Genomic, functional and structural analyses elucidate evolutionary innovation within the sea anemone 8 toxin family.

BACKGROUND: The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. RESULTS: We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. CONCLUSIONS: Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.

Venoms for all occasions: The functional toxin profiles of different anatomical regions in sea anemones are related to their ecological function.

The phylum Cnidaria is the oldest extant venomous group and is defined by the presence of nematocysts, specialized organelles responsible for venom production and delivery. Although toxin peptides and the cells housing nematocysts are distributed across the entire animal, nematocyte and venom profiles have been shown to differ across morphological structures in actiniarians. In this study, we explore the relationship between patterns of toxin expression and the ecological roles of discrete anatomical structures in Telmatactis stephensoni. Specifically, using a combination of proteomic and transcriptomic approaches, we examined whether there is a direct correlation between the functional similarity of regions and the similarity of their associated toxin expression profiles. We report that the regionalization of toxin production is consistent with the partitioning of the ecological roles of venom across envenomating structures, and that three major functional regions are present in T. stephensoni: tentacles, epidermis and gastrodermis. Additionally, we find that most structures that serve similar functions not only have comparable putative toxin profiles but also similar nematocyst types. There was no overlap in the putative toxins identified using proteomics and transcriptomics, but the expression patterns of specific milked venom peptides were conserved across RNA-sequencing and mass spectrometry imaging data sets. Furthermore, based on our data, it appears that acontia of T. stephensoni may be transcriptionally inactive and only mature nematocysts are present in the distal portions of the threads. Overall, we find that the venom profile of different anatomical regions in sea anemones varies according to its ecological functions.

Tentacle Morphological Variation Coincides with Differential Expression of Toxins in Sea Anemones.

Phylum Cnidaria is an ancient venomous group defined by the presence of cnidae, specialised organelles that serve as venom delivery systems. The distribution of cnidae across the body plan is linked to regionalisation of venom production, with tissue-specific venom composition observed in multiple actiniarian species. In this study, we assess whether morphological variants of tentacles are associated with distinct toxin expression profiles and investigate the functional significance of specialised tentacular structures. Using five sea anemone species, we analysed differential expression of toxin-like transcripts and found that expression levels differ significantly across tentacular structures when substantial morphological variation is present. Therefore, the differential expression of toxin genes is associated with morphological variation of tentacular structures in a tissue-specific manner. Furthermore, the unique toxin profile of spherical tentacular structures in families Aliciidae and Thalassianthidae indicate that vesicles and nematospheres may function to protect branched structures that host a large number of photosynthetic symbionts. Thus, hosting zooxanthellae may account for the tentacle-specific toxin expression profiles observed in the current study. Overall, specialised tentacular structures serve unique ecological roles and, in order to fulfil their functions, they possess distinct venom cocktails.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Sea Anemone Toxins: A Structural Overview.

Sea anemones produce venoms of exceptional molecular diversity, with at least 17 different molecular scaffolds reported to date. These venom components have traditionally been classified according to pharmacological activity and amino acid sequence. However, this classification system suffers from vulnerabilities due to functional convergence and functional promiscuity. Furthermore, for most known sea anemone toxins, the exact receptors they target are either unknown, or at best incomplete. In this review, we first provide an overview of the sea anemone venom system and then focus on the venom components. We have organised the venom components by distinguishing firstly between proteins and non-proteinaceous compounds, secondly between enzymes and other proteins without enzymatic activity, then according to the structural scaffold, and finally according to molecular target.

A process of convergent amplification and tissue-specific expression dominates the evolution of toxin and toxin-like genes in sea anemones.

Members of phylum Cnidaria are an ancient group of venomous animals and rely on a number of specialized tissues to produce toxins in order to fulfil a range of ecological roles including prey capture, defence against predators, digestion and aggressive encounters. However, limited comprehensive analyses of the evolution and expression of toxin genes currently exist for cnidarian species. In this study, we use genomic and transcriptomic sequencing data to examine gene copy number variation and selective pressure on toxin gene families in phylum Cnidaria. Additionally, we use quantitative RNA-seq and mass spectrometry imaging to understand expression patterns and tissue localization of toxin production in sea anemones. Using genomic data, we demonstrate that the first large-scale expansion and diversification of known toxin genes occurs in phylum Cnidaria, a process we also observe in other venomous lineages, which we refer to as convergent amplification. Our analyses of selective pressure on sea anemone toxin gene families reveal that purifying selection is the dominant mode of evolution for these genes and that phylogenetic inertia is an important determinant of toxin gene complement in this group. The gene expression and tissue localization data revealed that specific genes and proteins from toxin gene families show strong patterns of tissue and developmental-phase specificity in sea anemones. Overall, convergent amplification and phylogenetic inertia have strongly influenced the distribution and evolution of the toxin complement observed in sea anemones, while the production of venoms with different compositions across tissues is related to the functional and ecological roles undertaken by each tissue type.

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold.

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (KV) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric ß-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type KV channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.

Revisiting venom of the sea anemone Stichodactyla haddoni: Omics techniques reveal the complete toxin arsenal of a well-studied sea anemone genus.

More than a century of research on sea anemone venoms has shown that they contain a diversity of biologically active proteins and peptides. However, recent omics studies have revealed that much of the venom proteome remains unexplored. We used, for the first time, a combination of proteomic and transcriptomic techniques to obtain a holistic overview of the venom arsenal of the well-studied sea anemone Stichodactyla haddoni. A purely search-based approach to identify putative toxins in a transcriptome from tentacles regenerating after venom extraction identified 508 unique toxin-like transcripts grouped into 63 families. However, proteomic analysis of venom revealed that 52 of these toxin families are likely false positives. In contrast, the combination of transcriptomic and proteomic data enabled positive identification of 23 families of putative toxins, 12 of which have no homology known proteins or peptides. Our data highlight the importance of using proteomics of milked venom to correctly identify venom proteins/peptides, both known and novel, while minimizing false positive identifications from non-toxin homologues identified in transcriptomes of venom-producing tissues. This work lays the foundation for uncovering the role of individual toxins in sea anemone venom and how they contribute to the envenomation of prey, predators, and competitors. BIOLOGICAL SIGNIFICANCE: Proteomic analysis of milked venom combined with analysis of a tentacle transcriptome revealed the full extent of the venom arsenal of the sea anemone Stichodactyla haddoni. This combined approach led to the discovery of 12 entirely new families of disulfide-rich peptides and proteins in a genus of anemones that have been studied for over a century.

Melt With This Kiss: Paralyzing and Liquefying Venom of The Assassin Bug Pristhesancus plagipennis (Hemiptera: Reduviidae).

Assassin bugs (Hemiptera: Heteroptera: Reduviidae) are venomous insects, most of which prey on invertebrates. Assassin bug venom has features in common with venoms from other animals, such as paralyzing and lethal activity when injected, and a molecular composition that includes disulfide-rich peptide neurotoxins. Uniquely, this venom also has strong liquefying activity that has been hypothesized to facilitate feeding through the narrow channel of the proboscis-a structure inherited from sap- and phloem-feeding phytophagous hemipterans and adapted during the evolution of Heteroptera into a fang and feeding structure. However, further understanding of the function of assassin bug venom is impeded by the lack of proteomic studies detailing its molecular composition.By using a combined transcriptomic/proteomic approach, we show that the venom proteome of the harpactorine assassin bug Pristhesancus plagipennis includes a complex suite of >100 proteins comprising disulfide-rich peptides, CUB domain proteins, cystatins, putative cytolytic toxins, triabin-like protein, odorant-binding protein, S1 proteases, catabolic enzymes, putative nutrient-binding proteins, plus eight families of proteins without homology to characterized proteins. S1 proteases, CUB domain proteins, putative cytolytic toxins, and other novel proteins in the 10-16-kDa mass range, were the most abundant venom components. Thus, in addition to putative neurotoxins, assassin bug venom includes a high proportion of enzymatic and cytolytic venom components likely to be well suited to tissue liquefaction. Our results also provide insight into the trophic switch to blood-feeding by the kissing bugs (Reduviidae: Triatominae). Although some protein families such as triabins occur in the venoms of both predaceous and blood-feeding reduviids, the composition of venoms produced by these two groups is revealed to differ markedly. These results provide insights into the venom evolution in the insect suborder Heteroptera.

