Laboratory of Peptide Chemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences: forty years of research on peptides and proteins of sea anemones

Cover Page

Cite item

Full Text

Abstract

The review briefly describes a research carried out over the past 40 years at the Laboratory of Peptide Chemistry of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS (LPCh of PIBOC FEB RAS), in collaboration with Russian and foreign colleagues. The results of search, identification, and study of the structure, the biological activity, and the mechanisms of an interaction with the biological targets of peptides and polypeptides produced by the tropical sea anemone Heteractis crispa (=Heteractis magnifica, formerly Radianthus macrodactylus) are discussed. One of the main achievements of LPCh over the past years is the discovery of new structural type 2 neurotoxins, namely, six toxins that were not identified among the representatives of so-called long anemonotoxins in the first decade of foreign research (70–80s of the last century), and among them the first, previously unknown, double-chain neurotoxin. In addition, the presence of several multigene families expressing α-pore-forming toxins (actinoporins), serine protease inhibitors (Kunitz-type peptides), and APETx-like peptides forming the combinatorial libraries of the several dozen of highly homologous family members has been established. Using in silico methods (homologous modeling, alanine mutagenesis, full-atom molecular dynamics (MD) simulation), the spatial structures of the studied peptides and complexes with biological targets were predicted for the first time, and their structure-functional relationships were analyzed. This was the foundation for the further production of recombinant and mutant analogues on the basis of the combinatorial libraries for the purpose of conducting the electrophysiological studies of the mechanisms of their molecular interaction with targets as well as determining the pharmacological potential. In the review the most important results of recent years are presented. They are related to the discovery of analgesic, anti-inflammatory, and antitumor activity in a number of the studied peptides.

Full Text

Restricted Access

About the authors

Margarita M. Monastyrnaya

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS

Author for correspondence.
Email: rita1950@mail.ru
ORCID iD: 0000-0003-3157-0930

Doctor of Chemical Sciences, Leading Researcher

Russian Federation, Vladivostok

Emma P. Kozlovskaya

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS

Email: kozempa@mail.ru
ORCID iD: 0000-0002-8110-0382

Doctor of Chemical Sciences, Chief Researcher, Professor

Russian Federation, Vladivostok

References

  1. Prentis P. J., Pavasovic A., Norton R. S. Sea anemones: quiet achievers in the field of peptide toxins. Toxins. 2018;10:36.
  2. Béress L. Biologically active compounds from coelenterates. Pure Appl. Chem. 1982;54:1981–1994.
  3. Norton R. S. Structure and structure-function relationships of sea anemone proteins that interact with the sodium channel. Toxicon. 1991;29:1051–1084.
  4. Zykova T. A., Vinokurov L. M., Kozlovskaya E. P., Elyakov G. B. Amino acid sequence of neurotoxin III from the sea anemone Radianthus macrodactylus. Bioorg. Khim. 1985;11:302–310.
  5. Zykova T. A., Kozlovskaya E. P. Amino acid sequence of a neurotoxin from the sea anemone Radianthus macrodactylus. Bioorg. Khim. 1989;15:1301–1306.
  6. Zykova T. A., Kozlovskaya E. P., Elyakov G. B. Amino acid sequence of neurotoxin II from the sea anemone Radianthus macrodactylus. Bioorg. Khim. 1988;14:878–882.
  7. Zykova T. A., Kozlovskaya E. P., Elyakov G. B. Amino acid sequence of neurotoxins IV and V from the sea anemone Radianthus macrodactylus. Bioorg. Khim. 1988;14:1489–1494.
  8. Monastyrnaya M. M., Kalina R. S. Kozlovskaya E. P. The Sea anemone neurotoxins modulating sodium channels: an insight at structure and functional activity after four decades of investigation. Toxins. 2023;15:8.
  9. Schweitz H., Bidard J. N., Frelin C., Pauron D., Vijverberg H. P.M., Mahasneh D. M., Lazdunski M., Vibois F., Tsugita A. Purification, sequence, and pharmacological properties of sea anemone toxins from Radianthus paumotensis. A new class of sea anemone toxins acting on the sodium channel. Biochemistry. 1985;24:3554–3561.
  10. Honma T., Kawahata S., Ishida M., Nagai H., Nagashima Y., Shiomi K. Novel peptide toxins from the sea anemone Stichodactyla haddoni. Peptides. 2008;29:536–544.
  11. Kem W. R., Parten B., Pennington M. W., Price D. A., Dunn B. M. Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus. Biochemistry. 1989;28:3483–3489.
  12. Wunderer G., Fritz H., Wachter E., Machleidt W. Amino acid sequence of a coelenterate toxin: Toxin II from Anemonia sulcata. Eur. J. Biochem. 1976;8:193–198.
  13. Tanaka M., Hainu M., Yasunobu K. T., Norton T. R. Amino acid sequence of the Anthopleura xanthogrammica heart stimulant anthopleurin-A. Biochemistry. 1977;16:204–208.
  14. Reimer N. S., Yasunobu C. L., Yasunobu K. T., Norton T. R. Amino acid sequence of the Anthopleura xanthogrammica heart stimulant, anthopleurin B. J. Biol. Chem. 1985;260:8690–8693.
  15. Kalina R. S., Peigneur S., Zelepuga E. A., Dmitrenok P. S., Kvetkina A. N., Kim N. Y., Leychenko E. V., Tytgat J., Kozlovskaya E. P., Monastyrnaya M. M., Gladkikh I. N. New insights into the type II toxins from the sea anemone Heteractis crispa. Toxins. 2020;12:44.
  16. Baydan L. V., Kozlovskaya E. P., Tishkin S. M., Shuba M. F., Elyakov G. B. Effect of anemonotoxin on neuromuscular transmission in skeletal and smooth muscles. Dokl. Acad. Sci. USSR. 1981;259:1000–1002.
  17. Sorokina Z. A., Chizhmakov I. V., Elyakov G. B., Kozlovskaya E. P., Vozhzhova E. I. Investigation of inactivation mechanism of fast sodium channels using neurotoxin from the sea anemone Radianthus macrodactylus and various chemical reagents. Physiol. J. 1984;30:571–579.
  18. Linder R., Bernheimer A. W., Kim K. S. Interaction between sphingomyelin and a cytolysin from the sea anemone Stoichactis helianthus. Biochim. Biophys. Acta. 1977;467(3):290–300.
  19. Varanda W., Finkelstein A. Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone, Stoichactis helianthus. J. Membr. Biol. 1980;55(3):203–211.
  20. Chanturia A. N., Shatursky O. Ya., Lishko V. K., Monastyrnaya M. M., Kozlovskaya E. P. Vsaimodejstvie toxina morskoj aktinii Radianthus macrodactylus s bislojnimi phospholipidnimi membranami = [Interaction of the sea anemone toxin Radianthus macrodactylus with bilayer phospholipid membranes]. Biol. membrani. 1990;7(7):763–769. (In Russ.).
  21. Monastyrnaya M. M., Zykova T. A., Apalikova O. V., Shwets T. V., Kozlovskaya E. P. Biologically active polypeptides from the tropical sea anemone Radianthus macrodactylus. Toxicon. 2002;40:1197–1217.
  22. Ilyina A. P., Monastyrnaya M. M., Sokotun I. N., Egorov T. A., Nazarenko Yu.A., Likhatskaya G. N. Aktinoporini is aktinii Japonskogo morja Oulactis orientalis: videlenie i chastichnaja charakteristika = [Actinoporins from the Sea of Japan sea anemone Oulactis orientalis: isolation and partial characterization]. Bioorg. chimija. 2005;31:39–48. (In Russ.).
  23. Klyshko E. V., Issaeva M. P., Monastyrnaya M. M., Il’ina A.P., Guzev K. V., Vakorina T. I., Dmitrenok P. S., Zykova T. A., Kozlovskaya E. P. Isolation, properties and partial amino acid sequence of a new actinoporin from the sea anemone Radianthus macrodactylus. Toxicon. 2004;44:315–324.
  24. Monastyrnaya M., Leychenko E., Issaeva M., Likhatskaya G., Zelepuga E., Kostina E., Trifonov E., Nurminski E., Kozlovskaya E. Actinoporins from the sea anemones, tropical Radianthus macrodactylus and northern Oulactis orientalis: Comparative analysis of structure-function relationships. Toxicon. 2010;56:1299–1314.
  25. Il’ina A.P., Monastyrnaya M. M., Issaeva M. P., Guzev K. V., Rasskasov V. A., Kozlovskaya E. P. Primary structure of actinoporins from the sea anemone Oulactis orientalis. Bioorg. Chem. 2005;31:357–362.
  26. Il’ina A., Lipkin A., Barsova E., Issaeva M., Leychenko E., Guzev K., Monastyrnaya M., Lukyanov S., Kozlovskaya E. Amino acid sequence of RTX-A’s isoform actinoporin from the sea anemone Radianthus macrodactylus. Toxicon. 2006;47:517–520.
  27. Leychenko E., Isaeva M., Tkacheva E., Zelepuga E., Kvetkina A., Gusev K., Monastyrnaya M., Kozlovskaya E. Multigene family of pore-forming toxins from sea anemone Heteractis crispa. Mar. Drugs. 2018;16. 183 [1−18].
  28. Ivanov А. S., Molnar А. А., Monastyrnaya М. М., Kozlovskaya E. P. Dejstvie toksina is Radianthus macrodactylus na pronizaemost biologicheskich i modelnich membran = [Effect of toxin from Radianthus macrodactylus on the permeability of biological and model membranes]. Biol. Membrani. 1987;4(3):243–248. (In Russ.).
  29. Kozlovskaya E. P., Ivanov А. S., Molnar А. А., Grigoriev P. A., Monastyrnaya М. М., Chalilov E. М., Elyakov G. B. Ionnie kanali v membanach, induzirovannie gemolisinom is aktinii Radianthus macrodactylus = [Ion channels in membranes induced by hemolysin from the sea anemone Radianthus macrodactylus]. Dokl. AN SSSR. 1984;277(6):1491–1493. (In Russ.).
  30. Bakrač B., Gutiérrez-Aguirre I., Podlesek Z., Sonnen A. F.-P., Gilbert R. J.C., Maček P., Lakey J. H., Anderluh G. Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin. J. Biol. Chem. 2008;283:18665–18677.
  31. Mechaly A. E., Bellomio A., Gil-Carton D., Morante K., Valle M., González-Mañas J.M., Guérin D. M. Structural insights into the oligomerization and architecture of eukaryotic membrane pore-forming toxins. Structure. 2011;19:181–191.
  32. Bellomio A., Morante K., Barlič A., Gutiérrez-Aguirre I., Viguera A. R., González-Mañas J. M. Purification, cloning and characterization of fragaceatoxin C, a novel actinoporin from the sea anemone Actinia fragacea. Toxicon. 2009;54:869–880.
  33. Mancheño J. M., Martin-Benito J., Martínez M., Gavilanes J. G., Hermoso J. A. Crystal and electron microscopy structures of Sticholysin II actinoporin reveal insights into the mechanism of membrane pore formation. Structure. 2003;11:1–20.
  34. Rudnev V. С., Likhatskaya G. N., Kozlovskaya E. P., Monastyrnaya М. М., Elyakov G. B. Vlijanie gemolisina is morskoj aktinii Radianthus macrodactylus na pronizaemost lipidnich membrane = [Effect of hemolysin from the sea anemone Radianthus macrodactylus on the permeability of lipid membranes]. Biol. Membrani. 1984;1(10):1019–1023. (In Russ.).
  35. Fedorov S., Dyshlovoy S., Monastyrnaya M., Shubina L., Leychenko E., Kozlovskaya E., Jin J. O., Kwak J. Y., Bode A. M., Dong Z., Stonik V. The anticancer effects of actinoporin RTX-A from the sea anemone Heteractis crispa (=Radianthus macrodactylus). Toxicon. 2010;55:811–817.
  36. Kvetkina A., Malyarenko O., Pavlenko A., Dyshlovoy S., von Amsberg G., Ermakova S., Leychenko E. Sea anemone Heteractis crispa actinoporin demonstrates in vitro anticancer activities and prevents HT-29 colorectal cancer cell migration. Molecules. 2020;25:5979.
  37. Brezhestovsky P. D., Monastyrnaya M. M., Kozlovskaya E. P., Elyakov G. B. Dejstvie gemolisina is morskoj aktinii Radianthus macrodactylus na membranu eritrozitov = [Effect of hemolysin from the sea anemone Radianthus macrodactylus on the erythrocyte membrane]. Dokl. AN USSR. 1988;299(3):748–750. (In Russ.)
  38. Leichenko E. V., Monastirnaya M. M., Zelepuga E. A., Tkacheva E. S., Isaeva M. P., Likhatskaya G. N., Anastyuk S. D., Kozlovskaya E. P. Hct-A is a new actinoporin family from the Heteractis crispa sea anemone. Acta Naturae. 2014;6:89–98.
  39. Monastyrnaya M. M., Agafonova I. G., Tabakmakher V. M., Kozlovskaya E. P. The sea anemone pore-forming toxins (PFTs): from mechanism of action to perspectives in pharmacology as antitumor agents. EC Pharmacol. and Toxicol. 2023;11(4):20–26.
  40. Shnyrov V. L., Monastyrnaya M. M., Zhadan G. G., Kuznetsova S. M., Kozlovskaya E. P. Calorimetric study of interaction of toxin from Radianthus macrodactylus with erythrocyte membrane. Biochem. Intеrn. 1992;26(2):219–229.
  41. Zykova T. A., Vinokurov L. M., Markova L. F., Kozlovskaya E. P., Elyakov G. B. Aminokislotnaya posledovatelnost ingibitora tripsins IV is Radianthus macrodactylus = [Amino acid sequence of trypsin inhibitor IV from Radianthus macrodactylus]. Bioorg. Khimia. 1985;11:293–301. (In Russ.).
  42. Isaeva M. P., Chausova V. E., Zelepuga E. A., Guzev K. V., Tabakmakher V. M., Monastyrnaya M. M., Kozlovskaya E. P. A new multigene superfamily of Kunitz-type protease inhibitors from sea anemone Heteractis crispa. Peptides. 2012;34:88–97.
  43. Kvetkina A., Leychenko E., Chausova V., Zelepuga E., Chernysheva N., Guzev K., Pislyagin E., Yurchenko E., Menchinskaya E., Aminin D., Kaluzhskiy L., Ivanov A., Peigneur S., Tytgat J., Kozlovskaya E. P., Isaeva M. P. A new multigene HCIQ subfamily from the sea anemone Heteractis crispa encodes Kunitz-peptides exhibiting neuroprotective activity against 6-hydroxydopamine. Scientific Reports. 2020;10:4205.
  44. Sokotun I. N., Gnedenko O. V., Leichenko E. V., Monastyrnaya M. M., Kozlovskaya E. P., Molnar A. A., Ivanov A. S. Issledovanie vsaimodejstvija ingibitora tripsina is aktinii Radianthus macrodactylus s raslichnimi proteinasami = [Study of the interaction of trypsin inhibitor from the sea anemone Radianthus macrodactylus with various proteinases]. Biomed. Khimia. 2006;52(6):595–600. (In Russ.).
  45. Gladkikh I., Monastyrnaya M., Leychenko E., Zelepuga E., Chausova V., Isaeva M., Anastyuk S., Andreev Y., Peigneur S., Tytgat J., Kozlovskaya E. Atypical reactive center Kunitz-type inhibitor from the sea anemone Heteractis crispa. Mar. Drugs. 2012;10:1545–1565.
  46. Gladkikh I., Monastyrnaya M., Zelepuga E., Sintsova O., Tabakmakher V., Gnedenko O., Ivanov A., Hua K.-F., Kozlovskaya E. New Kunitz-type HCRG polypeptides from the sea anemone Heteractis crispa. Mar. Drugs. 2015;13:6038–6063.
  47. Gladkikh I., Peigneur S., Sintsova O., Pinheiro-Junior E.L., Klimovich A., Menshov A., Kalinovsky A., Isaeva M., Monastyrnaya M., Kozlovskaya E., Tytgat J., Leychenko E. Kunitz-type peptides from the sea anemone Heteractis crispa demonstrate potassium channel blocking and anti-inflammatory activities. Biomedicines. 2020;8:473.
  48. Andreev Y. A., Kozlov S. A., Koshelev S. G., Ivanova E. A., Monastyrnaya M. M., Kozlovskaya E. P., Grishin E. V. Analgesic compound from sea anemone Heteractis crispa is the first polypeptide inhibitor of vanilloid receptor 1 (TRPV1). J. Biol. Chem. 2008;283:23914–23921.
  49. Monastyrnaya M., Peigneur S., Zelepuga E., Sintsova O., Gladkikh I., Leychenko E., Isaeva M., Tytgat J., Kozlovskaya E. Kunitz-type peptide HCRG21 from the sea anemone Heteractis crispa is a full peptide antagonist of the TRPV1 receptor. Mar. Drugs. 2016;14. 229 [1–20].
  50. Sintsova О. V., Monastyrnaya М. М., Pislyagin E. A., Menchinskaya E. S., Leychenko Е. V., Aminin D. L., Kozlovskaya E. P. Anti-inflammatory activity of the polypeptide of the sea Anemone, Heteractis crispa. Bioorg. Chem. 2015;941:590–596.
  51. Sintsova O. V., Palikov V. A., Palikova Y. A., Klimovich A. A., Gladkikh I. N., Andreev Y. A., Monastyrnaya M. M., Kozlovskaya E. P., Dyachenko I. A., Kozlov S. A., Leychenko E. V. Peptide blocker of ion channel TRPV1 exhibits a long analgesic effect in the heat stimulation model. Dokl. Biochem. Biophys. 2020;493:215–217.
  52. Sintsova О. V., Pislyagin E. A., Gladkikh I. N., Monastyrnaya М. М., Menchinskaya E. S., Leychenko Е. V., Aminin D. L., Kozlovskaya E. P. Kunitz-type peptides of the sea anemone Heteractis crispa – potential anti-inflammatory compounds. Bioorg. Chem. 2017;43:105–112.
  53. Sintsova O., Gladkikh I., Chausova V., Monastyrnaya M., Anastyuk S., Chernikov O., Yurchenko E., Aminin D., Isaeva M., Leychenko E., Kozlovskaya E. Peptide fingerprinting of the sea anemone Heteractis magnifica mucus revealed neurotoxins, Kunitz-type proteinase inhibitors and a new β-defensin α-amylase inhibitor. Journal of Proteomics. 2018;173:12–21.
  54. Sintsova O., Gladkikh I., Kalinovskii A., Zelepuga E., Monastyrnaya M., Kim N., Shevchenko L., Peigneur S., Tytgat J., Kozlovskaya E., Leychenko E. Magnificamide, a a-defensin-like peptide from the mucus of the sea anemone Heteractis magnifica, is a strong inhibitor of mammalian a-amylases. Mar. Drugs. 2019;17:542.
  55. Tysoe C., Williams L. K., Keyzers R., Nguyen N. T., Tarling C., Wicki J., Goddard-Borger E.D., Aguda A. H., Perry S., Foster L. J., Andersen R. J., Brayer G. D., Wither S. G. Potent human α-amylase inhibition by the β-defensin-like protein Helianthamide. ACS Cent. Sci. 2016;2(3):154–161.
  56. Kalina R., Gladkikh I., Dmitrenok P., Chernikov O., Koshelev S., Kvetkina A., Kozlov S., Kozlovskaya E., Monastyrnaya M. New APETx-like peptides from sea anemone Heteractis crispa modulate ASIC1a channels. Peptides. 2018;104:41–49.
  57. Diochot S., Baron A., Rash L.D., Deval E., Escoubas P., Scarzello S., Salinas M., Lazdunski M. A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensoryneurons. EMBO J. 2004;23:1516–1525.
  58. Osmakov D.I., Andreev Y.A., Kozlov S.A. Acid-sensing ion channels and their modulators. Biochemistry. 2014;79:1528–1545.
  59. Kalina R.S., Koshelev S.G., Zelepuga E.A., Kim N.Y., Kozlov S.A., Kozlovskaya E.P., Monastyrnaya M.M., Gladkikh I.N. APETx-like peptides from the sea anemone Heteractis crispa, diverse in their effect on ASIC1a and ASIC3 ion channels. Toxins. 2020;12(4). 266 [1–20].

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. 1. Multiple alignment of amino acid sequences of neurotoxins: structural type 2 – RTX-I (P30831) [5], RTX-II (P30783) [6], RTX-III (P30832) [4], RTX-IV (P30784) and RTX-V (P30785) [7] from H. crispa, Rp-II (P01534) from R. paumotensis [9], SHTX-IV (B1B519) [10] from Stichodactyla haddoni, Sh1 (H19651) [8, 11] from Stichodactyla helianthus (a); structural type 1 – ATX-II (P01528) [12] from Anemonia sulcata, ApA (P01530) [13], ApB (P01531) [14] from Anthopleura xanthogrammica (b). Identical amino acid residues are shown on a gray background, point substitutions of residues in sequences are shown on a white background, and Arg13 residue, functionally significant for binding to NaV, is shown in red [8]. The straight lines in the upper part of the figure show the C1-C5, C2-C4, and C3-C6 compounds in disulfide bridges between cysteine residues (shown in bold). The alignment was performed using the Vector NTI program

Download (343KB)
Indexing metadata
3. 2. Multiple alignment of highly homologous amino acid sequences of Heteractis neurotoxins RTX-VI, RTX-III and δ-SHTX-Hcr1f (=Rp-II).Identical residues in sequences are shown on a gray background, dotted residue substitutions are shown on a white background, and Arg13 residue, functionally significant for binding to NaV, is shown in red [8]. The straight lines in the upper part of the figure show the connection of disulfide bonds C1-C5 (a.o. 3-43), C2-C4 (a.o. 5-33), C3-C6 (a.o. 26-44) between cysteine residues (shown in bold). The alignment was performed using the Vector NTI program

Download (164KB)
Indexing metadata
4. Fig. 3. Ribbon diagrams of models of 3D structures of type 1 and type 2 neurotoxins: ApA (type 1), Sh-1 and RTX-III (type 2) having a β-defensin-like fold (created on the basis of homologous modeling of the 3D structure of the neurotoxin Sh-1).The arrows show the dipole moments; their magnitudes are indicated in Bars (D): 183, 133, 109 [8, 15]

Download (61KB)
Indexing metadata
5. 4. Effect of δ-SHTX-Hcr1f (=Rp-II), RTX-III, and RTX-VI on mammalian and arachnid NaVs currents.Recordings of currents through the cell membrane of the X. laevis oocyte caused by changes in the membrane potential are shown under control conditions and in the presence of 10 microns of toxin [15] (scales of changes in the amplitudes of currents through the channels correspond to 0.5–1 µA per 5 ms)

Download (161KB)
Indexing metadata
6. Fig. 5. Interaction of δ-SHTX-Hcr1f (=Rp-II) with rNaV1.2: ribbon diagram of the complex δ-SHTX-Hcr1f–rNaV1.2 in the DPFC lipid bilayer; the molecular surfaces of δ-SHTX-Hcr1f are colored yellow, rNaV1.2 – in maroon-brown, DPFC – in blue (a); intermolecular interactions in the complex δ-SHTX-Hcr1f–rNaV1.2 is a yellow-greenish color, showing hydrogen bonds involved in the binding of neurotoxin side chains and the channel (green dotted line), ionic and π-cationic interactions (blue and orange contours, respectively) (b) [15]

Download (206KB)
Indexing metadata
7. 6. Multiple alignment of amino acid sequences of Radianthus and Oulactis actinoporins. The N-terminal fragment and the POC binding site to the membrane are highlighted, and the RGD/GD tripeptide is emphasized.

Download (326KB)
Indexing metadata
8. 7. Three-stage mechanism of pore formation (stages 1-3). Ribbon diagram of the spatial structure of actinoporin with functionally significant fragments (T-terminus, POV site) (a), binding to membrane sphingomyelin (CM) and anchoring in the membrane of the T-terminus (b), schematic representation of the toroidal pore (c) x33]

Download (271KB)
Indexing metadata
9. 8. Diagram of a fragment of the erythrocyte membrane, membrane proteins and cytoskeleton proteins (a), calorimetric curves (b), densitograms of SDS electrophoresis of human erythrocyte shadows (intact and modified with actinoporin RTX-A) (c) [40]

Download (115KB)
Indexing metadata
10. 9. Fertilization of the egg of the sea urchin S. intermedius by sperm (a), inhibition of egg fertilization by actinoporin RTX-Ala (b)

Download (92KB)
Indexing metadata
11. 10. Multiple alignment of amino acid sequences of the Kunitz peptides of the sea anemone H. crispa: APHC1, APHC2, APHC3, InhVJ, HCRG1, HCRG2, HCRG21, HCGS 1.10, HCGS 1.19, HCGS 1.20, HCGS 1.36, HCTX1, toxins of the anemone Stichodactyla mertensii SMTX1 and SMTX A49E, Stichodactyl of helianthus SHTXIII, as well as a trypsin inhibitor BPTI from B. taurus; identical a.o. are shown on a dark gray background, conservative ones on a light gray (a). A phylogenetic tree of Kunitz-type peptides from sea anemones of the families Actiniidae and Stichodactylidae, constructed by the method of minimal evolution; representatives of the H. crispa combinatorial library form four clusters on the tree: I–III are represented by HCGS/HCRG peptides, IV - HCTX peptides; HCGS peptides are indicated by red circles, HCRG peptides are green, HCTX/SMTX peptides are blue; sequences of peptides with P1Lys are shown on a blue background, with P1Thr – on a pink background, with P1Arg – on a green background [42] (b)

Download (553KB)
Indexing metadata
12. 11. Electrophysiological analysis of the inhibitory effect of the rHCRG21 peptide on TRPV1. Evaluation of TRPV1 activity under control conditions and in the presence of rHCRG21 (10 µm) (a); evaluation of TRPV1 conductivity upon coapplication of capsaicin (CAP) (2 µm) with rHCRG21 (10 µm) (b) (capsazepine (CZP) (10 µm) was used as a standard antagonist (n ≥ 3)) [49]

Download (57KB)
Indexing metadata
13. Fig. 12. Amino acid sequences of defensins of the sea anemones N. magnifica [54] and S. helianthus [55]. Identical amino acids are shown on a light gray background, dotted residue substitutions are shown on a white background, and cysteine residues are highlighted in bold. The reactive site of interaction with α-amylases is marked with asterisks.

Download (51KB)
Indexing metadata
14. 13. Multiple alignment of amino acid sequences of APETx-like peptides from H. crispa: Hcr 1b-1 (P0DL87), Hcr 1b-2 (C0HL52), Hcr 1b-3 (C0HL53), Hcr 1b-4 (C0HL54) [56], APETx1 (P61541) and APETx2 (P61542) from A. elegantissima [57]. Identical residues are depicted on a light gray background, and dotted replacements of residues are shown on a white background. The alignment was performed using the Vector NTI program [56]

Download (140KB)
Indexing metadata
15. Fig. 14. 3D structure model of the Hcr 1b-1–ASIC complex (a). Ribbon diagrams of 3D structure models of APETx2, Hcr 1b-2, Hcr 1b-3 and Hcr 1b-4 molecules (b) [56, 59]. The dipole moments of the molecules are shown by arrows, the equipotential surfaces (molecular electrostatic potentials – MEP) are colored pink (negative potential) and blue (positive), made using the programs SPDBV and Chimera

Download (187KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences