Identification of Novel Conopeptides and Distinct Gene Superfamilies in the Marine Cone Snail Conus quercinus

Conopeptides from the marine cone snails are a mixture of cysteine-rich active peptides, representing a unique and fertile resource for neuroscience research and drug discovery. The ConoServer database includes 8,134 conopeptides from 122 Conus species, yet many more natural conopeptides remain to be discovered. Here, we identified 517 distinct conopeptide precursors in Conus quercinus using de novo deep transcriptome sequencing. Ten of these precursors were verified at the protein level using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The combined gene and protein analyses revealed two novel gene superfamilies (Que-MNCLQ and Que-MAMNV), and three other gene superfamilies (N, P, and I1) were reported for the first time in C. quercinus. From the Que-MAMNV superfamily, a novel conotoxin, Que-0.1, was obtained via cloning and prokaryotic expression. We also documented a new purification process that can be used to induce the expression of conopeptides containing multiple pairs of disulfide bonds. The animal experiments showed that Que-0.1 strongly inhibited neuroconduction; the effects of Que-1.0 were 6.25 times stronger than those of pethidine hydrochloride. In addition, a new cysteine framework (CC-C-C-C-C-C-CC-C-C-C-C-C) was found in C. quercinus. These discoveries accelerate our understanding of conopeptide diversity in the genus, Conus and supply promising materials for medical research.

Reconstructing the Origins of a Neuropeptide Signaling System Using the Accelerated Evolution of Biodiverse Cone Snail Venoms

Somatostatin and its related peptides (SSRPs) form a 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 homologous 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 experience strong positive selection and repeated gene duplications resulting in the formation of a hyper-diverse 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. Comprehensive sequence mining enabled the identification of orthologous SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results establish the existence of SSRP-like sequences in many major branches of bilaterians, including xenacoelomorphs, a phylum believed to have emerged before the divergence of protostomes and deuterostomes, ~ 600 My ago. Finally, having a large set of predator-prey SSRP sequences available, we show that while the cone snail's signaling SSRP-like genes are under purifying selection, in striking contrast, the consomatin genes experience rapid directional selection to target receptors in a changing mix of prey.

Successful Decannulation of Hybrid ECMO Circuitry: The First Philippine Experience

We report the first VA-VAV-VV ECMO conversion in a 57-year-old Filipino female with persistent coronary insufficiency from toxic shock syndrome due to Streptococcus pyogenes bacteremia, acute respiratory distress syndrome, and Harlequin syndrome with progressive acute limb ischemia from cone snail venom poisoning. The patient came in via air ambulance transport because deteriorating clinical status after having stepped on a cone snail 3 days prior and developing severe dehydration from vomiting and passage of voluminous watery stools after eating a local delicacy. The patient was admitted at the ICU where after 3 days of treatment, the patient developed cardiac tamponade and underwent stat pleuropericardial windowing with pericardiocentesis. Due to increasing pressor requirements from the combined shock, Swan-Ganz catheterization was inserted and a VA-ECMO system was set up. There was gradual improvement in cardiac hemodynamics, however oxygen requirement was increasing and both lower extremities became progressively violaceous with decreasing pulses. Harlequin syndrome was ruled in, the multidisciplinary team decided to convert to VAV hybrid circuit by adding a venous access by way of the right internal jugular vein. Improvement in oxygenation lead to eventual conversion from VAV to purely VV dedicated circuit. Ultrafiltration via ECMO was likewise done because of worsening azotemia and oliguria. The patient was then gradually weaned off from ECMO and was successfully decannulated after 8 days. Below knee amputation was done to address the progressive acute limb ischemia. The patient was nutritionally built up and physically rehabilitated and was eventually discharged improved on the 28th hospital day.

Voltage-Gated Sodium Channels: A Prominent Target of Marine Toxins

Voltage-gated sodium channels (VGSCs) are considered to be one of the most important ion channels given their remarkable physiological role. VGSCs constitute a family of large transmembrane proteins that allow transmission, generation, and propagation of action potentials. This occurs by conducting Na+ ions through the membrane, supporting cell excitability and communication signals in various systems. As a result, a wide range of coordination and physiological functions, from locomotion to cognition, can be accomplished. Drugs that target and alter the molecular mechanism of VGSCs’ function have highly contributed to the discovery and perception of the function and the structure of this channel. Among those drugs are various marine toxins produced by harmful microorganisms or venomous animals. These toxins have played a key role in understanding the mode of action of VGSCs and in mapping their various allosteric binding sites. Furthermore, marine toxins appear to be an emerging source of therapeutic tools that can relieve pain or treat VGSC-related human channelopathies. Several studies documented the effect of marine toxins on VGSCs as well as their pharmaceutical applications, but none of them underlined the principal marine toxins and their effect on VGSCs. Therefore, this review aims to highlight the neurotoxins produced by marine animals such as pufferfish, shellfish, sea anemone, and cone snail that are active on VGSCs and discuss their pharmaceutical values.

Identification of Novel Conotoxin Precursors from the Cone Snail Conus spurius by High-Throughput RNA Sequencing

Marine gastropods of the genus Conus, comprising more than 800 species, have the characteristic of injecting worms and other prey with venom. These conopeptide toxins, highly diverse in structure and action, are highly potent and specific for their molecular targets (ion channels, receptors, and transporters of the prey’s nervous system), and thus are important research tools and source for drug discovery. Next-generation sequencing technologies are speeding up the discovery of novel conopeptides in many of these species, but only limited information is available for Conus spurius, which inhabits sandy mud. To search for new precursor conopeptides, we analyzed the transcriptome of the venous ducts of C. spurius and identified 55 putative conotoxins. Seven were selected for further study and confirmed by Sanger sequencing to belong to the M-superfamily (Sr3.M01 and Sr3.M02), A-superfamily (Sr1.A01 and Sr1.A02), O-superfamily (Sr15.O01), and Con-ikot-ikot (Sr21.CII01 and Sr22.CII02). Six of these have never been reported. To our knowledge, this report is the first to use high-throughput RNA sequencing for the study of the diversity of C. spurius conotoxins.

A Combined Transcriptomics and Proteomics Approach Reveals the Differences in the Predatory and Defensive Venoms of the Molluscivorous Cone Snail Cylinder ammiralis (Caenogastropoda: Conidae)

Venoms are complex mixtures of proteins that have evolved repeatedly in the animal kingdom. Cone snail venoms represent one of the best studied venom systems. In nature, this venom can be dynamically adjusted depending on its final purpose, whether to deter predators or hunt prey. Here, the transcriptome of the venom gland and the proteomes of the predation-evoked and defensive venoms of the molluscivorous cone snail Cylinder ammiralis were catalogued. A total of 242 venom-related transcripts were annotated. The conotoxin superfamilies presenting more different peptides were O1, O2, T, and M, which also showed high expression levels (except T). The three precursors of the J superfamily were also highly expressed. The predation-evoked and defensive venoms showed a markedly distinct profile. A total of 217 different peptides were identified, with half of them being unique to one venom. A total of 59 peptides ascribed to 23 different protein families were found to be exclusive to the predatory venom, including the cono-insulin, which was, for the first time, identified in an injected venom. A total of 43 peptides from 20 protein families were exclusive to the defensive venom. Finally, comparisons of the relative abundance (in terms of number of peptides) of the different conotoxin precursor superfamilies showed that most of them present similar abundance regardless of the diet.

A phylogeny-aware approach reveals unexpected venom components in divergent lineages of cone snails

Marine gastropods of the genus Conus are renowned for their remarkable diversity and deadly venoms. While Conus venoms are increasingly well studied for their biomedical applications, we know surprisingly little about venom composition in other lineages of Conidae. We performed comprehensive venom transcriptomic profiling for Conasprella coriolisi and Pygmaeconus traillii , first time for both respective genera. We complemented reference-based transcriptome annotation by a de novo toxin prediction guided by phylogeny, which involved transcriptomic data on two additional ‘divergent’ cone snail lineages, Profundiconus , and Californiconus . We identified toxin clusters (SSCs) shared among all or some of the four analysed genera based on the identity of the signal region—a molecular tag present in toxins. In total, 116 and 98 putative toxins represent 29 and 28 toxin gene superfamilies in Conasprella and Pygmaeconus , respectively; about quarter of these only found by semi-manual annotation of the SSCs. Two rare gene superfamilies, originally identified from fish-hunting cone snails, were detected outside Conus rather unexpectedly, so we further investigated their distribution across Conidae radiation. We demonstrate that both these, in fact, are ubiquitous in Conidae, sometimes with extremely high expression. Our findings demonstrate how a phylogeny-aware approach circumvents methodological caveats of similarity-based transcriptome annotation.

Visualization of Insulin Receptor Activation by a Novel Insulin Analog with Elongated A Chain and Truncated B Chain

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 a fully active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy and protein engineering to elucidate its interactions with the human insulin receptor ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of native insulin but also compromise therapeutic utility by delaying the onset action, suggesting approaches to develop improved therapeutic insulins. Curiously, a receptor conformation present in low abundance adopts a highly asymmetric structure that displays novel coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.