Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1–APHC3 from H. crispa, and clusters with the peptides from so named “analgesic cluster” of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10⁻⁷ and 7.0 × 10⁻⁷ M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21–TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides.

The Kunitz/BPTI peptide family includes unique representatives demonstrating various biological activities. Electrophysiological screening of peptides HCRG1 and HCRG2 from the sea anemone Heteractis crispa on six Kv1.x channel isoforms and insect Shaker IR channel expressed in Xenopus laevis oocytes revealed their potassium channels blocking activity. HCRG1 and HCRG2 appear to be the first Kunitz-type peptides from sea anemones blocking Kv1.3 with IC₅₀ of 40.7 and 29.7 nM, respectively. In addition, peptides mainly vary in binding affinity to the Kv1.2 channels. It was established that the single substitution, Ser5Leu, in the TRPV1 channel antagonist, HCRG21, induces weak blocking activity of Kv1.1, Kv1.2, and Kv1.3. Apparently, for the affinity and selectivity of Kunitz-fold toxins to Kv1.x isoforms, the number and distribution along their molecules of charged, hydrophobic, and polar uncharged residues, as well as the nature of the channel residue at position 379 (Tyr, Val or His) are important. Testing the compounds in a model of acute local inflammation induced by the introduction of carrageenan administration into mice paws revealed that HCRG1 at doses of 0.1–1 mg/kg reduced the volume of developing edema during 24 h, similar to the effect of the nonsteroidal anti-inflammatory drug, indomethacin, at a dose of 5 mg/kg. ELISA analysis of the animals blood showed that the peptide reduced the synthesis of TNF-α, a pro-inflammatory mediator playing a leading role in the development of edema in this model.

We obtained two novel draft genomes of type Zobellia strains with estimated genome sizes of 5.14 Mb for Z. amurskyensis KMM 3526^Т and 5.16 Mb for Z. laminariae KMM 3676^Т. Comparative genomic analysis has been carried out between obtained and known genomes of Zobellia representatives. The pan-genome of Zobellia genus is composed of 4853 orthologous clusters and the core genome was estimated at 2963 clusters. The genus CAZome was represented by 775 GHs classified into 62 families, 297 GTs of 16 families, 100 PLs of 13 families, 112 CEs of 13 families, 186 CBMs of 18 families and 42 AAs of six families. A closer inspection of the carbohydrate-active enzyme (CAZyme) genomic repertoires revealed members of new putative subfamilies of GH16 and GH117, which can be biotechnologically promising for production of oligosaccharides and rare monomers with different bioactivities. We analyzed AA3s, among them putative FAD-dependent glycoside oxidoreductases (FAD-GOs) being of particular interest as promising biocatalysts for glycoside deglycosylation in food and pharmaceutical industries.

Sea anemones produce pore-forming toxins, actinoporins, which are interesting as tools for cytoplasmic membranes study, as well as being potential therapeutic agents for cancer therapy. This investigation is devoted to structural and functional study of the Heteractis crispa actinoporins diversity. Here, we described a multigene family consisting of 47 representatives expressed in the sea anemone tentacles as prepropeptide-coding transcripts. The phylogenetic analysis revealed that actinoporin clustering is consistent with the division of sea anemones into superfamilies and families. The transcriptomes of both H. crispa and Heteractis magnifica appear to contain a large repertoire of similar genes representing a rapid expansion of the actinoporin family due to gene duplication and sequence divergence. The presence of the most abundant specific group of actinoporins in H. crispa is the major difference between these species. The functional analysis of six recombinant actinoporins revealed that H. crispa actinoporin grouping was consistent with the different hemolytic activity of their representatives. According to molecular modeling data, we assume that the direction of the N-terminal dipole moment tightly reflects the actinoporins’ ability to possess hemolytic activity.

The primary structure of a new Kunitz-type protease inhibitor InhVJ from the sea anemone Heteractis crispa (Radianthus macrodactylus) was determined by protein sequencing and cDNA cloning. InhVJ amino acid sequence was shown to share high sequence identity (up to 98%) with the other known Kunitz-type sea anemones sequences. It was determined that the P1 Thr at the reactive site resulted in a decrease of the K_i of InhVJ to trypsin and α-chymotrypsin (7.38 × 10⁻⁸ M and 9.93 × 10⁻⁷ M, respectively). By structure modeling the functional importance of amino acids at the reactive site as well as at the weak contact site were determined. The significant role of Glu45 for the orientation and stabilization of the InhVJ-trypsin complex was elucidated. We can suggest that there has been an adaptive evolution of the P1 residue at the inhibitor reactive site providing specialization or functional diversification of the paralogs. The appearance of a key so-called P1 Thr residue instead of Lys might lead to refinement of inhibitor specificity in the direction of subfamilies of serine proteases. The absence of Kv channel and TRPV1-receptor modulation activity was confirmed by electrophysiological screening tests.