed the presence of glacontryphan-M, a peptide toxin identified only in the sea snail genus Conus, in the wings of Hebomoia glaucippe (HG). The wings and body of HG were homogenized and the proteins were extracted and analyzed by 2D gel electrophoresis with subsequent in-gel digestion. Posttranslational protein modifications were detected and analyzed by nano-LC-MS/MS. An antibody was generated against glacontryphan-M, and protein extracts from the wings of HG samples from Malaysia, Indonesia, and the Philippines were tested by immunoblotting. Glacontryphan-M was unambiguously identified in the wings of HG containing the following posttranslational protein modifications: monoglutamylation at E55, methylation at E53, quinone modification at W61, cyanylation at C56, and amidation of the C terminus at G63. Immunoblotting revealed the presence of the toxin in the wings of HG from all origins, showing a single band for glacontryphan-M in HG samples from Malaysia and Philippines and a double band in HG samples from Indonesia. Intriguingly, sequence analysis indicated that the Conus glacontryphan is identical to that of HG. The toxin may function as a defense against diverse predators, including ants, mantes, spiders, lizards, green frogs, and birds.

Metal ion determinants of conantokin dimerization as revealed in the X-ray crystallographic structure of the Cd(2+)/Mg (2+)-con-T[K7gamma] complex.

Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in gamma-carboxyglutamic acid (Gla) residues. As potent and selective antagonists of the N-methyl-D: -aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10, and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca(2+) and Mg(2+) can fulfill this role, Ca(2+) induces dimerization of con-G, whereas the Mg(2+)-complexed peptide remains monomeric. A variant of con-T, con-T[K7gamma] (gamma is Gla), contains Gla residues at the same five positions as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca(2+) ions and two Mg(2+) ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca(2+) for dimer formation, we report here the structure of the monomeric Cd(2+)/Mg(2+)-con-T[K7gamma] complex, and, by comparison with the previously published con-T[K7gamma]/Ca(2+) dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization.

A turripeptide from Polystira nobilis venom inhibits human α3ß2 and α7 nicotinic acetylcholine receptors.

Almost all marine snails within superfamily Conoidea produce venoms containing numerous neuroactive peptides. Most toxins characterized from members of this superfamily are produced by species belonging to family Conidae. These toxins (conotoxins) affect diverse membrane proteins, such as voltage- and ligand-gated ion channels, including nicotinic acetylcholine receptors (nAChRs). Family Turridae has been considerably less studied than their Conidae counterpart and, therefore, turrid toxins (turritoxins) have just been barely described. Consequently, in this work the most prominent chromatographic (RP-HPLC) fractions from the East Pacific species Polystira nobilis venom duct extract were isolated. The biological activity of six selected fractions was assayed on human (h) α7 AChRs expressed in Xenopus laevis oocytes. One of these fractions, F21, inhibited the acetylcholine-elicited response by 62 ± 12%. Therefore, this fraction was further purified and the F21-2 peptide was obtained. This peptide (at 5.6 µM) strongly and irreversibly inhibited the acetylcholine-induced response on hα7 and hα3ß2 nAChRs, by 55 ± 4 and 91 ± 1%, respectively. Electrospray mass spectrometry indicates that the average molecular mass of this toxin is 12 358.80 Da. The affinity for hα3ß2 nAChRs is high (IC50 of 566.2 nM). A partial sequence without cysteines was obtained by automated Edman degradation: WFRSFKSYYGHHGSVYRPNEPNFRSFAS…; blastp search revealed that this sequence has low similarity to some non-Cys-containing turripeptides. This is the first report of a turritoxin from a species of the American Pacific and the second description of a turripeptide inhibiting nAChRs.

Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin.

Maculotoxin, a potent neurotoxin isolated from the posterior salivary glands of the blue-ringed octopus. Hapalochlaena maculosa, has now been identified as tetrodotoxin. This is the first reported case in which tetrodotoxin has been found to occur in a venom.

Peptide neurotoxins from fish-hunting cone snails.

