Study of the interaction of trypsin inhibitor from the sea anemone Radianthus macrodactylus with proteases

The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: K_D = 7.38 × 10^?8 M and 9.93 × 10^?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively.     

A sea anemone polypeptide toxin inhibiting the ASIC3 acid-sensitive channel

A polypeptide toxin ??-AnmTX Hcr 1b-1 with a molecular mass of 4537 Da was isolated from the whole extract of the sea anemone Heteractis crispa by multistage liquid chromatography. According to a homology search using the BLAST algorithm, the novel toxin was referred to the group of the known sea anemone toxins BDS and APETx with the homology of the amino acid sequence not exceeding 50%. In electrophysiological studies on the receptors expressed in Xenopus laevis oocytes the toxin inhibited the amplitude of the fast component of the integral ASIC3 current. The calculated IC₅₀ value was 5.5 ± 1.0 ??M. Among the known polypeptide blockers of ASIC3 channels the ??-AnmTX Hcr 1b-1 toxin was the least potent inhibitor, which can be explained, in our opinion, by a small amount of charged amino acid residues in its structure.     

Peptide Modulators of ASIC Channels of the Sea Anemone Urticina aff. coriacea (Cuvier, 1798) from the Sea of Okhotsk

Russian Journal of Marine Biology - A search for biologically active peptides has been performed in an aqueous extract of the sea anemone Urticina aff. coriacea (Cuvier, 1798) (Actiniidae) from the...     

Actinoporins from the Sea of Japan anemone <Emphasis Type="Italic">Oulactis orientalis</Emphasis>: Isolation and partial characterization

Two cytolytic toxins (cytolysins Or-A and Or-G) were isolated from the Sea of Japan anemone Oulactis orientalis and characterized. Their purification scheme involved a hydrophobic chromatography on Polychrom-1, a gel filtration on Akrilex P-4, a cation-exchange chromatography on CM-32 cellulose, and a reverse-phase HPLC on a Nucleosil C₁₈ column. The molecular masses of Or-A and Or-G were determined by SDS-PAGE in 14% PAG to be ca. 18 kDa. The absence of Cys residues and a high content of basic amino acid residues are characteristic of their amino acid compositions. The hemolytic activities of Or-A and Or-G were found to be 295.86 and 322.58 HU/mg, respectively; these are by three orders of magnitude lower than those of sphingomyelin-inhibitable cytolysins from the tropic sea anemones. The amino acid sequences of the N-terminal fragments of Or-A and Or-G were determined to be ATFRVLAK and GAIIAGAA, respectively. Action of the cytolysins on the erythrocyte membrane is inhibited by exogenous sphingomyelin. They form ion channels in bilayer lipid membranes with the conductivity of 16, 32, and 40 pSm in 0.1 M NaCl and 168, 240, and 320 pSm in 1 M NaCl at pH 7.2. Therefore, they were attributed to the group of actinoporins.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 39–48.Original Russian Text Copyright © 2005 by Ilina, Monastyrnaya, Sokotun, Egorov, Nazarenko, Likhatskaya, Kozlovskaya.     

Primary Structures of Actinoporins from Sea Anemone <Emphasis Type="Italic">Oulactis orientalis</Emphasis>

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with primers specific to the N-terminal regions of the O. orientalis actinoporin sequences and to the C-terminal region, which is highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were elucidated by sequencing the cDNA clones obtained by the rapid amplification of 3′-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain the lower hemolytic activities of Or-A and Or-G compared to those of actinoporins from the tropical species.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 357–362.Original Russian Text Copyright © 2005 by Il’ina, Monastyrnaya, Isaeva, Guzev, Rasskazov, Kozlovskaya     

Biologically active polypeptides of sea anemones: Structure, function, and prospects for application

This paper presents the results of a structure-functional study of neurotoxins, actinoporins, and proteinase inhibitors from the tropical sea anemone Heteractis crispa (= Radianthus macrodactylus) and the low boreal sea anemone Oulactis orientalis. The new data significantly contribute to the knowledge of the mechanisms of action of polypeptides from marine coelenterates and can be useful for solving practical problems of the biotechnological development of medicines.     

