On the importance of oxidative folding in the evolution of conotoxins: cysteine codon preservation through gene duplication and adaptation.
ABSTRACT: Conotoxin genes are among the most rapidly evolving genes currently known; however, despite the well-established hypervariability of the intercysteine loops, the cysteines demonstrate significant conservation, with a site-specific codon bias for each cysteine in a family of conotoxins. Herein we present a novel rationale behind the codon-level conservation of the cysteines that comprise the disulfide scaffold. We analyze cysteine codon conservation using an internal reference and phylogenetic tools; our results suggest that the established codon conservation can be explained as the result of selective pressures linked to the production efficiency and folding of conotoxins, driving the conservation of cysteine at the amino-acid level. The preservation of cysteine has resulted in maintenance of the ancestral codon in most of the daughter lineages, despite the hypervariability of adjacent residues. We propose that the selective pressures acting on the venom components of cone snails involve an interplay of biosynthetic efficiency, activity at the target receptor and the importance of that activity to effective prey immobilization. Functional redundancy in the venom can thus serve as a buffer for the energy expenditure of venom production.
Project description:The mini-M conotoxins are peptidic scaffolds found in the venom of cones snails. These scaffolds are tightly folded structures held together by three disulfide bonds with a CC-C-C-CC arrangement (conotoxin framework III) and belong to the M Superfamily of conotoxins. Here, we describe mini-M conotoxins from the venom of Conus regius, a Western Atlantic worm-hunting cone snail species using transcriptomic and peptidomic analyses. These C. regius conotoxins belong to three different subtypes: M1, M2, and M3. The subtypes show little sequence homology, and their loop sizes (intercysteine amino acid chains) vary significantly. The mini-Ms isolated from dissected venom contains preferentially hydroxylated proline residues, thus augmenting the structural reach of this conotoxin class. Using 2D-NMR methods, we have determined the 3D structure of reg3b, an M2 subtype conotoxin, which shows a constrained multi-turn scaffold. The structural diversity found within mini-M conotoxin scaffolds of C. regius is indicative of structural hypervariability of the conotoxin M superfamily that is not seen in other superfamilies. These stable minimalistic scaffolds may be investigated for the development of engineered peptides for therapeutic applications. DATABASES:Sequences are available in GenBank under accession numbers MF588935-MF588952. Structural data are available in the RCSB protein database under the accession code 6BX9.
Project description:Two novel conotoxins from vermivorous cone snails Conus pulicarius and Conus tessulatus, designated as Pu14.1 and ts14a, were identified by cDNA cloning and peptide purification, respectively. The signal sequence of Pu14.1 is identical to that of ?-conotoxins, while its predicted mature peptide, pu14a, shares high sequence similarity with ts14a, with only one residue different in their first intercysteine loop, which contains 10 residues and is rich in proline. Both pu14a and ts14a contain four separate cysteines in framework 14 (C-C-C-C). Peptide pu14a was chemically synthesized, air oxidized, and the connectivity of its two disulfide bonds was determined to be C1-C3, C2-C4, which is the same as found in ?-conotoxins. The synthetic pu14a induced a sleeping symptom in mice and was toxic to freshwater goldfish upon intramuscular injection. Using the Xenopus oocyte heterologous expression system, 1?M of pu14a demonstrated to inhibit the rat neuronal ?3?2-containing as well as the mouse neuromuscular ?1?1?? subtypes of nicotinic acetylcholine receptors, and then rapidly dissociated from the receptors. However, this toxin had no inhibitory effect on potassium channels in mouse superior cervical ganglion neurons. According to the identical signal sequence to ?-conotoxins, the unique cysteine framework and molecular target of pu14a, we propose that pu14a and ts14a may represent a novel subfamily in the A-superfamily, designated as ?1-conotoxins.
Project description:Alpha-conotoxins isolated from Conus venoms contain 11-19 residues and preferentially fold into the globular conformation that possesses a specific disulfide pairing pattern (C1-3, C2-4). We and others isolated a new family of chi-conotoxins (also called lambda conotoxins) with the conserved cysteine framework of alpha-conotoxins but with alternative disulfide pairing (C1-4, C2-3) resulting in the ribbon conformation. In both families, disulfide pairing and hence folding are important for their biological potency. By comparing the structural differences, we identified potential structural determinants responsible for the folding tendencies of these conotoxins. We examined the role of conserved proline in the first intercysteine loop and the conserved C-terminal amide on folding patterns of synthetic analogues of ImI conotoxin by comparing the isoforms with the regiospecifically synthesized conformers. Deamidation at the C-terminus and substitution of proline in the first intercysteine loop switch the folding pattern from the globular form of alpha-conotoxins to the ribbon form of chi/lambda-conotoxins. The findings are corroborated by reciprocal folding of CMrVIA chi/lambda-conotoxins. Substitution of Lys-6 from the first intercysteine loop of CMrVIA conotoxin with proline, as well as the inclusion of an amidated C-terminal shifted the folding preference of CMrVIA conotoxin from its native ribbon conformation toward the globular conformation. Binding assays of ImI conotoxin analogues with Aplysia and Bulinus acetylcholine binding protein indicate that both these substitutions and their consequent conformational change substantially impact the binding affinity of ImI conotoxin. These results strongly indicate that the first intercysteine loop proline and C-terminal amidation act as conformational switches in alpha- and chi/lambda-conotoxins.