To paralyze their more agile prey, the venomous fish-hunting cone snails (Conus) have developed a potent biochemical strategy. They produce several classes of toxic peptides (conotoxins) that attack a series of successive physiological targets in the neuromuscular system of the fish. The peptides include presynaptic omega-conotoxins that prevent the voltage-activated entry of calcium into the nerve terminal and release of acetylcholine, postsynaptic alpha-conotoxins that inhibit the acetylcholine receptor, and muscle sodium channel inhibitors, the mu-conotoxins, which directly abolish muscle action potentials. These distinct peptide toxins share several common features: they are relatively small (13 to 29 amino acids), are highly cross-linked by disulfide bonds, and strongly basic. The fact that they inhibit sequential steps in neuromuscular transmission suggests that their action is synergistic rather than additive. Five new omega-conotoxins that block presynaptic calcium channels are described. They vary in their activity against different vertebrate classes, and also in their actions against different synapses from the same animal. There are susceptible forms of the target molecule in peripheral synapses of fish and amphibians, but those of mice are resistant. However, the mammalian central nervous system is clearly affected, and these toxins are thus of potential significance for investigating the presynaptic calcium channels.

Diversity of Conus neuropeptides.

Conus venoms contain a remarkable diversity of pharmacologically active small peptides. Their targets are ion channels and receptors in the neuromuscular system. The venom of Conus geographus contains high-affinity peptides that act on voltage-sensitive calcium channels, sodium channels, N-methyl-D-aspartate (NMDA) receptors, acetylcholine receptors, and vasopressin receptors; many more peptides with still uncharacterized receptor targets are present in this venom. It now seems that the Conus species (approximately 500 in number) will each use a distinctive assortment of peptides and that the pharmacological diversity in Conus venoms may be ultimately comparable to that of plant alkaloids or secondary metabolites of microorganisms. The cone snails may generate this diverse spectrum of venom peptides by a "fold-lock-cut" synthetic pathway. These peptides are specific enough to discriminate effectively between closely related receptor subtypes and can be used for structure-function correlations.

Inactivation of a serotonin-gated ion channel by a polypeptide toxin from marine snails.

The venom of predatory marine snails is a rich source of natural products that act on specific receptors and ion channels within the mammalian nervous system. A 41-amino acid peptide, final sigma-conotoxin GVIIIA, was purified on the basis of its ability to inactivate the 5-HT3 receptor, an excitatory serotonin-gated ion channel. final sigma-Conotoxin contains a brominated tryptophan residue, which may be important for peptide activity because the endogenous ligand for the 5-HT3 receptor is a hydroxylated derivative of tryptophan. final sigma-Conotoxin inactivates the 5-HT3 receptor through competitive antagonism and is a highly selective inhibitor of this receptor. Serotonin receptors can now be included among the molecular targets of natural polypeptide neurotoxins.

Novel conorfamides from Conus austini venom modulate both nicotinic acetylcholine receptors and acid-sensing ion channels.

Conorfamides are a poorly studied family of cone snail venom peptides with broad biological activities, including inhibition of glutamate receptors, acid-sensing ion channels, and voltage-gated potassium channels. The aim of this study was to characterize the pharmacological activity of two novel linear conorfamides (conorfamide_As1a and conorfamide_As2a) and their non-amidated counterparts (conopeptide_As1b and conopeptide_As2b) that were isolated from the venom of the Mexican cone snail Conus austini. Although As1a, As2a, As1b and As2b were identified by activity-guided fractionation using a high-throughput fluorescence imaging plate reader (FLIPR) assay assessing α7 nAChR activity, sequence determination revealed activity associated with four linear peptides of the conorfamide rather than the anticipated α-conotoxin family. Pharmacological testing revealed that the amidated peptide variants altered desensitization of acid-sensing ion channels (ASICs) 1a and 3, and the native lysine to arginine mutation differentiating As1a and As1b from As2a and As2b introduced ASIC1a peak current potentiation. Surprisingly, these conorfamides also inhibited α7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. This is the first report of conorfamides with dual activity, with the nAChR activity being the most potent molecular target of any conorfamide discovered to date.

