Venom Peptides From Cone Snails: Pharmacological Probes for Voltage-Gated Sodium Channels.

The venoms of cone snails provide a rich source of neuroactive peptides (conotoxins). Several venom peptide families have been identified that are either agonists (ι- and δ-conotoxins) or antagonists (µ- and µO-conotoxins) of voltage-gated sodium channels (VGSCs). Members of these conotoxin classes have been integral in identifying and characterizing specific neurotoxin binding sites on the channel. Furthermore, given the specificity of some of these peptides for one sodium channel subtype over another, conotoxins have also proven useful in exploring differences between VGSC subtypes. This chapter summarizes the current knowledge of the structure and function based on the results of conotoxin interactions with VGSCs and correlates the peptides with the phylogeny of the Conus species from which they were derived.

One, four or 100 genera? A new classification of the cone snails.

We present a new classification for the genus Conus sensu lato (family Conidae), based on molecular phylogenetic analyses of 329 species. This classification departs from both the traditional classification in only one genus and from a recently proposed shell- and radula-based classification scheme that separates members of this group into five families and 115 genera. Roughly 140 genus-group names are available for Recent cone snails. We propose to place all cone snails within a single family (Conidae) containing four genera-Conus, Conasprella, Profundiconus and Californiconus (with Conus alone encompassing about 85% of known species)-based on the clear separation of cone snails into four distinct and well-supported groups/lineages in molecular phylogenetic analyses. Within Conus and Conasprella, we recognize 57 and 11 subgenera, respectively, that represent well-supported subgroupings within these genera, which we interpret as evidence of intrageneric distinctiveness. We allocate the 803 Recent species of Conidae listed as valid in the World Register of Marine Species into these four genera and 71 subgenera, with an estimate of the confidence for placement of species in these taxonomic categories based on whether molecular or radula and/or shell data were used in these determinations. Our proposed classification effectively departs from previous schemes by (1) limiting the number of accepted genera, (2) retaining the majority of species within the genus Conus and (3) assigning members of these genera to species groups/subgenera to enable the effective communication of these groups, all of which we hope will encourage acceptance of this scheme.

Molecular phylogeny and evolution of the cone snails (Gastropoda, Conoidea).

We present a large-scale molecular phylogeny that includes 320 of the 761 recognized valid species of the cone snails (Conus), one of the most diverse groups of marine molluscs, based on three mitochondrial genes (COI, 16S rDNA and 12S rDNA). This is the first phylogeny of the taxon to employ concatenated sequences of several genes, and it includes more than twice as many species as the last published molecular phylogeny of the entire group nearly a decade ago. Most of the numerous molecular phylogenies published during the last 15years are limited to rather small fractions of its species diversity. Bayesian and maximum likelihood analyses are mostly congruent and confirm the presence of three previously reported highly divergent lineages among cone snails, and one identified here using molecular data. About 85% of the species cluster in the single Large Major Clade; the others are divided between the Small Major Clade (∼12%), the Conus californicus lineage (one species), and a newly defined clade (∼3%). We also define several subclades within the Large and Small major clades, but most of their relationships remain poorly supported. To illustrate the usefulness of molecular phylogenies in addressing specific evolutionary questions, we analyse the evolution of the diet, the biogeography and the toxins of cone snails. All cone snails whose feeding biology is known inject venom into large prey animals and swallow them whole. Predation on polychaete worms is inferred as the ancestral state, and diet shifts to molluscs and fishes occurred rarely. The ancestor of cone snails probably originated from the Indo-Pacific; rather few colonisations of other biogeographic provinces have probably occurred. A new classification of the Conidae, based on the molecular phylogeny, is published in an accompanying paper.

[Conotoxins: from the biodiversity of gastropods to new drugs].

A review describes general trends in research of conotoxins that are peptide toxins isolated from sea gastropods of the Conus genus, since the toxins were discovered in 1970th. There are disclosed a conotoxin classification, their structure diversity and different ways of action to their molecular targets, mainly, ion channels. In the applied aspect of conotoxin research, drug discovery and development is discussed, the drugs being based on conotoxin structure. A first exemplary drug is a ziconotide, which is an analgesic of new generation.

