ABSTRACT
Venom systems are complex traits that have independently emerged multiple times in diverse plant and animal phyla. Within each venomous lineage there typically exists interspecific variation in venom composition where several factors have been proposed as drivers of variation, including phylogeny and diet. Understanding these factors is of broad biological interest and has implications for the development of antivenom therapies and venom-based drug discovery. Because of their high species richness and the presence of several major evolutionary prey shifts, venomous marine cone snails (genus Conus) provide an ideal system to investigate drivers of interspecific venom variation. Here, by analyzing the venom gland expression profiles of â¼3,000 toxin genes from 42 species of cone snail, we elucidate the role of prey-specific selection pressures in shaping venom variation. By analyzing overall venom composition and individual toxin structures, we demonstrate that the shifts from vermivory to piscivory in Conus are complemented by distinct changes in venom composition independent of phylogeny. In vivo injections of venom from piscivorous cone snails in fish further showed a higher potency compared with venom of nonpiscivores demonstrating a selective advantage. Together, our findings provide compelling evidence for the role of prey shifts in directing the venom composition of cone snails and expand our understanding of the mechanisms of venom variation and diversification.
Subject(s)
Conus Snail , Mollusk Venoms , Animals , Conus Snail/genetics , Mollusk Venoms/genetics , Predatory Behavior , Biological Evolution , Phylogeny , Evolution, MolecularABSTRACT
Persistent pain affects one in five people worldwide, often with severely debilitating consequences. Current treatment options, which can be effective for mild or acute pain, are ill-suited for moderate-to-severe persistent pain, resulting in an urgent need for new therapeutics. In recent years, the somatostatin receptor 4 (SSTR 4 ), which is expressed in sensory neurons of the peripheral nervous system, has emerged as a promising target for pain relief. However, the presence of several closely related receptors with similar ligand-binding surfaces complicates the design of receptor-specific agonists. In this study, we report the discovery of a potent and selective SSTR 4 peptide, consomatin Fj1, derived from extensive venom gene datasets from marine cone snails. Consomatin Fj1 is a mimetic of the endogenous hormone somatostatin and contains a minimized binding motif that provides stability and drives peptide selectivity. Peripheral administration of synthetic consomatin Fj1 provided analgesia in mouse models of postoperative and neuropathic pain. Using structure-activity studies, we designed and functionally evaluated several Fj1 analogs, resulting in compounds with improved potency and selectivity. Our findings present a novel avenue for addressing persistent pain through the design of venom-inspired SSTR 4 -selective pain therapeutics. One Sentence Summary: Venom peptides from predatory marine mollusks provide new leads for treating peripheral pain conditions through a non-opioid target.
ABSTRACT
Peptide hormones and neuropeptides form a diverse class of bioactive secreted molecules that control essential processes in animals. Despite breakthroughs in peptide discovery, many signaling peptides remain undiscovered. Recently, we demonstrated the use of somatostatin-mimicking toxins from cone snails to identify the invertebrate ortholog of somatostatin. Here, we show that this toxin-based approach can be systematically applied to discover other unknown secretory peptides that are likely to have signaling function. Using large sequencing datasets, we searched for homologies between cone snail toxins and secreted proteins from the snails' prey. We identified and confirmed expression of five toxin families that share strong similarities with unknown secretory peptides from mollusks and annelids and in one case also from ecdysozoans. Based on several lines of evidence we propose that these peptides likely act as signaling peptides that serve important physiological functions. Indeed, we confirmed that one of the identified peptides belongs to the family of crustacean hyperglycemic hormone, a peptide not previously observed in Spiralia. We propose that this discovery pipeline can be broadly applied to other systems in which one organism has evolved molecules to manipulate the physiology of another.
ABSTRACT
Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1-NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure-activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.
Subject(s)
Conotoxins , Conus Snail , Toxins, Biological , Humans , Animals , Conotoxins/pharmacology , Membrane Proteins , Sodium Channels/geneticsABSTRACT
Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.
Subject(s)
Conotoxins , Mice , Animals , Conotoxins/pharmacology , Conotoxins/chemistry , Calcium Channels , Peptides/chemistry , Sensory Receptor Cells/metabolism , SnailsABSTRACT
Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.
Subject(s)
Conotoxins , Conus Snail , Receptors, Nicotinic , Mice , Humans , Animals , Conotoxins/pharmacology , Conus Snail/metabolism , Venoms , Receptors, Nicotinic/metabolism , Peptides/metabolism , Nicotinic Antagonists/pharmacologyABSTRACT
We isolated a new dimeric conotoxin with inhibitory activity against neuronal nicotinic acetylcholine receptors. Edman degradation and transcriptomic studies indicate a homodimeric conotoxin composed by two chains of 47 amino acid in length. It has the cysteine framework XX and 10 disulfide bonds. According to conotoxin nomenclature, it has been named as αD-FrXXA. The αD-FrXXA conotoxin inhibited the ACh-induced response on nAChR with a IC50 of 125 nM on hα7, 282 nM on hα3ß2, 607 nM on α4ß2, 351 nM on mouse adult muscle, and 447 nM on mouse fetal muscle. This is first toxin characterized from C. fergusoni and, at the same time, the second αD-conotoxin characterized from a species of the Eastern Pacific.
Subject(s)
Conotoxins , Conus Snail , Receptors, Nicotinic , Amino Acid Sequence , Animals , Conotoxins/chemistry , Conus Snail/chemistry , Mice , Nicotinic Antagonists/metabolism , Nicotinic Antagonists/pharmacology , Receptors, Nicotinic/metabolism , Snails/metabolismABSTRACT
Insulin and its related peptides are found throughout the animal kingdom, in which they serve diverse functions. This includes regulation of glucose homeostasis, neuronal development and cognition. The surprising recent discovery that venomous snails evolved specialized insulins to capture fish demonstrated the nefarious use of this hormone in nature. Because of their streamlined role in predation, these repurposed insulins exhibit unique characteristics that have unraveled new aspects of the chemical ecology and structural biology of this important hormone. Recently, insulins were also reported in other venomous predators and pathogenic viruses, demonstrating the broader use of insulin by one organism to manipulate the physiology of another. In this Review, we provide an overview of the discovery and biomedical application of repurposed insulins and other hormones found in nature and highlight several unique insights gained from these unusual compounds.
Subject(s)
Insulin , Insulins , AnimalsABSTRACT
Somatostatin and its related peptides (SSRPs) form an important family of hormones with diverse physiological roles. The ubiquitous presence of SSRPs in vertebrates and several invertebrate deuterostomes suggests an ancient origin of the SSRP signaling system. However, the existence of SSRP genes outside of deuterostomes has not been established, and the evolutionary history of this signaling system remains poorly understood. Our recent discovery of SSRP-like toxins (consomatins) in venomous marine cone snails (Conus) suggested the presence of a related signaling system in mollusks and potentially other protostomes. Here, we identify the molluscan SSRP-like signaling gene that gave rise to the consomatin family. Following recruitment into venom, consomatin genes experienced strong positive selection and repeated gene duplications resulting in the formation of a hyperdiverse family of venom peptides. Intriguingly, the largest number of consomatins was found in worm-hunting species (>400 sequences), indicating a homologous system in annelids, another large protostome phylum. Consistent with this, comprehensive sequence mining enabled the identification of SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results established the existence of SSRP-like peptides in many major branches of bilaterians and challenge the prevailing hypothesis that deuterostome SSRPs and protostome allatostatin-C are orthologous peptide families. Finally, having a large set of predator-prey SSRP sequences available, we show that although the cone snail's signaling SSRP-like genes are under purifying selection, the venom consomatin genes experience rapid directional selection to target receptors in a changing mix of prey.
Subject(s)
Conotoxins , Conus Snail , Animals , Conotoxins/genetics , Conus Snail/genetics , Neuropeptides , Peptides/genetics , Somatostatin/genetics , VenomsABSTRACT
Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.
Subject(s)
Conus Snail , Somatostatin , Venoms , Animals , Conus Snail/chemistry , Phylogeny , Predatory Behavior , Somatostatin/chemistry , Venoms/chemistryABSTRACT
The venomous marine snails are conventionally divided into three groups, the cone snails (family Conidae), the auger snails (family Terebridae) and the turrids (formerly all assigned to a single family, Turridae). In this study, a library of venom peptides from species conventionally assigned to the genus Turris was correlated to a phylogenetic analysis. Nucleotide sequences of multiple genes from transcriptomes were used to assess the phylogenetic relationships across a diverse set of species. The resulting tree shows that as conventionally defined, the conoidean genus Turris, is polyphyletic. We describe a new genus, Purpuraturris gen. nov., that comprises the outlier species. In addition to morphological distinctions, molecular data reveal that this group is divergent from Turris sensu stricto. The correlation between phylogenetic information and a family of peptide sequences was used to highlight those peptides mostly likely to be unique and intimately associated with biological diversity. The plethora of peptide sequences available requires two prioritization decisions: which subset of peptides to initially characterize, and after these are characterized, which to comprehensively investigate for potential biomedical applications such as drug developments. Life Science Identifiers: urn:lsid:zoobank.org; pub: 60D46561-28F0-4C39-BAC4-66DC8B4EAEA4.
ABSTRACT
Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.
Subject(s)
Insulin , Mollusk Venoms , Cryoelectron Microscopy , Humans , Insulin/metabolism , Mollusk Venoms/chemistry , Mollusk Venoms/metabolism , Peptides , Protein ConformationABSTRACT
Secreted proteins and peptides hold large potential both as therapeutics and as enzyme catalysts in biotechnology. The high stability of many secreted proteins helps maintain functional integrity in changing chemical environments and is a contributing factor to their commercial potential. Disulphide bonds constitute an important post-translational modification that stabilizes many of these proteins and thus preserves the active state under chemically stressful conditions. Despite their importance, the discovery and applications within this group of proteins and peptides are limited by the availability of synthetic biology tools and heterologous production systems that allow for efficient formation of disulphide bonds. Here, we refine the design of two DisCoTune (Disulphide bond formation in E. coli with tunable expression) plasmids that enable the formation of disulphides in the highly popular Escherichia coli T7 protein production system. We show that this new system promotes significantly higher yield and activity of an industrial protease and a conotoxin, which belongs to a group of disulphide-rich venom peptides from cone snails with strong potential as research tools and pharmacological agents.
Subject(s)
Disulfides , Escherichia coli , Escherichia coli/genetics , Peptides/genetics , Plasmids/genetics , Protein FoldingABSTRACT
Venomous molluscs (Superfamily Conoidea) comprise a substantial fraction of tropical marine biodiversity (>15,000 species). Prior characterization of cone snail venoms established that bioactive venom components used to capture prey, defend against predators and for competitive interactions were relatively small, structured peptides (10-35 amino acids), most with multiple disulfide crosslinks. These venom components ("conotoxins, conopeptides") have been widely studied in many laboratories, leading to pharmaceutical agents and probes. In this review, we describe how it has recently become clear that to varying degrees, cone snail venoms also contain bioactive non-peptidic small molecule components. Since the initial discovery of genuanine as the first bioactive venom small molecule with an unprecedented structure, a broad set of cone snail venoms have been examined for non-peptidic bioactive components. In particular, a basal clade of cone snails (Stephanoconus) that prey on polychaetes produce genuanine and many other small molecules in their venoms, suggesting that this lineage may be a rich source of non-peptidic cone snail venom natural products. In contrast to standing dogma in the field that peptide and proteins are predominantly used for prey capture in cone snails, these small molecules also contribute to prey capture and push the molecular diversity of cone snails beyond peptides. The compounds so far characterized are active on neurons and thus may potentially serve as leads for neuronal diseases. Thus, in analogy to the incredible pharmacopeia resulting from studying venom peptides, these small molecules may provide a new resource of pharmacological agents.
