Diversity of the neurotoxic Conus peptides: a model for concerted pharmacological discovery

Predatory cone snails (genus Conus) produce a rich array of venoms that collectively contain an estimated 100,000 small, disulfide-rich peptides (i.e., conotoxins, or conopeptides). Over the last few decades, the conopeptides have revealed a remarkable diversity of pharmacological function and utility. An evolutionary rationale for the existence of such a large and pharmacologically diverse set of gene products can be premised on the complexity of intra- and interspecies interactions that define the ecology of Conus snails. Insights into these evolutionary trends, moreover, have been exploited with great neuropharmacological success, so that research into the Conus snails effectively recapitulates a new concerted discovery approach, which we discuss here, for developing unique ligands for both laboratory and therapeutic applications. The Conus peptides thus serve as a model system for reaping the pharmacological potential of biodiverse animal lineages.     

Post-translationally modified neuropeptides from Conus venoms.

Predatory cone snails (genus Conus) comprise what is arguably the largest living genus of marine animals (500 species). All Conus use complex venoms to capture prey and for other biological purposes. Most biologically active components of these venoms are small disulfide-rich peptides, generally 7-35 amino acids in length. There are probably of the order of 100 different peptides expressed in the venom of each of the 500 Conus species [1,2]. Peptide sequences diverge rapidly between Conus species, resulting in a distinct peptide complement for each species. Thus, the genus as a whole has probably generated approximately 50 000 different peptides, which can be organized into families and superfamilies with shared sequence elements [3]. In this minireview, we provide a brief overview of the neuropharmacological, molecular and cell-biological aspects of the Conus peptides. However, the major focus of the review will be the remarkable array of post-translational modifications found in these peptides.     

Direct cDNA cloning of novel conopeptide precursors of the O-superfamily

Conotoxins from the venom of marine cone snails (genus Conus) represent large families of proteins exhibiting a similar precursor organization, but highly diverse pharmacological activities. A directed PCR-based approach using primers according to the conserved signal sequence was applied to investigate the diversity of conotoxins from the O-superfamily. Using 3' RACE, cDNA sequences encoding precursor peptides were identified in five Conus species (Conus capitaneus, Conus imperialis, Conusstriatus, Conus vexillum and Conus virgo). In all cases, the sequence of the signal region exhibited high conservancy, whereas the sequence of the mature peptides was either almost identical or highly divergent among the five species. These findings demonstrate that beside a common genetic pattern divergent evolution of toxins occurred in a highly mutating peptide family.     

Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity.

Conotoxins (CTX) from the venom of marine cone snails (genus Conus) represent large families of proteins, which show a similar precursor organization with surprisingly conserved signal sequence of the precursor peptides, but highly diverse pharmacological activities. By using the conserved sequences found within the genes that encode the alpha-conotoxin precursors, a technique based on RT-PCR was used to identify, respectively, two novel peptides (LiC22, LeD2) from the two worm-hunting Conus species Conus lividus, and Conus litteratus, and one novel peptide (TeA21) from the snail-hunting Conus species Conus textile, all native to Hainan in China. The three peptides share an alpha4/7 subfamily alpha-conotoxins common cysteine pattern (CCX(4)CX(7)C, two disulfide bonds), which are competitive antagonists of nicotinic acetylcholine receptor (nAChRs). The cDNA of LiC22N encodes a precursor of 40 residues, including a propeptide of 19 residues and a mature peptide of 21 residues. The cDNA of LeD2N encodes a precursor of 41 residues, including a propeptide of 21 residues and a mature peptide of 16 residues with three additional Gly residues. The cDNA of TeA21N encodes a precursor of 38 residues, including a propeptide of 20 residues and a mature peptide of 17 residues with an additional residue Gly. The additional residue Gly of LeD2N and TeA21N is a prerequisite for the amidation of the preceding C-terminal Cys. All three sequences are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The properties of the alpha4/7 conotoxins known so far were discussed in detail. Phylogenetic analysis of the new conotoxins in the present study and the published homologue of alpha4/7 conotoxins from the other Conus species were performed systematically. Patterns of sequence divergence for the three regions of signal, proregion, and mature peptides, both nucleotide acids and residue substitutions in DNA and peptide levels, as well as Cys codon usage were analyzed, which suggest how these separate branches originated. Percent identities of the DNA and amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the proregion was also high, with an intermediate percentage of divergence between that observed in the signal and in the toxin regions. The data presented are new and are of importance, and should attract the interest of researchers in this field. The elucidated cDNAs of these toxins will facilitate a better understanding of the relationship of their structure and function, as well as the process of their evolutionary relationships.     

Unraveling the peptidome of the South African cone snails Conus pictus and Conus natalis

Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails are considered to be specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology system for their own biological purposes by producing venoms which contains a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus species occurring along the coast of South Africa. Due to their scarceness, these species remain, therefore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus, a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total, 26 novel peptides were identified. Comparing the venoms of both snails, interesting differences were observed regarding venom composition and molecular characteristics of these components.     