Structural characterization, teratogenicity and in vitro avian antimicrobial activity of posterior salivary gland (PSG) toxin from cuttlefish, Sepia prashadi.

A low molecular weight posterior salivary gland (PSG) toxin was isolated and purified from the cuttlefish Sepia prashadi by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The protein and neutral sugar content of the PSG toxin was determined to be 1.033 mg/g and 282 µg/g. Fourier Transform Infrared (FT-IR) spectroscopy revealed the presence of υ-OH, υ-CO and δ-NH functional groups. Circular Dichroism (CD) spectroscopy and K2D2 analysis quantified the presence of 38.39% α-helix and 9.25% ß-sheet and 52.36% of ß-turn. Matrix Assisted Laser Desorption/Ionization-Time-of Flight/Mass Spectrometry (MALDI-TOF/MS) and MASCOT analysis revealed the amino acid sequence of MEMQSKQQNSKAPANRKIFPWMKTSAVATASKRVEMASLLNLQERQIKIWFQNRMKQKSQQPQTR (1.92 kDa) homologous to homeobox protein H4 of pufferfish, T. rubripes. The PSG toxin showed differential stability with pH and induced premature hatching in Zebrafish eggs and dose dependant developmental malformations in embryos with a Maximum tolerated dose of 1.85 µM. The PSG toxin exhibited significant antibacterial activity with pronounced zone of inhibition against S. typhimurium (12.94 mm) and inhibited avian RBC binding of Newcastle Disease virus (NDV) at a titre value of 1/4. The present study strongly advocates the biomedical potential of the PSG toxin from S. prashadi and illustrates its promise as a potential avian antimicrobial agent of the future.

Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks.

A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPM gammaDPLgammaIIRI-nh2, (gamma, gamma-carboxyglutamate; -nh2, amidated C-terminus; calculated monoisotopic mass, 1468.72Da; experimental monoisotopic mass, 1468.70Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.

Purification and molecular cloning of SE-cephalotoxin, a novel proteinaceous toxin from the posterior salivary gland of cuttlefish Sepia esculenta.

Cephalopods contain toxins in their salivary glands, presumably to paralyze prey animals such as crabs and bivalves. Proteinaceous toxins (called cephalotoxins) with crab lethality have previously been purified from three species of octopodiform cephalopods (octopuses) but their detailed properties and primary structures have remained unknown. In this study, salivary glands of six species of decapodiform cephalopods were newly found to be toxic; three species of cuttlefish were lethal only to crabs and three species of squid to both mice and crabs. A proteinaceous toxin (named SE-cephalotoxin) in the salivary gland of cuttlefish Sepia esculenta was soluble only in high-salt solvents. This unique solubility enabled us to purify SE-cephalotoxin by gel filtration HPLC and hydroxyapatite HPLC. SE-cephalotoxin was shown to be a 100 kDa monomeric glycoprotein with an LD(50) (against crabs) of 2 microg/kg. Based on the determined partial amino acid sequence, a full-length cDNA (3402 bp) coding for SE-cephalotoxin was cloned by RT-PCR and RACE. The SE-cephalotoxin precursor protein (1052 amino acid residues) is composed of a signal peptide (region 1-21), propeptide (region 22-29) and mature protein (region 30-1052). A database search failed to find any proteins sharing homology with SE-cephalotoxin.

Isolation and characterization of Conohyal-P1, a hyaluronidase from the injected venom of Conus purpurascens.

