Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa 2. Three-dimensional solution structure.

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D 1H NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and muO-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18+/-0.05 A for the backbone atoms and 1.39+/-0.33 A for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.     

Expression and characterization of recombinant vitamin K-dependent gamma-glutamyl carboxylase from an invertebrate, Conus textile.

The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, gamma-carboxyglutamic acid, have been identified. To examine its biosynthesis of gamma-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and approximately 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin K-dependent epoxidase activity. The recombinant carboxylase has a molecular mass of approximately 130 kDa. The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues -18 to +10 of human proprothrombin and proFactor IX with Km values of 420 micro m, 1.7 micro m and 6 micro m, respectively; the Km for vitamin K is 52 micro m. The Km values for peptides based on the sequence of the conotoxin epsilon-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 micro m, 75 micro m and 74 micro m, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent gamma-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of gamma-carboxyglutamic acid synthesis in diverse biological systems.     

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.     

Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning

Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a 3' RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1-Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.     

Conopressin-T from Conus tulipa reveals an antagonist switch in vasopressin-like peptides

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V 1a receptor.     

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.     

Discovery of a novel class of conotoxin from Conus litteratus, lt14a, with a unique cysteine pattern

Conus litteratus is a worm-hunting cone snail with a highly sophisticated neuropharmacological defense strategy using small peptides in its venom. By analyzing different clones in the cDNA library of venom ducts from C. litteratus, we identified the peptide lt14a which displays a characteristic signal peptide sequence in its precursor and a unique arrangement of Cys residues (-C-C-C-C-) in its mature peptide region. RT-PCR analysis suggested that lt14a is abundantly expressed throughout the whole venom duct. An intensive analysis in sequence suggested that lt14a is similar to alpha-conotoxin qc1.1 cloned from Conus quercinus. We conducted the chemical synthesis of lt14a. The synthetic lt14a has a remarkable biological activity to suppress pain and inhibits the neuronal-type nicotinic acetylcholine receptors.     

Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-d-aspartate antagonist activity

Conantokin-T, a 21-amino acid peptide which induces sleep-like symptoms in young mice was purified from the venom of the fish-hunting cone snail, Conus tulipa. The amino acid sequence of the peptide was determined and verified by chemical synthesis. The peptide has 4 residues of the modified amino acid, gamma-carboxyglutamate (Gla). The sequence of the peptide is: Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys- Lys-Asn-Ala-NH2. Conantokin-T inhibits N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx in central nervous system neurons. This observation suggests that like conantokin-G (a homologous Conus peptide with recently identified NMDA antagonist activity) conantokin-T has NMDA antagonist activity. A sequence comparison of conantokins-T and -G identifies the 4 Gla residues and the N-terminal dipeptide sequence as potential key elements for the biological activity of this peptide.     

织锦芋螺ο家族芋螺毒素的序列分析

为了从织锦芋螺（Ｃｏｎｕｓｔｅｘｔｉｌｅ）中尽可能多地分离出ο家族的毒素序列和研究其应用价值,在克隆了织锦芋螺α芋螺毒素的基础上进行了织锦芋螺ο家族芋螺毒素基因的分离工作．从织锦芋螺毒管中提取ｍ ＲＮＡ,通过ＲＡＣＥ（ｒａｐｉｄ ａｍ ｐｌｉｆｉｃａｔｉｏｎ ｏｆｃＤＮＡ ｅｎｄｓ,ｃＤＮＡ 末端的快速扩增）－ＰＣＲ方法扩增获得ο家族芋螺毒素ｃＤＮＡ 片段,并进行克隆和序列分析．从织锦芋螺毒液中获得了６种新的芋螺毒素序列,且毒素序列的成熟肽部分均符合Ｃ－ Ｃ－ ＣＣ－ Ｃ－ Ｃ的保守半胱氨酸框架．这些是新的ο家族芋螺毒素序列,新序列的阐明为进一步研究其生物活性和应用打下了基础．     

Hydrophobic amino acids define the carboxylation recognition site in the precursor of the gamma-carboxyglutamic-acid-containing conotoxin epsilon-TxIX from the marine cone snail Conus textile.

To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxIX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K(M) substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K(M) substrates (5 microM) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K(M) of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K(M) substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.     

Differentiation of Venoms of Predatory Marine Gastropods: Divergence of Orthologous Toxin Genes of Closely Related Conus Species with Different Dietary Specializations

Venoms of Conus are remarkably diverse among species and the genes that encode conotoxins show high rates of evolution. Yet no prior studies have specifically explored how conotoxin gene evolution contributes to the differentiation of venoms of closely related Conus species. Previous investigations of four-loop conotoxin expression patterns of six closely related Conus species identified 12 sets of putative orthologous loci from these species, including eight pairs of loci that are coexpressed by two of these six species, C. abbreviatus and C. miliaris. Here I analyze the molecular evolution of orthologous conotoxin loci of these species and specifically examine the divergence of the eight orthologous counterparts of C. abbreviatus and C. miliaris. Tree and maximum likelihood-based analyses of these sequences reveal that positive selection promotes the divergence of orthologous genes among species and that the evolution of orthologues of C. abbreviatus and C. miliaris is asymmetric among species. The asymmetric evolution of conotoxin loci among species may result from lineage-specific dietary shifts or interspecific differences in the impact of selection from predator-prey interactions on conotoxin loci.     

从织锦芋螺中克隆α芋螺毒素序列

为了从我国南海产织锦芋螺（Ｃｏｎｕｓｔｅｘｔｉｌｅ）中分离新的毒素序列并研究其应用价值,进行了织锦芋螺毒素基因的分离工作．从织锦芋螺毒管中提取ｍＲＮＡ,以Ａ族芋螺毒素的信号肽编码部分和３′端非翻译部分的保守序列为引物,通过ＲＴ－ＰＣＲ扩增和序列分析方法获得新的芋螺毒素序列．结果得到两种不同的α芋螺毒素序列,两者都属于α４／７亚型芋螺毒素,预测其成熟肽序列分别为Ｐｒｏ－Ｇｌｕ－Ｃｙｓ－Ｃｙｓ－Ｓｅｒ－Ａｓｐ－Ｐｒｏ－Ａｒｇ－Ｃｙｓ－Ａｓｎ－Ｓｅｒ－Ｓｅｒ－Ｈｉｓ－Ｐｒｏ－Ｇｌｕ－Ｌｅｕ－Ｃｙｓ－Ｇｌｙ（Ｃ端Ｇｌｙ可能被酰胺化）和Ｐｒｏ－Ｇｌｕ－Ｃｙｓ－Ｃｙｓ－Ｓｅｒ－Ｈｉｓ－Ｐｒｏ－Ａｌａ－Ｃｙｓ－Ａｓｎ－Ｖａｌ－Ａｓｐ－Ｈｉｓ－Ｐｒｏ－Ｇｌｕ－Ｉｌｅ－Ｃｙｓ－Ａｒｇ．采用传统的生化分离手段尚未从织锦芋螺中获得过α芋螺毒素序列,这两种α芋螺毒素作用的种属特异性、受体类型特异性和在小细胞肺癌的诊断和治疗中的应用价值有待进一步研究     

A novel sodium channel inhibitor from Conus geographus: purification, structure, and pharmacological properties

A novel toxin, tentatively named conotoxin GS (CGS), has been isolated from a marine snail, Conus geographus. CGS was found to exist as a single polypeptide chain, consisting of 34 amino acid residues, cross-linked by three disulfide bonds. Its amino acid sequence was shown to be Ala-Cys-Ser-Gly-Arg-Gly-Ser-Arg-Cys-Hyp-Hyp-Gln-Cys-Cys-Met-Gly-Leu-Arg- Cys-Gly - Arg-Gly-Asn-Pro-Gln-Lys-Cys-Ile-Gly-Ala-His-Gla-Asp-Val. In competition experiments, CGS inhibited the bindings of [3H]Lys-tetrodotoxin ([3H]Lys-TTX) and [3H]propionylconotoxin GIIIA to Electrophorus electricus electroplax membranes, with Ki values of 34 nM and 24 nM, respectively. The toxin inhibited the binding of [3H]Lys-TTX (1 nM) to rat skeletal muscle homogenates with an IC50 value of 880 nM but showed very little effect on this binding to the rat brain P2 fraction at 10 microM. These binding studies indicate that CGS belongs to the same group of Na channel inhibitors as TTX, STX (saxitoxin), and mu-conotoxins. Although CGS, like the mu-conotoxins, is a pharmacological probe for distinguishing between neuronal and muscle Na channel subtypes, the homology in the sequences of CGS and mu-conotoxins is very limited.     

Alpha S-conotoxin RVIIIA: a structurally unique conotoxin that broadly targets nicotinic acetylcholine receptors

We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT3 receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.     

Isolation and cloning of a conotoxin with a novel cysteine pattern from Conus caracteristicus

A new conotoxin, ca16a, containing 8 cysteine residues was purified, sequenced, and cloned from a worm-hunting snail, Conus caracteristicus. This conotoxin is an extremely hydrophilic peptide comprising 34 residues, with 4 acidic and 4 basic residues. It is rich in polar Gly, Ser, and Thr residues and includes a hydroxylated Pro residue. The cysteine arrangement pattern of ca16a (-C-C-CC-C-CC-C-, designated as framework #16) is distinct from that of other known conotoxins. Furthermore, the signal peptide sequence of this conotoxin does not share any homology with those of other conotoxins. Leu residues account for almost 50% of its 20-residue signal peptide. The unique cysteine framework and signal peptide sequence of ca16a suggest that it belongs to a new conotoxin superfamily.