Purification and characterization of a novel excitatory peptide from Conus distans venom that defines a novel gene superfamily of conotoxins.

An excitatory peptide, di16a, with 49 amino acids and 10 cysteine residues was purified and characterized from the venom of Conus distans. Five AA residues were modified: one gamma-carboxyglutamate (Gla), and four hydroxyproline (Hyp) residues. A cDNA clone encoding the precursor for the peptide was characterized; the peptide has a novel cysteine framework and a distinctive signal sequence that differs from any other conotoxin superfamily. The peptide was chemically synthesized and folded, and synthetic and native materials were shown to co-elute. Injection of the synthetic peptide causes a hyperexcitable phenotype in mice greater than 3 weeks of age at lower doses, and lethargy at higher doses. The peptide defines both a previously uncharacterized gene superfamily of conopeptides, and a new Cys pattern with three vicinal Cys residues.

Structural and functional diversities among mu-conotoxins targeting TTX-resistant sodium channels.

mu-Conotoxins are peptides that block sodium channels. Molecular cloning was used to identify four novel mu-conotoxins: CnIIIA, CnIIIB, CIIIA, and MIIIA from Conus consors, C. catus and C. magus. A comparison of their sequences with those of previously characterized mu-conotoxins suggested that the new mu-conotoxins were likely to target tetrodotoxin-resistant (TTX-r) sodium channels. The four peptides were chemically synthesized, and their biological activities were characterized. The new conotoxins all blocked, albeit with varying potencies, TTX-r sodium currents in frog dorsal-root-ganglion (DRG) neurons. The more potent of the four new mu-conotoxins, CnIIIA and CIIIA, exhibited a strikingly different selectivity profile in blocking TTX-r versus TTX-sensitive channels, as determined by their ability to block extracellularly recorded action potentials in three preparations from frog: skeletal muscle, cardiac muscle and TTX-treated C-fibers. CnIIIA was highly specific for TTX-r sodium channels, whereas CIIIA was nonselective. Both peptides appeared significantly less potent in blocking TTX-r sodium currents in rat and mouse DRG neurons. When CnIIIA and CIIIA were injected intracranially into mice, both induced seizures, but only CIIIA caused paralysis. This is the most comprehensive characterization to date of the structural and functional diversities of an emerging group of mu-conotoxins targeting TTX-r sodium channels.

Production of recombinant Conkunitzin-S1 in Escherichia coli.

Conkunitzin-S1 from the cone snail Conus striatus is the first member of a new neurotoxin family with a canonical Kunitz domain fold. Conk-S1 is 60 amino acids long and lacks one of the three conserved disulfide bonds typically found in Kunitz domain modules. It binds specifically to voltage activated potassium channels of the Shaker family. The peptide was expressed in insoluble form in fusion with an N-terminal intein. Refolding in the presence of glutathione followed by pH shift-induced cleavage of the fusion protein resulted in a functional toxin as demonstrated by voltage-clamp measurements.

Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms.

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.

Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels.

The peptides isolated from venoms of predatory marine Conus snails ("conotoxins") are well-known to be highly potent and selective pharmacological agents for voltage-gated ion channels and receptors. We report the discovery of two novel TTX-resistant sodium channel blockers, mu-conotoxins SIIIA and KIIIA, from two species of cone snails. The two toxins were identified and characterized by combining molecular techniques and chemical synthesis. Both peptides inhibit TTX-resistant sodium currents in neurons of frog sympathetic and dorsal root ganglia but poorly block action potentials in frog skeletal muscle, which are mediated by TTX-sensitive sodium channels. The amino acid sequences in the C-terminal region of the two peptides and of the previously characterized mu-conotoxin SmIIIA (which also blocks TTX-resistant channels) are similar, but the three peptides differ in the length of their first N-terminal loop. We used molecular dynamics simulations to analyze how altering the number of residues in the first loop affects the overall structure of mu-conotoxins. Our results suggest that the naturally occurring truncations do not affect the conformation of the C-terminal loops. Taken together, structural and functional differences among mu-conotoxins SmIIIA, SIIIA, and KIIIA offer a unique insight into the "evolutionary engineering" of conotoxin activity.

Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family. Structural and functional characterization.

Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.

Biochemical and gene expression analyses of conotoxins in Conus textile venom ducts.

Each Conus snail species produces 50-200 unique peptide-based conotoxins, derived from a number of different gene superfamilies. Conotoxins are synthesized and secreted in a long venom duct, but biochemical and molecular aspects of their biosynthesis remain poorly understood. Here, we analyzed expression patterns of conotoxin genes belonging to different superfamilies in Conus textile venom ducts. The results demonstrate that specific gene families are expressed in particular regions of the venom duct. Biochemical analysis using liquid chromatography and mass spectrometry revealed an even more localized accumulation of individual conotoxins. This study demonstrates for the first time that specialization of gene expression, processing, and secretion of conotoxins occurs in different regions of the venom duct.

Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. Alpha-conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related alpha3 and alpha6 subunits. Structure-function analysis of alpha-conotoxin MII was performed in an attempt to generate analogs with preference for alpha6-containing [alpha6(*) (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at alpha3(*) versus alpha6(*) nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC(50) at alpha6beta4 versus alpha3beta4 nAChRs. The peptide had a 590-fold lower IC(50) for alpha6/alpha3beta2 versus alpha3beta2 and a 2020-fold lower IC(50) for alpha6/alpha3beta2beta3 versus alpha3beta2 nAChRs. MII[H9A;L15A] had little or no activity at alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nAChRs. Functional block by MII[H9A;L15A] of rat alpha6/alpha3beta2beta3 nAChRs (IC(50) = 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [(125)I]alpha-conotoxin MII binding to putative alpha6beta2(*) nAChRs in mouse brain homogenates (K(i) = 3.3 nM). Thus, structure-function analysis of alpha-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing alpha3 and alpha6 subunits.

Efficient oxidative folding of conotoxins and the radiation of venomous cone snails.

The 500 different species of venomous cone snails (genus Conus) use small, highly structured peptides (conotoxins) for interacting with prey, predators, and competitors. These peptides are produced by translating mRNA from many genes belonging to only a few gene superfamilies. Each translation product is processed to yield a great diversity of different mature toxin peptides (approximately 50,000-100,000), most of which are 12-30 aa in length with two to three disulfide crosslinks. In vitro, forming the biologically relevant disulfide configuration is often problematic, suggesting that in vivo mechanisms for efficiently folding the diversity of conotoxins have been evolved by the cone snails. We demonstrate here that the correct folding of a Conus peptide is facilitated by a posttranslationally modified amino acid, gamma-carboxyglutamate. In addition, we show that multiple isoforms of protein disulfide isomerase are major soluble proteins in Conus venom duct extracts. The results provide evidence for the type of adaptations required before cone snails could systematically explore the specialized biochemical world of "microproteins" that other organisms have not been able to systematically access. Almost certainly, additional specialized adaptations for efficient microprotein folding are required.

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.

Mu-conotoxin SmIIIA, a potent inhibitor of tetrodotoxin-resistant sodium channels in amphibian sympathetic and sensory neurons.

Mu-conotoxins are a family of peptides from the venoms of predatory cone snails. Previously characterized mu-conotoxins preferentially block skeletal muscle voltage-gated sodium channels. We report here the discovery (via cloning), synthesis, and electrophysiological characterization of a new peptide in this family, mu-conotoxin SmIIIA from Conus stercusmuscarum. Although mu-conotoxin SmIIIA shares several biochemical characteristics with other mu-conotoxins (the arrangement of cysteine residues and a conserved arginine believed to interact with residues near the channel pore), it has distinctive features such as the absence of hydroxyproline. In voltage-clamped dissociated neurons from frog sympathetic and dorsal root ganglia, the peptide inhibited the majority of tetrodotoxin-resistant sodium currents irreversibly; in contrast, tetrodotoxin-sensitive sodium currents were largely unaffected by the peptide. We believe that mu-conotoxin SmIIIA is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be discovered. Thus, the peptide provides a new and potentially useful tool to investigate the functional roles of tetrodotoxin-resistant voltage-gated sodium channels, including those that are found in sensory nerves that convey nociceptive information.

Alpha-conotoxin GIC from Conus geographus, a novel peptide antagonist of nicotinic acetylcholine receptors.

Many venomous organisms produce toxins that disrupt neuromuscular communication to paralyze their prey. One common class of such toxins comprises nicotinic acetylcholine receptor antagonists (nAChRs). Thus, most toxins that act on nAChRs are targeted to the neuromuscular subtype. The toxin characterized in this report, alpha-conotoxin GIC, is a most striking exception. The 16-amino acid peptide was identified from a genomic DNA clone from Conus geographus. The predicted mature toxin was synthesized, and synthetic toxin was used in all studies described. alpha-Conotoxin GIC shows no paralytic activity in fish or mice. Furthermore, even at concentrations up to 100 microm, the peptide has no detectable effect on the human muscle nicotinic receptor subtype heterologously expressed in Xenopus oocytes. In contrast, the toxin has high affinity (IC(50) approximately 1.1 nm) for the human alpha3beta2 subunit combination, making it the most neuronally selective nicotinic antagonist characterized thus far. Although alpha-conotoxin GIC shares some sequence similarity with alpha-conotoxin MII, which is also a potent alpha3beta2 nicotinic antagonist, it is much less hydrophobic, and the kinetics of channel block are substantially different. It is noteworthy that the nicotinic ligands in C. geographus venom fit an emerging pattern in venomous predators, with one nicotinic antagonist targeted to the muscle subtype (thereby causing paralysis) and a second nicotinic antagonist targeted to the alpha3beta2 nAChR subtype (possibly inhibiting the fight-or-flight response).

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

The posttranslational gamma-carboxylation of glutamate residues in secreted proteins to gamma-carboxyglutamate is carried out by the vitamin K-dependent enzyme gamma-glutamyl carboxylase. gamma-Carboxylation has long been thought to be a biochemical specialization of vertebrates, essential for blood clotting. Recently, a gamma-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 gamma-glutamyl carboxylase from the marine mollusc, Conus, the only nonvertebrate organism for which gamma-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 gamma-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 gamma-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 gamma-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 gamma-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.