Hyaluronidases are ubiquitous enzymes commonly found in venom and their main function is to degrade hyaluran, which is the major glycosaminoglycan of the extracellular matrix in animal tissues. Here we describe the purification and characterization of a 60kDa hyaluronidase found in the injected venom from Conus purpurascens, Conohyal-P1. Using a combined strategy based on transcriptomic and proteomic analysis, we determined the Conohyal-P1 sequence. Conohyal-P1 has conserved consensus catalytic and positioning domain residues characteristic of hyaluronidases and a C-terminus EGF-like domain. Additionally, the enzyme is expressed as a mixture of glycosylated isoforms at five asparagine sites. The activity of the native Conohyal-P1 was assess MS-based methods and confirmed by classical turbidimetric methods. The MS-based assay is particularly sensitive and provides the first detailed analysis of a venom hyaluronidase activity monitored with this method. The discovery of new hyaluronidases and the development of techniques to evaluate their performance can advance several therapeutic procedures, as these enzymes are widely used for enhanced drug delivery applications. BIOLOGICAL SIGNIFICANCE: Cone snail venom is a remarkable source of therapeutically important molecules, as is the case of conotoxins, which have undergone extensive clinical trials for several applications. In addition to the conotoxins, a large array of proteins have been reported in the venom of several species of cone snails, including enzymes that were found in dissected and injected Conus venom. Here we describe the isolation and characterization of the hyaluronidase Conohyal-P1 from the injected venom of C. purpurascens. We employed a combined transcriptomic and proteomic analysis to obtain the full sequence of this hyaluronidase. The activity of Conohyal-P1 was assessed by a mass spectrometry-based method, which provide the first detailed venom hyaluronidase activity analysis monitored by mass spectrometry allowing the visualization of the substrate degradation by the enzyme.

Enhancing the therapeutic potential of peptide toxins.

INTRODUCTION: Peptide toxins are potent and often exquisitely selective probes of the structure and function of ion channels and receptors, and as such are of significant interest to the pharmaceutical and biotech industries as both therapeutic leads and pharmacological tools. Their progression as clinical candidates, however, faces many of the challenges that are common to peptide drugs generally. Areas covered: The attributes of peptide toxins as therapeutic leads are outlined, as well as some of the limiting factors that have hampered the clinical development of many promising candidates. Strategies to overcome or circumvent these limitations are described, and their applications to peptide toxins from cone snails, sea anemones and scorpions are exemplified. Expert opinion: Peptide toxins have exceeded their promise as valuable pharmacological tools but have yet to yield the anticipated bounty of therapeutic leads. As the number of new peptides identified in venom transcriptomes and proteomes expands rapidly, screening approaches that capture those with genuine therapeutic potential are required, along with methods for enhancing the stability, pharmacokinetics and pharmacodynamics of these peptides.

Isolation of yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'-->1'')-beta-L-arabinofuranoside] from Protoceratium reticulatum.

Yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'-->1'')-beta-L-arabinofuranoside] (3) was isolated from extracts of Protoceratium reticulatum during a large scale isolation of yessotoxin (1). The structure was characterized by mass spectrometry and NMR spectroscopy. Di-glycoside-3, along with the corresponding mono-glycoside (2) were detected in cultures of P. reticulatum originating from Europe and New Zealand, suggesting that production of arabinosides of 1 is a normal feature of this alga. Formation of multiply charged anions and fragmentation of 3 occurred much more readily than for 1 and 2 under the LC-MS conditions used in this study.

Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors.

Until now, there have been no antagonists to discriminate between heteromeric nicotinic acetylcholine receptors (nAChRs) containing the very closely related alpha6 and alpha3 subunits. nAChRs containing alpha3, alpha4, or alpha6 subunits in combination with beta2, occasionally beta4, and sometimes beta3 or alpha5 subunits, are thought to play important roles in cognitive function, pain perception, and the reinforcing properties of nicotine. We cloned a novel gene from the predatory marine snail Conus purpurascens. The predicted peptide, alpha-conotoxin PIA, potently blocks the chimeric alpha6/alpha3beta2beta3 subunit combination as expressed in oocytes but neither the muscle nor the major neuronal nAChR alpha4beta2. Additionally, this toxin is the first described ligand to discriminate between nAChRs containing alpha6 and alpha3 subunits. Exploiting the unusual intron conservation of conotoxin genes may represent a more general approach for defining conotoxin ligand scaffolds to discriminate among closely related receptor populations.

Discovery by proteogenomics and characterization of an RF-amide neuropeptide from cone snail venom.

In this study, a proteogenomic annotation strategy was used to identify a novel bioactive peptide from the venom of the predatory marine snail Conus victoriae. The peptide, conorfamide-Vc1 (CNF-Vc1), defines a new gene family. The encoded mature peptide was unusual for conotoxins in that it was cysteine-free and, despite low overall sequence similarity, contained two short motifs common to known neuropeptides/hormones. One of these was the C-terminal RF-amide motif, commonly observed in neuropeptides from a range of organisms, including humans. The mature venom peptide was synthesized and characterized structurally and functionally. The peptide was bioactive upon injection into mice, and calcium imaging of mouse dorsal root ganglion (DRG) cells revealed that the peptide elicits an increase in intracellular calcium levels in a subset of DRG neurons. Unusually for most Conus venom peptides, it also elicited an increase in intracellular calcium levels in a subset of non-neuronal cells. BIOLOGICAL SIGNIFICANCE: Our findings illustrate the utility of proteogenomics for the discovery of novel, functionally relevant genes and their products. CNF-Vc1 should be useful for understanding the physiological role of RF-amide peptides in the molluscan and mammalian nervous systems.

A new Conus peptide ligand for Ca channel subtypes.

A cDNA clone encoding a new omega-conotoxin was identified from Conus magus. The predicted peptide was chemically synthesized using a novel strategy that efficiently yielded the biologically active disulfide-bonded isomer. This peptide, omega-conotoxin MVIID, targets other voltage-gated calcium channels besides the N-subtype and exhibits greater discrimination against the N-channel subtype than any other omega-conotoxin variant to date. Consequently, omega-conotoxin MVIID may be a particularly useful ligand for calcium channel subtypes that are not of the L- or N-subclasses. Of the eight major sequence variants of omega-conotoxins that have been elucidated, four come from Conus magus venom. We suggest that sequence variants from the same venom may be designed to optimally interact with different molecular variants of calcium channels; such omega-conotoxin sets from a single venom may therefore be useful for helping to identify novel calcium channel subtypes.

Inhibition of evoked neurotransmitter release from rat hippocampus by a polypeptide toxin isolated from the marine snail Conus distans.

The active fraction, isolated and partially purified from the crude venom of the marine snail Conus distans, with a molecular mass of about 25 kDa, inhibits neurotransmitter release in rat hippocampus. This toxin (distans Toxin) inhibits the electrically evoked tritium labelled noradrenaline release from rat hippocampal slices in a dose and time dependent manner. The neurotransmitter release is mainly regulated by N-type of voltage sensitive Ca(2+)-channels. The distans toxin behaves as a partial antagonist of calcium in the buffer, possibly by competing with calcium for this type of voltage sensitive Ca(2+)-channels.

A toxin from the venom of the predator snail Conus textile modulates ionic currents in Aplysia bursting pacemaker neuron.

Conus textile crude venom and a peptide component ('King Kong' toxin) purified from this venom, alter membrane excitability of Aplysia neurons. Venom, applied to the medium bathing an abdominal ganglion, changes dramatically the electrical activity of bursting pacemaker neuron. The effects on bursting neuron R15 was examined in current-clamp and voltage-clamp modes. A dual phase effect of both the venom and the purified toxin were observed. The first phase starts immediately after venom or toxin application and is observed as an increase in membrane excitability, resulting in an enhancement of bursting. The second phase begins about 15 min later and consists of a long-lasting hyperpolarization. The dual phase effect of the venom and the toxin persists even when synaptic input is eliminated either by axotomy, or by recording from freshly dissociated neurons or from neurons in primary cell culture. The ionic currents affected are an inward current, INSR, which is activated upon depolarization and an anomalously rectifying potassium current, IR, which is activated upon hyperpolarization. In the first phase of toxin action INSR is increased. In the second phase both the venom and the toxin block INSR and increase IR. The toxin effects may be due to complex alteration of one or more second messenger cascades rather than a direct action on ion channels.