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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 Ki 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.

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the Kunitz/Bpti-type peptides are ubiquitous in numerous organisms including marine venomous animals. the peptides demonstrate various biological activities and therefore they are the subject of a number of investigations. We have discovered a new HciQ subfamily belonging to recently described multigene HcGS family of Heteractis crispa Kunitz-peptides. the uniqueness of this subfamily is that the HciQ precursors contain a propeptide terminating in Lys-Arg (endopeptidase cleavage site) the same as in the neuro-and cytotoxin ones. Moreover, the HCIQ genes contain two introns in contrast to HCGS genes with one intron. As a result of Sanger and amplicon deep sequencings, 24 HCIQ isoforms were revealed. The recombinant peptides for the most prevalent isoform (HCIQ2c1) and for the isoform with the rare substitution Gly17Glu (HCIQ4c7) were obtained. They can inhibit trypsin with K i 5.2 × 10 −8 M and K i 1.9 × 10 −7 M, respectively, and interact with some serine proteinases including inflammatory ones according to the SPR method. For the first time, Kunitz-peptides have shown to significantly increase neuroblastoma cell viability in an in vitro 6-OHDA-induced neurotoxicity model being a consequence of an effective decrease of ROS level in the cells. Kunitz-type proteinase inhibitors are present in various living organisms and in viruses. They are widely distributed and well-characterized in animals including marine invertebrates, snakes, spiders, ticks, flies and mammals 1. In spider, snake, scorpion, cone snail and sea anemone venoms Kunitz-peptides may exist in multiple isoforms possessing conserved BPTI-like fold but exhibit different biological activities 2-9. This phenomenon is associated with gene duplication and their diversification throughout adaptive evolution leading to the formation of families of evolutionarily related but functionally distinct genes 10. Among sea anemone transcriptomes, such multigene families have been discovered in Anemonia viridis 11 , Stichodactyla haddoni 12 , and Heteractis crispa 5. HCGS multigene family of H. crispa has been found to be divided in four distinct subfamilies (GS, RG, GG, and GN) forming the combinatory library of Kunitz/BPTI peptides 5. The group of HCGN peptides was presented by one sequence, different from other sequences that are characterized by a propeptide insertion containing the cleavage site Lys-Arg, and additional residues Ile-Gln at the N-terminus of a mature peptide. Its full-length homolog HMIQ3c1, containing a mature peptide with the same residues at N-terminus, was derived from the cDNA of the sea anemone Heteractis magnifica 13. The most abundant HCGS and HCRG peptides are being actively studied nowadays 14-17. The main targets of Kunitz-peptides are serine proteinases, such as trypsin and α-chymotrypsin.

Six Gram-stain-negative, aerobic, rod-shaped, and motile by gliding bacterial strains were isolated from Pacific green and red algae. Phylogenetic analysis based on 16S rRNA gene sequences placed the novel strains into the genus Zobellia as a distinct evolutionary lineage close to Zobellia nedashkovskayae Asnod2-B07-BT and Zobellia laminariae KMM 3676T sharing the highest similarity of 99.7% and 99.5%, respectively. The average nucleotide identity and the average amino acid identity values between strains 36-CHABK-3-33T and Z. nedashkovskayae Asnod2-B07-BT and Z. laminariae KMM 3676T were 89.7%/92.9% and 94.2%/95.8%, respectively. The digital DNA–DNA hybridization values based on the draft genomes between strains 36-CHABK-3-33T and Z. nedashovskayae Asnod2-B07-BT and Z. laminariae KMM 3676T were 39.5 ± 2.5% and 59.6 ± 2.7%, respectively. Multilocus sequence analysis based on house-keeping genes (dnaK, gyrB, pyrH, recA and topA) assigned the alga-associated isolates to the same species, which clustered separately from the recognized species of the genus Zobellia. The strains under study grew at 4–32 °C and with 0.5–8% NaCl and decomposed aesculin, gelatin, DNA, and Tweens 20 and 80, and weakly agar. The DNA G+C content was 36.7% calculated from genome sequence analysis for the strain 36-CHABK-3-33T. The predominant fatty acids of strain 36-CHABK-3-33T (> 5% of the total fatty acids) were iso-C17:0 3-OH, summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH fatty acids), iso-C15:0, iso-C15:1 G, and C15:0. The major polar lipids were phosphatidylethanolamine, three unidentified lipids, and two unidentified aminolipids. The only detected respiratory quinone was MK-6. The significant molecular distinctiveness between the novel isolates and their nearest neighbor was strongly supported by differences in physiological and biochemical tests. Therefore, the six novel strains represent a novel species of the genus Zobellia, for which the name Zobellia barbeyronii sp. nov. is proposed. The type strain is 36-CHABK-3-33T (= KACC 21790T = KMM 6746T).