ConoServer, a database for conopeptide sequences and structures

SUMMARY: ConoServer is a new database dedicated to conopeptides, a large family of peptides found in the venom of marine snails of the genus Conus. These peptides have an exceptional diversity of sequences and chemical modifications and their ability to block ion channels makes them important as drug leads and tools for physiological studies. ConoServer uses standardized names and a genetic and structural classification scheme to present data retrieved from SwissProt, GenBank, the Protein DataBank and the literature. The ConoServer web site incorporates specialized features like the graphic display of post-translational modifications that are extensively present in conopeptides. Currently, ConoServer manages 1214 nucleic sequences (from 54 Conus species), 2258 proteic sequences (from 66 Conus species) and 99 3D structures. AVAILABILITY: http://research1t.imb.uq.edu.au/conoserver/.     

Novel excitatory Conus peptides define a new conotoxin superfamily

A new class of Conus peptides, the I-superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified from Conus radiatus venom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the 'lightning-strike cabal' of toxins that effect instant immobilization of fish prey. A parallel analysis of Conus cDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in a Conus radiatus venom duct library; several C. radiatus clones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I-superfamily peptides within a single Conus species is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I-conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I-superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.     

Adaptive radiation of venomous marine snail lineages and the accelerated evolution of venom peptide genes

An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, each containing approximately 100 biologically active, disulfide-rich peptides. In the genus Conus, the most intensively investigated toxoglossan lineage (～500 species), a small set of venom gene superfamilies undergo rapid sequence hyperdiversification within their mature toxin regions. Each major lineage of Toxoglossa has its own distinct set of venom gene superfamilies. Two recently identified venom gene superfamilies are expressed in the large Turridae clade, but not in Conus. Thus, as major venomous molluscan clades expand, a small set of lineage-specific venom gene superfamilies undergo accelerated evolution. The juxtaposition of extremely conserved signal sequences with hypervariable mature peptide regions is unprecedented and raises the possibility that in these gene superfamilies, the signal sequences are conserved as a result of an essential role they play in enabling rapid sequence evolution of the region of the gene that encodes the active toxin.     

Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms

The vasopressin-oxytocin family of peptides is of very ancient lineage, found in organisms as diverse as hydra and man. Although these peptides have been intensively studied in vertebrates, the presumably more extensive invertebrate series was defined primarily by immunological methods. In this report, we describe the purification and structures of two peptides of the vasopressin-oxytocin family from molluscs ("Conopressins"), which were found in the venom of fish-hunting marine snails of the genus Conus. The biological activity observed when the two snail peptides are injected intracerebrally into mice is very similar to that elicited by the vertebrate neurohypophyseal hormones and presumably reflects their actions upon a common receptor in the brain. The sequences of the purified peptides reveal unique features not found in the vertebrate peptide series, most notably an additional positive charge. These are the first members of the invertebrate series of the vasopressin-oxytocin family to be characterized biochemically. The sequences of these peptides are: from Conus geographus venom, Lys-conopressin-G, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; and from Conus striatus venom, Arg-conopressin-S, Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2.     

Diversity of the O-superfamily conotoxins from Conus miles.

Conopeptides display prominent features of hypervariability and high selectivity of large gene families that mediate interactions between organisms. Remarkable sequence diversity of O-superfamily conotoxins was found in a worm-hunting cone snail Conus miles. Five novel cDNA sequences encoding O-superfamily precursor peptides were identified in C. miles native to Hainan by RT-PCR and 3'-RACE. They share the common cysteine pattern of the O-superfamily conotoxin (C-C-CC-C-C, with three disulfide bridges). The predicted peptides consist of 27-33 amino acids. We then performed a phylogenetic analysis of the new and published homologue sequences from C. miles and the other Conus species. Sequence divergence (%) and residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were analyzed. Percentage divergence of the amino acid sequences of the prepro region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical with to highly divergent from inter- and intra-species. Despite the O-superfamily being a large and diverse group of peptides, widely distributed in the venom ducts of all major feeding types of Conus and discovered in several Conus species, it was for the first time that the newly found five O-superfamily peptides in this research came from the vermivorous C. miles. So far, conotoxins of the O-superfamily whose properties have been characterized are from piscivorous and molluscivorous Conus species, and their amino acid sequences and mode of action have been discussed in detail. The elucidated cDNAs of the five toxins are new and of importance and should attract the interest of researchers in the field, which would pave the way for a better understanding of the relationship of their structure and function.     

Novel alpha- and omega-conotoxins from Conus striatus venom.

