A novel 13 residue acyclic peptide from the marine snail, Conus monile, targets potassium channels

A novel 13-residue peptide Mo1659 has been isolated from the venom of a vermivorous cone snail, Conus monile. HPLC fractions of the venom extract yielded an intense UV absorbing fraction with a mass of 1659 Da. De novo sequencing using both matrix assisted laser desorption and ionization and electrospray MS/MS methods together with analysis of proteolytic fragments successfully yielded the amino acid sequence, FHGGSWYRFPWGY-NH2. This was further confirmed by comparison with the chemically synthesized peptide and by conventional Edman sequencing. Mo1659 has an unusual sequence with a preponderance of aromatic residues and the absence of apolar, aliphatic residues like Ala, Val, Leu, and Ile. Mo1659 has no disulfide bridges distinguishing it from the conotoxins and bears no sequence similarity with any of the acyclic peptides isolated thus far from the venom of cone snails. Electrophysiological studies on the effect of Mo1659 on measured currents in dorsal root ganglion neurons suggest that the peptide targets non-inactivating voltage-dependent potassium channels

Novel peptides of therapeutic promise from Indian conidae

Highly structured small peptides are the major toxic constituents of the venom of cone snails, a family of widely distributed predatory marine molluscs. These animals use the venom for rapid prey immobilization. The peptide components in the venom target a wide variety of membrane-bound ion channels and receptors. Many have been found to be highly selective for a diverse range of mammalian ion channels and receptors associated with pain-signaling pathways. Their small size, structural stability, and target specificity make them attractive pharmacologic agents. A select number of laboratories mainly from the United States, Europe, Australia, Israel, and China have been engaged in intense drug discovery programs based on peptides from a few snail species. Coastal India has an estimated 20-30% of the known cone species; however, few serious studies have been reported so far. We have begun a comprehensive program for the identification and characterization of peptides from cone snails found in Indian Coastal waters. This presentation reviews our progress over the last 2 years. As expected from the evolutionary history of these venom components, our search has yielded novel peptides of therapeutic promise from the new species that we have studied

Tyrosine-rich conopeptides affect voltage-gated K+ channels

Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12-18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom

Effect of ester chemical structure and peptide bond conformation in fragmentation pathways of differently metal cationized cyclodepsipeptides

Fragmentation behavior of two classes of cyclodepsipeptides, isariins and isaridins, obtained from the fungus Isaria, was investigated in the presence of different metal ions using multistage tandem mass spectrometry (MS(n)) with collision induced dissociation (CID) and validated by NMR spectroscopy. During MS(n) process, both protonated and metal-cationized isariins generated product ions belonging to the identical `b-ion' series, exhibiting initial backbone cleavage explicitly at the beta-ester bond. Fragmentation behavior for the protonated and metal-cationized acyclic methyl ester derivative of isariins was very similar. On the contrary, isaridins during fragmentation produced ions belonging to the `b' or/and the `y' ion series depending on the nature of interacting metal ions, due to initial backbone cleavages at the beta-ester linkage or/and at a specific amide linkage. Interestingly, independent of the nature of the interacting metal ions, the product ions formed from the acyclic methyl ester derivative of isaridins belonged only to the `y-type'. Complementary NMR data showed that, while all metal ions were located around the beta-ester group of isariins, the metal ion interacting sites varied across the backbone for isaridins. Combined MS and NMR data suggest that the different behavior in sequence specific charge-driven fragmentation of isariins and isaridins is predetermined because of the constituent beta-hydroxy acid residue in isariins and the cis peptide bond in isaridins

Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels

Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated $Ca^{2+}$ channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, $GCP^D WDPWC-NH_2$ and Am975, $GCO^D WDPWC-NH_2$ (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated $Ca^{2+}$ channels. While Lo959 increases the $Ca^{2+}$ current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels

Solution structure of δ\delta-Am2766: A highly hydrophobic δ \delta-conotoxin from Conus amadis that inhibits inactivation of neuronal voltage-gated sodium channels

The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM

Sodium channel modulating activity in a N-conotoxin from an Indian marine snail

Abstract A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH 2 has been isolated and puri¢ed from the venom of the molluscivorous snail, Conus amadis, collected o¡ the southeastern coast of India. Chemical modi¢cation and mass spectrometric studies establish that Am 2766 has three disul¢de bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the N N-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na + channel, stably expressed in Chinese hamster ovary cells. Unlike N N-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.

De Novo Sequencing and Disulfide Mapping of a Bromotryptophan-Containing Conotoxin by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, -CC-CC, and disulfide linkage as those of the T-1-family members. The complete sequence, $GN\underline{W}CCSARVCC$, in which $\underline{W}$ denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)-phosphine, followed by differential alkylation with Nethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond