Craig S. Walker

Speciation of Cone Snails and Interspecific Hyperdivergence of Their Venom Peptides: Potential Evolutionary Significance of Intronsa

ABSTRACT: All 500 species of cone snails (Conus) are venomous predators. From a biochemical/genetic perspective, differences among Conus species may be based on the 50‐200 different peptides in the venom of each species. Venom is used for prey capture as well as for interactions with predators and competitors. The venom of every species has its own distinct complement of peptides. Some of the interspecific divergence observed in venom peptides can be explained by differential expression of venom peptide superfamilies in different species and of peptide superfamily branching in various Conus lineages into pharmacologic groups with different targeting specificity. However, the striking interspecific divergence of peptide sequences is the dominant factor in the differences observed between venoms. The small venom peptides (typically 10‐35 amino acids in length) are processed from larger prepropeptide precursors (ca. 100 amino acids). If interspecific comparisons are made between homologous prepropeptides, the three different regions of a Conus peptide precursor (signal sequence, pro‐region, mature peptide) are found to have diverged at remarkably different rates. Analysis of synonymous and nonsynonymous substitution rates for the different segments of a prepropeptide suggests that mutation frequency varies by over an order of magnitude across the segments, with the mature toxin region undergoing the highest rate. The three sections of the prepropeptide which exhibit apparently different mutation rates are separated by introns. This striking segment‐specific rate of divergence of Conus prepropeptides suggests a role for introns in evolution: exons separated by introns have the potential to evolve very different mutation rates. Plausible mechanisms that could underlie differing mutational frequency in the different exons of a gene are discussed.

The T-superfamily of conotoxins.

We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conusspecies were identified; they share a consensus signal sequence, and a conserved arrangement of cysteine residues (- -CC- -CC-). T-superfamily peptides were found expressed in venom ducts of all major feeding types of Conus; the results suggest that the T-superfamily will be a large and diverse group of peptides, widely distributed in the 500 different Conusspecies. These peptides are likely to be functionally diverse; although the peptides are small (11–17 amino acids), their sequences are strikingly divergent, with different peptides of the superfamily exhibiting varying extents of post-translational modification. Of the three peptides tested for in vivo biological activity, only one was active on mice but all three had effects on fish. The peptides that have been extensively characterized are as follows: p5a, GCCPKQMRCCTL*; tx5a, γCCγDGW+CCT§AAO; and au5a, FCCPFIRYCCW (where γ = γ-carboxyglutamate, W+ = bromotryptophan, O = hydroxyproline, T§ = glycosylated threonine, and * = COOH-terminal amidation). We also demonstrate that the precursor of tx5a contains a functional γ-carboxylation recognition signal in the −1 to −20 propeptide region, consistent with the presence of γ-carboxyglutamate residues in this peptide.

γ-Glutamyl carboxylation: An extracellular posttranslational modification that antedates the divergence of molluscs, arthropods, and chordates

The posttranslational γ-carboxylation of glutamate residues in secreted proteins to γ-carboxyglutamate is carried out by the vitamin K-dependent enzyme γ-glutamyl carboxylase. γ-Carboxylation has long been thought to be a biochemical specialization of vertebrates, essential for blood clotting. Recently, a γ-carboxylase was shown to be expressed in Drosophila, although its function remains undefined in this organism. We have characterized both cDNA and genomic clones for the γ-glutamyl carboxylase from the marine mollusc, Conus, the only nonvertebrate organism for which γ-carboxyglutamate-containing proteins have been biochemically and physiologically characterized. The predicted amino acid sequence has a high degree of sequence similarity to the Drosophila and vertebrate enzymes. Although γ-carboxylases are highly conserved, the Conus and mammalian enzymes have divergent substrate specificity. There are striking parallels in the gene organization of Conus and human γ-carboxylases. Of the 10 Conus introns identified, 8 are in precisely the same position as the corresponding introns in the human enzyme. This remarkable conservation of intron/exon boundaries reveals that an intron-rich γ-carboxylase was present early in the evolution of the animal phyla; although specialized adaptations in mammals and molluscs that require this extracellular modification have been identified, the ancestral function(s) and wider biological roles of γ-carboxylation still need to be defined. The data raise the possibility that most introns in the genes of both mammals and molluscs antedate the divergence of these phyla.

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.

Conantokin-G precursor and its role in gamma-carboxylation by a vitamin K-dependent carboxylase from a Conus snail.

Conantokin-G isolated from the marine snailConus geographus is a 17-amino acid γ-carboxyglutamate (Gla)-containing peptide that inhibits theN-methyl-d-aspartate receptor. We describe the cloning and sequence of conantokin-G cDNA and the possible role of the propeptide sequence. The cDNA encodes a 100amino acid peptide. The N-terminal 80 amino acids constitute the prepro-sequence, and the mature peptide is derived from the remaining C-terminal residues after proteolysis, C-terminal amidation, and a unique post-translational modification, γ-carboxylation of glutamate residues to Gla. Mature conantokin-G peptide containing Glu residues (E.Con-G) in place of Gla is a poor substrate for the vitamin K-dependent γ-glutamyl carboxylase (apparentK m = 3.4 mm). Using peptides corresponding to different segments of the propeptide we investigated a potential role for the propeptide sequences in γ-carboxylation. Propeptide segment −20 to −1 covalently linked to E.Con-G or the synthetic pentapeptide FLEEL increased their apparent affinities 2 orders of magnitude. These substrates are not efficiently carboxylated by the bovine microsomal γ-glutamyl carboxylase, suggesting differences in specificities between the Conus and the mammalian enzyme. However, the role of propeptide in enhancing the efficiency of carboxylation is maintained.