Three neurotoxic peptides from the venom of Conus striatus have been purified, biochemically characterized, and chemically synthesized. One of these, an acetylcholine receptor blocker designated alpha-conotoxin SII, has the sequence GCCCNPACGPNYGCGTSCS. In contrast to all other alpha-conotoxins, SII has three disulfide bonds (instead of two), has no net positive charge, and has a free C-terminus. The other two paralytic peptides are Ca channel-targeted omega-conotoxins, SVIA and SVIB. omega-SVIA is the smallest natural omega-conotoxin so far characterized and has the sequence CRSSGSPCGVTSICCGRCYRGKCT-NH2. Although omega-conotoxin SVIA is a potent paralytic toxic in lower vertebrate species, it was much less effective in mammals. The third toxin, omega-conotoxin SVIB, has the sequence CKLKGQSCRKTSYDCCSGSCGRSGKC-NH2. This peptide has a different pharmacological specificity from other omega-conotoxins previously purified from Conus venoms; only omega-conotoxin SVIB has proven to be lethal to mice upon ic injection. Binding competition experiments with rat brain synaptosomal membranes indicate that the high-affinity binding site for omega-conotoxin SVIB is distinct from the high-affinity omega-conotoxin GVIA or MVIIA site.     

Conus venoms: a source of toxins which interact with membrane- potential-dependent sodium channels

Marine snails of the genus Conus, as they are carnivorous predators, have a venom apparatus used to capture their prey. The toxins contained in the venoms of Conidae, called conotoxins, are of a particular high degree of diversity and represent powerful tools in the neuroscience field. Indeed, these toxins specifically bind with a high affinity to receptors and ionic channels. Therefore, they provide original pharmacological tools which receive increasing investigation both to identify and study some functions of the nervous systems and to characterize new types and closely related subtypes of receptors or ionic channels. The voltage-gated sodium channel, because of its fundamental role in cell membrane excitability, is the specific target of a large number of animal and vegetal toxins. Actually, at least seven toxin receptor sites have been identified on this channel-protein. These toxins, and in particular conotoxins, are used to precise the role of different types and/or closely related subtypes of sodium channels in the peripheral and central nervous systems. The focus of the present review is to summarize our current knowledge of the consequences of physiological interactions between different conotoxin families and sodium channels.     

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.     

Origins of diverse feeding ecologies within Conus, a genus of venomous marine gastropods

Specialized predators on polychaetes, fishes, hemichordates or other molluscs, members of the predominantly tropical gastropod genus Conus diversified rapidly during the Miocene to constitute the most species-rich modern marine genus. We used DNA sequence data from mitochondrial and nuclear loci of 76 Conus species to generate species-level phylogenetic hypotheses for this genus and then mapped known diets onto the phylogenies to elucidate the origins and evolutionary histories of different feeding specializations. The results indicate that dramatically new feeding modes arose only a few times, that the most derived feeding modes likely arose in the Miocene, and that much of the known diversity of Conus that was generated during Miocene radiations has survived to the present.     

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.     

Patterns of cladogenesis in the venomous marine gastropod genus Conus from the Cape Verde islands

Isolated oceanic archipelagos are excellent model systems to study speciation, biogeography, and evolutionary factors underlying the generation of biological diversity. Despite the wealth of studies documenting insular speciation, few of them focused on marine organisms. Here, we reconstruct phylogenetic relationships among species of the marine venomous gastropod genus Conus from the Cape Verde archipelago. This small island chain located in the Central Atlantic hosts 10% of the worldwide species diversity of Conus. Analyses were based on mtDNA sequences, and a novel nuclear marker, a megalin-like protein, member of the low-density lipoprotein receptor gene family. The inferred phylogeny recovered two well-defined clades within Conus. One includes Cape Verde endemic species with larger shells, known as the "venulatus" complex together with C. pulcher from the Canary Islands. The other is composed of Cape Verde endemic and West Africa and Canary Island "small" shelled species. In both clades, nonendemic Conus were resolved as sister groups of the Cape Verde endemics, respectively. Our results indicate that the ancestors of "small" and "large" shelled lineages independently colonized Cape Verde. The resulting biogeographical pattern shows the grouping of most Cape Verde endemics in monophyletic island assemblages. Statistical tests supported a recent radiation event within the "small shell" clade. Using a molecular clock, we estimated that the colonization of the islands by the "small" shelled species occurred relatively close to the origin of the islands whereas the arrival of "large" shelled Conus is more recent. Our results suggest that the main factor responsible for species diversity in the archipelago may be allopatric speciation promoted by the reduced dispersal capacity of nonplanktonic lecithotrophic larvae.     

Alpha-conotoxins

alpha-Conotoxins (alpha-CgTxs) are a family of Cys-enriched peptides found in several marine snails from the genus Conus. These small peptides behave pharmacologically as competitive antagonists of the nicotinic acetylcholine receptor (AChR). The data indicate that (1) alpha-CgTxs are able to discriminate between muscle- and neuronal-type AChRs and even among distinct AChR subtypes; (2) the binding sites for alpha-CgTxs are located, like other cholinergic ligands, at the interface of alpha and non-alpha subunits (gamma, delta, and epsilon for the muscle-type AChR, and beta for several neuronal-type AChRs); (3) some alpha-CgTxs differentiate the high- from the low-affinity binding site found on either alpha/non-alpha subunit interface; and that (4) specific residues in the cholinergic binding site are energetically coupled with their corresponding pairs in the toxin stabilizing the alpha-CgTx-AChR complex. The alpha-CgTxs have proven to be excellent probes for studying the structure and function of the AChR family.