Project description:The primary objective of this study was to realize the large-scale discovery of conotoxin sequences from different organs (including the venom duct, venom bulb and salivary gland) of the vermivorous Oak cone snail, Conus quercinus. Using high-throughput transcriptome sequencing, we identified 133 putative conotoxins that belong to 34 known superfamilies, of which nine were previously reported while the remaining 124 were novel conotoxins, with 17 in new and unassigned conotoxin groups. A-, O?-, M-, and I?- superfamilies were the most abundant, and the cysteine frameworks XIII and VIII were observed for the first time in the A- and I?-superfamilies. The transcriptome data from the venom duct, venom bulb and salivary gland showed considerable inter-organizational variations. Each organ had many exclusive conotoxins, and only seven of all the inferred mature peptides were common in the three organs. As expected, most of the identified conotoxins were synthesized in the venom duct at relatively high levels; however, a number of conotoxins were also identified in the venom bulb and the salivary gland with very low transcription levels. Therefore, various organs have different conotoxins with high diversity, suggesting greater contributions from several organs to the high-throughput discovery of new conotoxins for future drug development.
Project description:Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the alpha-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain alpha-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson's disease and nociception. Structure/function characterization of alpha-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of alpha-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of alpha-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.
Project description:The venom of each Conus species consists of a diverse array of neurophysiologically active peptides, which are mostly unique to the examined species. In this study, we performed high-throughput transcriptome sequencing to extract and analyze putative conotoxin transcripts from the venom ducts of 3 vermivorous cone snails (C. caracteristicus, C. generalis, and C. quercinus), which are resident in offshore waters of the South China Sea. In total, 118, 61, and 48 putative conotoxins (across 22 superfamilies) were identified from the 3 Conus species, respectively; most of them are novel, and some possess new cysteine patterns. Interestingly, a series of 45 unassigned conotoxins presented with a new framework of C-C-C-C-C-C, and their mature regions were sufficiently distinct from any other known conotoxins, most likely representing a new superfamily. O- and M-superfamily conotoxins were the most abundant in transcript number and transcription level, suggesting their critical roles in the venom functions of these vermivorous cone snails. In addition, we identified numerous functional proteins with potential involvement in the biosynthesis, modification, and delivery process of conotoxins, which may shed light on the fundamental mechanisms for the generation of these important conotoxins within the venom duct of cone snails.
Project description:Conotoxins are small peptides present in the venom of cone snails. The snail uses this venom to paralyze and capture prey. The constituent conopeptides display a high level of chemical diversity and are of particular interest for scientists as tools employed in neurological studies and for drug development, because they target with exquisite specificity membrane receptors, transporters, and various ion channels in the nervous system. However, these peptides are known to contain a high frequency and variability of post-translational modifications-including sometimes O-glycosylation-which are of importance for biological activity. The potential application of specific conotoxins as neuropharmalogical agents and chemical probes requires a full characterization of the relevant peptides, including the structure of the carbohydrate part. In this review, the currently existing knowledge of O-glycosylation of conotoxins is described.
Project description:Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a gamma-carboxyglutamic acid-containing peptide, conotoxin epsilon-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca2+ channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca2+ in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two gamma-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region.
Project description:The venom of various Conus species is composed of a rich variety of unique bioactive peptides, commonly referred to as conotoxins (conopeptides). Most conopeptides have specific receptors or ion channels as physiologically relevant targets. In this paper, high-throughput transcriptome sequencing was performed to analyze putative conotoxin transcripts from the venom duct of a vermivorous cone snail species, Conus litteratus native to the South China Sea. A total of 128 putative conotoxins were identified, most of them belonging to 22 known superfamilies, with 43 conotoxins being regarded as belonging to new superfamilies. Notably, the M superfamily was the most abundant in conotoxins among the known superfamilies. A total of 15 known cysteine frameworks were also described. The largest proportion of cysteine frameworks were VI/VII (C-C-CC-C-C), IX (C-C-C-C-C-C) and XIV (C-C-C-C). In addition, five novel cysteine patterns were also discovered. Simple sequence repeat detection results showed that di-nucleotide was the major type of repetition, and the codon usage bias results indicated that the codon usage bias of the conotoxin genes was weak, but the M, O1, O2 superfamilies differed in codon preference. Gene cloning indicated that there was no intron in conotoxins of the B1- or J superfamily, one intron with 1273-1339 bp existed in a mature region of the F superfamily, which is different from the previously reported gene structure of conotoxins from other superfamilies. This study will enhance our understanding of conotoxin diversity, and the new conotoxins discovered in this paper will provide more potential candidates for the development of pharmacological probes and marine peptide drugs.
Project description:Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.