Molecular phylogeny, classification and evolution of conopeptides.

Conopeptides are toxins expressed in the venom duct of cone snails (Conoidea, Conus). These are mostly well-structured peptides and mini-proteins with high potency and selectivity for a broad range of cellular targets. In view of these properties, they are widely used as pharmacological tools and many are candidates for innovative drugs. The conopeptides are primarily classified into superfamilies according to their peptide signal sequence, a classification that is thought to reflect the evolution of the multigenic system. However, this hypothesis has never been thoroughly tested. Here we present a phylogenetic analysis of 1,364 conopeptide signal sequences extracted from GenBank. The results validate the current conopeptide superfamily classification, but also reveal several important new features. The so-called "cysteine-poor" conopeptides are revealed to be closely related to "cysteine-rich" conopeptides; with some of them sharing very similar signal sequences, suggesting that a distinction based on cysteine content and configuration is not phylogenetically relevant and does not reflect the evolutionary history of conopeptides. A given cysteine pattern or pharmacological activity can be found across different superfamilies. Furthermore, a few conopeptides from GenBank do not cluster in any of the known superfamilies, and could represent yet-undefined superfamilies. A clear phylogenetically based classification should help to disentangle the diversity of conopeptides, and could also serve as a rationale to understand the evolution of the toxins in the numerous other species of conoideans and venomous animals at large.

Conolysin-Mt: a conus peptide that disrupts cellular membranes.

Conus venoms are estimated to comprise over 100,000 distinct pharmacologically active peptides, the majority probably targeting ion channels. Through the characterization of a cytolytic peptide from the venom of Conus mustelinus, conolysin-Mt, we expand the known conopeptide mechanisms to include association with and destruction of cellular membranes. A new 23AA conopeptide, conolysin-Mt has potent hemolytic activity when tested on human erythrocytes. At a concentration of 0.25 microM, the peptide permeabilized both negatively charged prokaryotic (PE:PG) and zwitterionic eukaryotic (PC:cholesterol) model membranes. The affinity constants (KA) of conolysin-Mt for PE:PG and PC:cholesterol model membranes were 0.9 +/- 0.3 x 10(7) and 3 +/- 1 x 10(7) M-1, respectively. In contrast, conolysin-Mt exhibited low antimicrobial activity (MIC > 50 microM) against two Escherichia coli strains, with an MIC for the Gram-positive S. aureus of 25-50 microM. The specificity of conolysin-Mt for native eukaryotic membranes is a novel feature of the peptide compared to other well-characterized cytolytic peptides such as melittin.

Biochemical characterization of Drosophila gamma-glutamyl carboxylase and its role in fly development.

To investigate structure-function relationships in gamma-glutamyl carboxylases, the enzyme from Drosophila melanogaster was characterized. Four cysteine residues were shown to be important determinants for enzymatic activity. Native Drosophila substrates have not yet been identified, but propeptides of human prothrombin and factor IX are recognized by the Drosophila enzyme. The presence of the propeptide region increased apparent affinity by approximately 200-fold, and mutation of a hydrophobic residue of factor IX propeptide (F-16A) decreased carboxylation by 90%, as in the human enzyme. Substrate recognition appears to be highly conserved between the human and Drosophila gamma-glutamyl carboxylases. Inactivation of Drosophila gamma-glutamyl carboxylase by non-sense mutations or insertional mutagenesis by P-element insertion have no apparent effects on growth and fertility under laboratory conditions.

Conotoxins and the posttranslational modification of secreted gene products.

The venoms of predatory cone snails (genus Conus) have yielded a complex library of about 50-100,000 bioactive peptides, each believed to have a specific physiological target (although peptides from different species may overlap in their target specificity). Conus has evolved the equivalent of a drug development strategy that combines the accelerated evolution of toxin sequences with an unprecedented degree of posttranslational modification. Some Conus venom peptide families are the most highly posttranslationally modified classes of gene products known. We review the variety and complexity of posttranslational modifications documented in Conus peptides so far, and explore the potential of Conus venom peptides as a model system for a more general understanding of which secreted gene products may have modified amino acids. Although the database of modified conotoxins is growing rapidly, there are far more questions raised than answers provided about possible mechanisms and functions of posttranslational modifications in Conus.