ABSTRACT
Venomous animals hunt using bioactive peptides, but relatively little is known about venom small molecules and the resulting complex hunting behaviors. Here, we explored the specialized metabolites from the venom of the worm-hunting cone snail, Conus imperialis Using the model polychaete worm Platynereis dumerilii, we demonstrate that C. imperialis venom contains small molecules that mimic natural polychaete mating pheromones, evoking the mating phenotype in worms. The specialized metabolites from different cone snails are species-specific and structurally diverse, suggesting that the cones may adopt many different prey-hunting strategies enabled by small molecules. Predators sometimes attract prey using the prey's own pheromones, in a strategy known as aggressive mimicry. Instead, C. imperialis uses metabolically stable mimics of those pheromones, indicating that, in biological mimicry, even the molecules themselves may be disguised, providing a twist on fake news in chemical ecology.
Subject(s)
Conus Snail , Predatory Behavior , Animals , Conus Snail/chemistry , Peptides/chemistry , Pheromones/chemistry , SnailsABSTRACT
Natural products such as conotoxins have tremendous potential as tools for biomedical research and for the treatment of different human diseases. Conotoxins are peptides present in the venoms of predatory cone snails that have a rich diversity of pharmacological functions. One of the major bottlenecks in natural products research is the rapid identification and evaluation of bioactive molecules. To overcome this limitation, we designed a set of light-induced behavioral assays in zebrafish larvae to screen for bioactive conotoxins. We used this screening approach to test several unique conotoxins derived from different cone snail clades and discovered that a conorfamide from Conus episcopatus, CNF-Ep1, had the most dramatic alterations in the locomotor behavior of zebrafish larvae. Interestingly, CNF-Ep1 is also bioactive in several mouse assay systems when tested in vitro and in vivo. Our novel screening platform can thus accelerate the identification of bioactive marine natural products, and the first compound discovered using this assay has intriguing properties that may uncover novel neuronal circuitry.
Subject(s)
Larva/drug effects , Locomotion/drug effects , Mollusk Venoms/pharmacology , Neuropeptides/pharmacology , Zebrafish , Animals , Conus Snail/chemistry , Female , Male , MiceABSTRACT
The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, >20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Clavus davidgilmouri Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.
Subject(s)
Conus Snail , Mollusk Venoms/chemistry , Peptides , Amino Acid Sequence , Animals , Behavior, Animal/drug effects , Ganglia, Spinal/cytology , Mice , Neurons/drug effects , Peptides/chemistry , Peptides/isolation & purification , Peptides/toxicityABSTRACT
Conotoxins form a diverse group of peptide toxins found in the venom of predatory marine cone snails. Decades of conotoxin research have provided numerous measurable scientific and societal benefits. These include their use as a drug, diagnostic agent, drug leads, and research tools in neuroscience, pharmacology, biochemistry, structural biology, and molecular evolution. Human envenomations by cone snails are rare but can be fatal. Death by envenomation is likely caused by a small set of toxins that induce muscle paralysis of the diaphragm, resulting in respiratory arrest. The potency of these toxins led to concerns regarding the potential development and use of conotoxins as biological weapons. To address this, various regulatory measures have been introduced that limit the use and access of conotoxins within the research community. Some of these regulations apply to all of the ≈200,000 conotoxins predicted to exist in nature of which less than 0.05% are estimated to have any significant toxicity in humans. In this review we provide an overview of the many benefits of conotoxin research, and contrast these to the perceived biosecurity concerns of conotoxins and research thereof.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.