Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

Abstract

ShK fromStichodactyla helianthushas established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in actinarians 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 inActinia tenebrosaandTelmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom ofT. stephensoni. We identified ten SA8 genes in two clusters and six SA8 genes in five clusters forT. stephensoniandA. tenebrosa, respectively. Nine SA8T. stephensonigenes 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 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. 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 ofT. stephensoni.

Acontia, a Specialised Defensive Structure, Has Low Venom Complexity in Calliactis polypus

“…Similar to many sea anemone peptide toxins [ 11 , 38 ], multiple codons from the Unknown 12C protein were found to be evolving in a manner consistent with the action of negative selection, which was most prominent for the cysteine encoding codons. The cysteine framework has no similarity to any known toxin in sea anemones [ 45 , 46 ], although it has an identical motif at two locations in the scaffold that match the last three C-X frames of ShK toxins (C-X3-C-X2-C) [ 47 ] and sea anemone 8 toxins [ 48 ]. This scaffold, as well the presence of the Lys-Arg (KR) motif at location 71–72 of the protein sequence ( Figure 2 ), may indicate that Unknown 12C has two tandem domains with a scaffold of six cysteines each, and that it is similar to but divergent from ShK toxins.…”

Towards the Exploration and Evolution of Insulin-Derived Venoms in Actiniaria (Sea anemones)

“…Alternative splicing is a common genetic regulatory mechanism in Eukaryotes and is commonly part of the venom systems of many venomous species [54][55][56][57][58][59], where it has been suggested to increase the diversity of possible venoms in any one species. Alternative splicing has been identified in venom genes for sea anemones only for the sea anemone 8 (SA8) toxin gene family, which has alternative alternate splice isoforms derived from an inverted SA8 gene [60].…”

Structure–function relationships in ShKT domain peptides: ShKT‐Ts1 from the sea anemone Telmatactis stephensoni

“…The venoms of sea anemones are complex mixtures of biologically active compounds, including peptides, proteins, and low‐molecular weight molecules 1–4 . Peptides from the venom of sea anemones, as well as from other parts of these animals, 5 exhibit exceptional molecular diversity, with more than 17 structural scaffolds described, 3,6,7 and more being discovered 8 . A commonly detected scaffold is the ShKT domain, which was first identified in ShK toxin, from the Caribbean sea anemone Stichodactyla helianthus 9 .…”