A Novel Conus Snail Polypeptide Causes Excitotoxicity by Blocking Desensitization of AMPA Receptors

BACKGROUND Ionotropic glutamate receptors (iGluRs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the central nervous system. Based on both molecular and pharmacological criteria, iGluRs have been divided into two major classes, the non-NMDA class, which includes both AMPA and kainate subtypes of receptors, and the NMDA class. One evolutionarily conserved feature of iGluRs is their desensitization in the continued presence of glutamate. Thus, when in a desensitized state, iGluRs can be bound to glutamate, yet the channel remains closed. However, the relevance of desensitization to nervous system function has remained enigmatic. RESULTS Here, we report the identification and characterization of a novel polypeptide (con-ikot-ikot) from the venom of a predatory marine snail Conus striatus that specifically disrupts the desensitization of AMPA receptors (AMPARs). The stoichiometry of con-ikot-ikot appears reminiscent of the proposed subunit organization of AMPARs, i.e., a dimer of dimers, suggesting that it acts as a molecular four-legged clamp that holds the AMPAR channel open. Application of con-ikot-ikot to hippocampal slices caused a large and rapid increase in resting AMPAR-mediated current leading to neuronal death. CONCLUSIONS Our findings provide insight into the mechanisms that regulate receptor desensitization and demonstrate that in the arms race between prey and predators, evolution has selected for a toxin that blocks AMPAR desensitization, thus revealing the fundamental importance of desensitization for regulating neural function.

Evolutionary Conserved Role for TARPs in the Gating of Glutamate Receptors and Tuning of Synaptic Function

Neurotransmission in the brain is critically dependent on excitatory synaptic signaling mediated by AMPA-class ionotropic glutamate receptors (AMPARs). AMPARs are known to be associated with Transmembrane AMPA receptor Regulatory Proteins (TARPs). In vertebrates, at least four TARPs appear to have redundant roles as obligate chaperones for AMPARs, thus greatly complicating analysis of TARP participation in synaptic function. We have overcome this limitation by identifying and mutating the essential set of TARPs in C. elegans (STG-1 and STG-2). In TARP mutants, AMPAR-mediated currents and worm behaviors are selectively disrupted despite apparently normal surface expression and clustering of the receptors. Reconstitution experiments indicate that both STG-1 and STG-2 can functionally substitute for vertebrate TARPs to modify receptor function. Thus, we show that TARPs are obligate auxiliary subunits for AMPARs with a primary, evolutionarily conserved functional role in the modification of current kinetics.

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 γ-carboxylation of glutamate to γ-carboxyglutamate was originally well characterized in the mammalian blood clotting cascade. γ-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 γ-carboxylation. We demonstrate that an open reading frame from aDrosophila melanogaster cDNA clone encodes a protein with vitamin K-dependent γ-carboxylase activity. The open reading frame, 670 amino acids in length, is truncated at the C-terminal end compared with mammalian γ-carboxylase, which is 758 amino acids. The mammalian gene has 14 introns; inDrosophila 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 aConus 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.

Conantokin-L, a new NMDA receptor antagonist: Determinants for anticonvulsant potency

Conantokins are N-methyl-D-aspartate receptor antagonist peptides found in the venoms of marine cone snails. Current intense interest in this peptide family stems from the discovery of their therapeutic potential as anticonvulsants. It was recently reported that conantokin-R is a highly potent anticonvulsant compound, with a protective index of 17.5 when tested in the audiogenic mouse model of epilepsy. Conantokin-L was characterized from Conus lynceus and found to have extensive homology with conantokin-R, except For the C-terminal amino acids. Although conantokin-L appears almost as potent as conantokin-R in standard in vivo assays for conantokins and NMDA receptor binding assays, it is far less potent as an anticonvulsant, with a protective index of 1.2 in the audiogenic mouse model. The results suggest that the C-terminal sequences of conantokin-R and conantokin-L are a major determinant of their anticonvulsant potency.

Conantokin-Br from Conus brettinghami and selectivity determinants for the NR2D subunit of the NMDA receptor.

Conantokins are venom peptides from marine cone snails that are NMDA receptor antagonists. Here, we report the characterization of a 24 AA conantokin from Conus brettinghami Coomans , H. E. , Moolenbeek , R. G. and Wils , E. ( 1982 ) Basteria 46 ( 1/4 ), 3 - 67 , conantokin-Br (con-Br), the first conantokin that does not have the conserved glutamate residue at position 2. Molecular modeling studies suggest that con-Br has a helical structure between residues 2-13. In contrast to other characterized conantokins, con-Br has a high potency for NMDA receptors with NR2D subunits. To identify determinants for NR2D potency, we synthesized chimeras of con-Br and conantokin-R (con-R); the latter has a approximately 30-fold lower potency for the NR2D subtype. The characterization of two reciprocal chimeras (con-Br/R and con-R/Br), comprising the first 9-10 N-terminal AAs of each conantokin followed by the corresponding C-terminal AAs of the other conantokin demonstrates that determinants for NR2D selectivity are at the N-terminal region. Additional analogues comprising 1-3 amino acid substitutions from each peptide into the homologous region of the other led to the identification of a key determinant; a Tyr residue in position 5 increases potency for NR2D, while Val at this locus causes a decrease. The systematic definition of key determinants in the conantokin peptides for NMDA receptor subtype selectivity is an essential component in the development of conantokin peptides that are highly selective for each specific NMDA receptor subtype.