Conophysin-R, a Conus radiatus venom peptide belonging to the neurophysin family.

A novel Conus peptide, conophysin-R, was purified from the venom of Conus radiatus. The distinctive disulfide framework and sequence indicates that it is a member of the neurophysin peptide family. The complete sequence of the peptide is HPTKPCMYCSFGQCVGPHICCGPTGCEMGTAEANMCSEEDEDPIPCQVFGSDCALNNPDNIHGHCVADGICCVDDTCTTHLGCLThis is the first time a neurophysin-like peptide has been found in any venom. In addition, conophysin-R is the first neurophysin family member isolated and biochemically characterized from an invertebrate source.

Conorfamide, a Conus venom peptide belonging to the RFamide family of neuropeptides.

A novel Conus peptide, conorfamide-Sr1, has been characterized. The sequence of the natural peptide was determined using standard Edman sequencing methods and mass spectrometry, and confirmed by chemical synthesis. The peptide has 12 amino acids and no cysteine residues. The following sequence was obtained: GPMGWVPVFYRF-NH(2). No other peptide from a vermivorous Atlantic Conus species has previously been characterized. Conorfamide-Sr1 belongs to the RFamide neuropeptide family, and is the first RFamide peptide to be found in any venom. The presence of conorfamide-Sr1 as a major peptide in Conus spurius venom suggests that Conus lineages in the Atlantic may have evolved novel Conus venom peptide families.

Delta-conotoxin structure/function through a cladistic analysis.

Delta-conotoxins are Conus peptides that inhibit inactivation of voltage-gated sodium channels. The suggestion that delta-conotoxins might be an essential component of the venoms of fish-hunting cone snails which rapidly immobilize their prey [Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151] has not been tested. On the basis of cDNA cloning, all of the fish-hunting Conus analyzed yielded at least one delta-conotoxin sequence. In addition, one delta-conotoxin isolated from the venom of Conus striatus had an amino acid sequence identical to that predicted from cDNA cloning. This new peptide exhibited properties of delta-conotoxins: it targeted sodium channels and potentiated action potentials by slowing channel inactivation. Homologous sequences of delta-conotoxins from two groups (clades) of related fish-hunting Conus species share consensus features but differ significantly from the two known delta-conotoxins from mollusc-hunting Conus venoms. Three large hydrophobic amino acids were conserved; analogues of the previously described delta-conotoxin PVIA with alanine substituted for the conserved amino acids F9 and I12 lost substantial biological activity. In contrast, both the T8A and K13A delta-conotoxin PVIA analogues, where substitutions were at nonconserved loci, proved to be biologically active. Taken together, our results indicate that a cladistic approach can identify amino acids critical for the activity of conotoxins and provide extensive information as to which amino acid substitutions can be made without significant functional consequences.

Venomous cone snails: molecular phylogeny and the generation of toxin diversity.

In order to investigate the generation of conotoxin diversity, delta-conotoxin sequences from nine Conus species were analyzed in the context of their phylogeny. Using a standard molecular marker, mitochondrial 16S RNA, we determined that the delta-conotoxins were derived from three distinct species clades based on the phylogenetic reconstruction of a large set (>80) of Conus species and other toxoglossate molluscs. Four different mechanisms appear to have contributed to the diversity of the delta-conotoxins analyzed: (1) Speciation: Delta-conotoxins in different species diverge from each other (the prepro regions of orthologous genes somewhat more slowly than the reference rRNA rate, the mature toxin regions significantly faster). (2) Duplication: Intraspecific delta-conotoxin divergence is initiated by gene duplication events, some of which may have predated the species itself. (3) Recombination: A novel delta-conotoxin may arise through recombination of two parental delta-contoxin genes. (4) 'Focal hypermutation': This sudden, almost saltatory change in sequence is always restricted to the mature toxin region. The first three have been recognized previously as mechanisms important for the evolution of gene families in other phylogenetic systems; the last is a remarkable, mechanistically unexplained and specialized feature of Conus peptide diversification.

Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels.

It has previously been shown that nicotine-evoked dopamine release from rat striatal synaptosomes and nicotine-evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage-gated calcium channels was examined. Cd(2+) (200 microM), as well as omega-conotoxin MVIIC (5 microM), blocks approximately 85% of nicotine-evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin-susceptibility suggests that these calcium channels contain alpha(1A) and/or alpha(1B) subunits. Inhibition of nicotine-evoked dopamine release by conotoxins alpha-MII and omega-GVIA is additive and indicates that presynaptic alpha3beta2 nAChRs are functionally coupled to alpha(1A), but not alpha(1B), calcium channel subtypes. Conversely, insensitivity to alpha-AuIB and sensitivity to omega-MVIIC indicate that non-alpha3beta2/alpha3beta4-containing nAChRs are functionally coupled to alpha(1B)-containing calcium channels. In contrast, Cd(2+) blocks only 65% of nicotine-evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd(2+)-insensitive component of release is blocked by alpha-AuIB and therefore appears to be triggered by Ca(2+) flowing directly through the channels of presynaptic alpha3beta4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage-gated calcium channels.

Autoradiographic localization of N-type VGCCs in gerbil hippocampus and failure of omega-conotoxin MVIIA to attenuate neuronal injury after transient cerebral ischemia.

In the mammalian central nervous system, transient global ischemia of specific duration causes selective degeneration of CA1 pyramidal neurons in hippocampus. Many of the ischemia-induced pathophysiologic cascades that destroy the neurons are triggered by pre- and postsynaptic calcium entry. Consistent with this, many calcium channel blockers have been shown to be neuroprotective in global models of ischemia. omega-Conotoxin MVIIA, a selective N-type VGCC blocker isolated from the venom of Conus magus, protects CA1 neurons in the rat model of global ischemia, albeit transiently. The mechanism by which this peptide renders neuroprotection is unknown. We performed high-resolution receptor autoradiography with the radiolabeled peptide and observed highest binding in stratum lucidum of CA3 subfield, known to contain inhibitory neurons potentially important in the pathogenesis of delayed neuronal death. This finding suggested that the survival of stratum lucidum inhibitory neurons might be the primary event, leading to CA1 neuroprotection after ischemia. Testing of this hypothesis required the reproduction of its neuroprotective effects in the gerbil model of global ischemia. Surprisingly, we found that omega-MVIIA did not attenuate CA1 hippocampal injury after 5 min of cerebral ischemia in gerbil. Possible reasons are discussed. Lastly, we show that the peptide can be used as a synaptic marker in assessing short and long-term changes that occur in hippocampus after ischemic injury.

Contryphans from Conus textile venom ducts.

Contryphans are unusual Conus peptides which contain a distinctive post-translational modification, D-tryptophan or D-leucine. cDNA clones encoding new contryphans from the mollusc-hunting cone snail Conus textile were identified and the inferred mature peptides were synthesized: contryphan-Tx (Gly-Cys-Hyp-D-Trp-Gln-Pro-Tyr-Cys-NH(2)), Leu-contryphan-Tx (Cys-Val-D-Leu-Tyr-Pro-Trp-Cys-NH(2)) and contryphan R/Tx which is identical to contryphan-R [Jimenez et al., 1996. Contryphan is a D-tryptophan containing Conus peptide. J. Biol. Chem. 281, 28002-28005]. Leu-contryphan-Tx exhibits a single peak, but contryphan-Tx shows two peaks under reverse-phase high-performance liquid chromatography conditions. Ultraviolet resonance Raman spectroscopy demonstrates a difference in the D-tryptophan dihedral angle for the two contryphan-Tx equilibrium conformers. Both the sequences and in vivo effects of all contryphans isolated suggest that there are two major branches of the contryphan family.