Interaction of sea anemone <Emphasis Type="Italic">Heteractis crispa</Emphasis> Kunitz type polypeptides with pain vanilloid receptor TRPV1: In silico investigation

Using methods of molecular biology we defined the structures of the 31 sea anemone Heteractis crispa genes encoding polypeptides which are structurally homologous to the Kunitz protease inhibitor family. The identified sequences have single-point amino acid substitutions, a high degree of homology with sequences of known Kunitz family members from H. crispa, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by methods of homology modeling. Analysis of their molecular electrostatic potential allowed the division of the polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2, and APHC3 which have been shown to possess, in addition to their trypsin inhibitory activity, a unique property of inhibiting the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo. The spatial structure of the polypeptide complexes with TRPV1, the nature of the interactions, as well as functionally important structural elements involved in the complex formation, were established by molecular docking technique. The designed models allowed us to propose a hypothesis contributing to the understanding of how APHC1-APHC3 affect the pain signals transduction by TRPV1: apparently, relaxation time of the receptor increases due to binding of its two chains with a polypeptide molecule which disrupts functioning of TRPV1 and leads to partial inhibition of the signal transduction in electrophysiological experiments.     

Analgesic compound from sea anemone Heteractis crispa is the first polypeptide inhibitor of vanilloid receptor 1 (TRPV1)

Venomous animals from distinct phyla such as spiders, scorpions, snakes, cone snails, or sea anemones produce small toxic proteins interacting with a variety of cell targets. Their bites often cause pain. One of the ways of pain generation is the activation of TRPV1 channels. Screening of 30 different venoms from spiders and sea anemones for modulation of TRPV1 activity revealed inhibitors in tropical sea anemone Heteractis crispa venom. Several separation steps resulted in isolation of an inhibiting compound. This is a 56-residue-long polypeptide named APHC1 that has a Bos taurus trypsin inhibitor (BPTI)/Kunitz-type fold, mostly represented by serine protease inhibitors and ion channel blockers. APHC1 acted as a partial antagonist of capsaicin-induced currents (32 +/- 9% inhibition) with half-maximal effective concentration (EC(50)) 54 +/- 4 nm. In vivo, a 0.1 mg/kg dose of APHC1 significantly prolonged tail-flick latency and reduced capsaicin-induced acute pain. Therefore, our results can make an important contribution to the research into molecular mechanisms of TRPV1 modulation and help to solve the problem of overactivity of this receptor during a number of pathological processes in the organism.     

Biologically Active Polypeptides and Hydrolytic Enzymes in Sea Anemones of Northern Temperate Waters

The content of biologically active polypeptides in aqueous and ethanol extracts of seven sea anemone species collected near Sakhalin Island (Sea of Okhotsk) and in Posyet Bay (Sea of Japan) was analyzed. Water extracts of the sea anemone Cribrinopsis similis showed the highest hemolytic activity, while ethanol extracts proved to have toxic properties. The levels of toxic and hemolytic activity of extracts of sea anemones inhabiting northern temperate waters were 2 to 3 orders of magnitude lower, compared to tropic species. The reason for this is likely to be the differences in the habitat conditions and biological traits of these animals. The water extracts of all species possessed proteolytic, phospholipase A2, and low DNAase activities, except Actinostola sp., whose aqueous extract contained a high activity alkaline DNAase. The species studied contained a wide range of proteinase inhibitors, O-glycosyl hydrolases (glycosidases and polysaccharide hydrolases). Water extracts of C. similis and Stomphia coccinea possessed the highest laminarinase activity. High activity of N-galactopyranosidase was found in water extracts of S. coccinea and Oulactis orientalis.