Ancient Venom Systems: A Review on Cnidaria Toxins.

Cnidarians are the oldest extant lineage of venomous animals. Despite their simple anatomy, they are capable of subduing or repelling prey and predator species that are far more complex and recently evolved. Utilizing specialized penetrating nematocysts, cnidarians inject the nematocyst content or "venom" that initiates toxic and immunological reactions in the envenomated organism. These venoms contain enzymes, potent pore forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that catabolize prey tissues. Cnidarian pore forming toxins self-assemble to form robust membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion channel specific activities. In addition, certain cnidarian venoms contain or induce the release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and caissarone accelerating the pathogenic effects of other venom enzymes and porins. The cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation of humans may result in death, in some cases within a few minutes to an hour after sting. The complexity of venom components represents a unique therapeutic challenge and probably reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are invaluable as a therapeutic target for sting treatment or as lead compounds for drug design.

BcsTx3 is a founder of a novel sea anemone toxin family of potassium channel blocker.

Sea anemone venoms have become a rich source of peptide toxins which are invaluable tools for studying the structure and functions of ion channels. In this work, BcsTx3, a toxin found in the venom of a Bunodosoma caissarum (population captured at the Saint Peter and Saint Paul Archipelago, Brazil) was purified and biochemically and pharmacologically characterized. The pharmacological effects were studied on 12 different subtypes of voltage-gated potassium channels (K(V)1.1-K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; hERG and Shaker IR) and three cloned voltage-gated sodium channel isoforms (Na(V)1.2, Na(V)1.4 and BgNa(V)1.1) expressed in Xenopus laevis oocytes. BcsTx3 shows a high affinity for Drosophila Shaker IR channels over rKv1.2, hKv1.3 and rKv1.6, and is not active on NaV channels. Biochemical characterization reveals that BcsTx3 is a 50 amino acid peptide crosslinked by four disulfide bridges, and sequence comparison allowed BcsTx3 to be classified as a novel type of sea anemone toxin acting on K(V) channels. Moreover, putative toxins homologous to BcsTx3 from two additional actiniarian species suggest an ancient origin of this newly discovered toxin family.

Biochemical and electrophysiological characterization of two sea anemone type 1 potassium toxins from a geographically distant population of Bunodosoma caissarum.

Sea anemone (Cnidaria, Anthozoa) venom is an important source of bioactive compounds used as tools to study the pharmacology and structure-function of voltage-gated K+ channels (KV). These neurotoxins can be divided into four different types, according to their structure and mode of action. In this work, for the first time, two toxins were purified from the venom of Bunodosoma caissarum population from Saint Peter and Saint Paul Archipelago, Brazil. Sequence alignment and phylogenetic analysis reveals that BcsTx1 and BcsTx2 are the newest members of the sea anemone type 1 potassium channel toxins. Their functional characterization was performed by means of a wide electrophysiological screening on 12 different subtypes of KV channels (KV1.1-KV1.6; KV2.1; KV3.1; KV4.2; KV4.3; hERG and Shaker IR). BcsTx1 shows a high affinity for rKv1.2 over rKv1.6, hKv1.3, Shaker IR and rKv1.1, while Bcstx2 potently blocked rKv1.6 over hKv1.3, rKv1.1, Shaker IR and rKv1.2. Furthermore, we also report for the first time a venom composition and biological activity comparison between two geographically distant populations of sea anemones.