On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems. Evidence for a gamma-glutamyl carboxylase in Drosophila.

The vitamin K-dependent gamma-carboxylation of glutamate to gamma-carboxyglutamate was originally well characterized in the mammalian blood clotting cascade. gamma-Carboxyglutamate has also been found in a number of other mammalian proteins and in neuropeptides from the venoms of marine snails belonging to the genus Conus, suggesting wider prevalence of gamma-carboxylation. We demonstrate that an open reading frame from a Drosophila melanogaster cDNA clone encodes a protein with vitamin K-dependent gamma-carboxylase activity. The open reading frame, 670 amino acids in length, is truncated at the C-terminal end compared with mammalian gamma-carboxylase, which is 758 amino acids. The mammalian gene has 14 introns; in Drosophila there are two much shorter introns but in positions precisely homologous to two of the mammalian introns. In addition, a deletion of 6 nucleotides is observed when cDNA and genomic sequences are compared. In situ hybridization to fixed embryos indicated ubiquitous presence of carboxylase mRNA throughout embryogenesis. Northern blot analysis revealed increased mRNA levels in 12-24-h embryos. The continued presence of carboxylase mRNA suggests that it plays an important role during embryogenesis. Although the model substrate FLEEL is carboxylated by the enzyme, a substrate containing the propeptide of a Conus carboxylase substrate, conantokin G, is poorly carboxylated. Its occurrence in vertebrates, molluscan systems (i.e. Conus), and Drosophila and the apparently strong homology between the three systems suggest that this is a highly conserved and widely distributed post-translational modification in biological systems.

Structures of the contryphan family of cyclic peptides. Role of electrostatic interactions in cis-trans isomerism.

The contryphan family of cyclic peptides, isolated recently from various species of cone shell, has the conserved sequence motif NH(3)(+)-X(1)COD-WX(5)PWC-NH(2), where X(1) is either Gly or absent, O is 4-trans-hydroxyproline, and X(5) is Glu, Asp, or Gln. The solution structures described herein of two new naturally occurring contryphan sequences, contryphan-Sm and des[Gly1]-contryphan-R, are similar to those of contryphan-R, the structure of which has been determined recently [Pallaghy et al. (1999) Biochemistry 38, 11553-11559]. The (1)H NMR chemical shifts of another naturally occurring peptide, contryphan-P, indicate that it also adopts a similar structure. All of these contryphans exist in solution as a mixture of two conformers due to cis-trans isomerization about the Cys2-Hyp3 peptide bond. The lower cis-trans ratio for contryphan-Sm enabled elucidation of the 3D structure of both its major and its minor forms, for which the patterns of (3)J(H)(alpha)(HN) coupling constants are very different. As with contryphan-R, the structure of the major form of contryphan-Sm (cis Cys2-Hyp3 peptide bond) contains an N-terminal chain reversal and a C-terminal type I beta-turn. The minor conformer (trans peptide bond) forms a hairpin structure with sheetlike hydrogen bonds and a type II beta-turn, with the D-Trp4 at the 'Gly position' of the turn. The ratio of conformers arising from cis-trans isomerism around the peptide bond preceding Hyp3 is sensitive to both the amino acid sequence and the solution conditions, varying from 2.7:1 to 17:1 across the five sequences. The sequence and structural determinants of the cis-trans isomerism have been elucidated by comparison of the cis-trans ratios for these peptides with those for contryphan-R and an N-acetylated derivative thereof. The cis-trans ratio is reduced for peptides in which either the charged N-terminal ammonium or the X(5) side-chain carboxylate is neutralized, implying that an electrostatic interaction between these groups stabilizes the cis conformer relative to the trans. These results on the structures and cis-trans equilibrium of different conformers suggest a paradigm of 'locally determined but globally selected' folding for cyclic peptides and constrained protein loops, where the series of stereochemical centers in the loop dictates the favorable conformations and the equilibrium is determined by a small number of side-chain interactions.

Isolation and characterization of a novel conus peptide with apparent antinociceptive activity.

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.