Abstract
Sea anemones (Actiniaria) are intensely popular objects of study in venomics. Order Actiniaria includes more than 1,000 species, thus presenting almost unlimited opportunities for the discovery of novel biologically active molecules. The venoms of cold-water sea anemones are studied far less than the venoms of tropical sea anemones. In this work, we analysed the molecular venom composition of the cold-water sea anemone Cnidopus japonicus. Two sets of NGS data from two species revealed molecules belonging to a variety of structural classes, including neurotoxins, toxin-like molecules, linear polypeptides (Cys-free), enzymes, and cytolytics. High-throughput proteomic analyses identified 27 compounds that were present in the venoms. Some of the toxin-like polypeptides exhibited novel Cys frameworks. To characterise their function in the venom, we heterologously expressed 3 polypeptides with unusual Cys frameworks (designated CjTL7, CjTL8, and AnmTx Cj 1c-1) in E. coli. Toxicity tests revealed that the CjTL8 polypeptide displays strong crustacean-specific toxicity, while AnmTx Cj 1c-1 is toxic to both crustaceans and insects. Thus, an improved NGS data analysis algorithm assisted in the identification of toxins with unusual Cys frameworks showing no homology according to BLAST. Our study shows the advantage of combining omics analysis with functional tests for active polypeptide discovery.
Highlights
Organisms that produce venom have long been important for a number of applications
Large sets of possible toxin candidates derived from transcriptomics data may be validated using proteomics to determine the peptide and protein compositions of real venoms
This database was compared with proteomics data to identify the toxins expressed in the venom, similar to the method described by Nesvizhskii et al.[26]
Summary
Organisms that produce venom have long been important for a number of applications. Such organisms produce a wide range of compounds with different functions (e.g., neurotoxic, haemolytic)[1,2]. The NGS-based approach does not allow researchers to determine the biological activity of molecules, it provides comprehensive sequence information, helping to overcome the time and technical limitations associated with Edman degradation and shotgun MS-MS de novo sequencing. Using this approach, researchers can search for both new toxins and toxins that are homologous to those described previously. We employed an approach based on the NGS sequencing of transcriptomes (RNA-seq) to analyse the diversity of expressed peptides and protein molecules in 2 specimens of the cold-water sea anemone C. japonicus and validated the results using proteomics. We functionally characterised 3 polypeptides from C. japonicus venom through toxicity tests on crustaceans and insects
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