Elena Leychenko

The anticancer effects of actinoporin RTX-A from the sea anemone Heteractis crispa (=Radianthus macrodactylus)

Four isoforms of actinoporins were isolated in 2002-2004 from the tropical sea anemone Heteractis crispa (=Radianthus macrodactylus). Their potent hemolytic activities and effects on Ehrlich ascites carcinoma bearing mice were also studied. In this study, the individual actinoporin (RTX-A) demonstrated potential cancer-preventive activity at extremely low and non-cytotoxic concentrations. The substance suppressed the malignant transformation of mouse JB6 P(+) Cl41 cells stimulated by epidermal growth factor (EGF) in soft agar with the inhibition of number of the colonies C(50) (INCC(50))=0.034 nM. Actinoporin RTX-A also was shown to inhibit the phenotype expression of HeLa human cancer cells with an INCC(50)=0.03 nM. The cytotoxic effect of RTX-A against JB6 P(+) Cl41 cells and HeLa, THP-1, MDA-MB-231, and SNU-C4 human tumor cell lines was high (IC(50)=0.57, 2.26, 1.11, 30.0 and 4.66 nM), but significantly less than their capacity to suppress tumor cell colony formation or phenotype expression. RTX-A also induced apoptosis and inhibited basal AP-1, NF-kappaB, and p53-dependent transcriptional activity in JB6 Cl41 cells. These results confirmed that actinoporin RTX-A from H. crispa, at least partially, might exhibit cancer-preventive and anticancer cytotoxic properties through the induction of p53-independent apoptosis and inhibition of the oncogenic AP-1 and NF-kappaB nuclear factors activity.

Atypical reactive center Kunitz-type inhibitor from the sea anemone Heteractis crispa.

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.

New actinoporins from sea anemone Heteractis crispa: Cloning and functional expression

A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10–27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins.

[Anti-Inflammatory Activity of the Polypeptide of the Sea Anemone, Heteractis crispa].

The anti-inflammatory activity of the HCGS 1.20 recombinant polypeptide (a Kunitz-type serine protease inhibitor from the Heteractis crispa sea anemone) was investigated. The polypeptide was shown to inhibit the histamine-induced increase in the concentration of calcium ions and the lipopolysaccharidestimulated increase in the concentration of nitric oxide (II) in macrophages. A possible mechanism of this anti-inflammatory activity of the polypeptide was discussed.

Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis crispa Is a Full Antagonist of the TRPV1 Receptor

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.

Peptide fingerprinting of the sea anemone Heteractis magnifica mucus revealed neurotoxins, Kunitz-type proteinase inhibitors and a new β-defensin α-amylase inhibitor

Sea anemone mucus, due to its multiple and vital functions, is a valuable substance for investigation of new biologically active peptides. In this work, compounds of Heteractis magnifica mucus were separated by multistage liquid chromatography and resulting fractions were analyzed by MALDI-TOF MS. Peptide maps constructed according to the molecular masses and hydrophobicity showed presence of 326 both new and known peptides. Several major peptides from mucus were identified, including the sodium channel toxin RpII isolated earlier from H. magnifica, and four Kunitz-type proteinase inhibitors identical to H. crispa ones. Kunitz-type transcript diversity was studied and sequences of mature peptides were deduced. New β-defensin α-amylase inhibitor, a homolog of helianthamide from Stichodactyla helianthus, was isolated and structurally characterized. Overall, H. magnifica is a source of biologically active peptides with great pharmacological potential. BIOLOGICAL SIGNIFICANCE Proteinase and α-amylase inhibitors along with toxins are major components of H. magnifica mucus which play an important role in the successful existence of sea anemones. Obtained peptide maps create a basis for more accurate identification of peptides during future transcriptomic/genomic studies of sea anemone H. magnifica.

Kunitz-type peptides of the sea anemone Heteractis crispa: Potential anti-inflammatory compounds

Antihistamine activity of recombinant peptides, Kunitz-type serine protease inhibitors of the sea anemone Heteractis crispa, was studied. It was shown that the peptides rHCGS1.19 and rHCGS1.36 at a concentration 10 μM inhibit an increase in the calcium ion concentration in macrophages elicited by histamine at 62.2 and 84.0%, respectively. The anti-inflammatory effect can be seen as the result of mediated reactions between peptides and proteases involved in these processes, as with the histamine H1-type receptor blocking.

Multigene Family of Pore-Forming Toxins from Sea Anemone Heteractis crispa

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.

A New Iq-Peptide of the Kunitz Type from the Heteractis magnifica Sea Anemone Exhibits Neuroprotective Activity in a Model of Alzheimer’s Disease

The HMIQ3c1 recombinant peptide (6434 Da), the Kunitz-type protease inhibitor, which belonged to the new group of the IQ-peptides from the Heteractis magnifica sea anemone was prepared. The inhibitory constant of trypsin by this peptide proved to be 5.0 × 10–8 M. rHMIQ3c1 was shown to have no interaction with eight isoforms of Kv1 channels. This peptide exhibited neuroprotective activity in a concentration of 10 μM, and the peptide increased the viability of cells of the Neuro2a murine neuroblastoma by 39.4 ± 6.6% in the presence of β-amyloid. However, the peptide did not influence the viability of the cells during their incubation with 6-hydroxydofamine.