Biologically active polypeptides from the tropical sea anemone Radianthus macrodactylus
Introduction
Sea anemones, like other coelenterates, produce many biologically active polypeptides and proteins, including neurotoxins, pore-forming toxins (or cytolysins), phospholipases, and proteinase inhibitors. Anemone neurotoxins (polypeptides with relative low molecular weight 3000–5000) are very important tools in neurophysiological and pharmacological research as blockers and modulators of K+ and Na+ channels. Their structure-function relationships were studied and discussed (Schweitz et al., 1985, Kem, 1988, Kalman et al., 1998, Kem et al., 1999, Rauer et al., 1999). Nearly 30 pore-forming toxins (called actinoporins, due to their ability to forming pores in the lipid membranes of the host organism) have been isolated and characterized from more than 20 different species of sea anemones. Actinoporins produced by Stichodactyla helianthus (Devlin, 1974, Bernheimer and Avigad, 1976, Kem and Dunn, 1988, Tejica et al., 1996, De los Rios et al., 1998, De los Rios et al., 1999), Actinia equina (Ferlan and Lebez, 1974, Maček and Lebez, 1998, Belmonte et al., 1994, Malavašič et al., 1996, Anderluh et al., 1997, Pungerčar et al., 1997), Actinia tenebrosa (Thomson et al., 1987, Norton et al., 1990), Heteractis magnifica (Khoo et al., 1993, Samejima et al., 2000, Wang et al., 2000) and some other sea anemones (Shiomi et al., 1985, Senčič and Maček, 1990, Samejima et al., 2000) were the most studied. All these cytolysins are basic proteins (pI in the range of 8–12), with Mrs of 16,000–20,000 and a high degree of homology of their amino acid sequence. They can be inhibited by exogenous sphingomyelin, and it is their characteristic feature. Only two cytolytic toxins differ from sphingomyelin-inhibitable cytolysins: a high molecular weight metridiolysin (Mr 80,000) from Metridium senile (Bernheimer et al., 1979, Monastyrnaya et al., 1988) and a low molecular weight tealiatoxin (Mr 7800) from Tealia felina (Aldeen et al., 1981). The membranolytic action of metridiolysin is inhibited by cholesterol (Bernheimer and Avigad, 1978). Tealiatoxin was shown to have both hemolytic and histaminolytic activities (Elliott et al., 1986).
The interest to pore-forming proteins, extensively studied in the last time, is determined by the variety of their pharmacological properties, such as cardiostimulatory, antitumor, antimicrobial, antiparasite and some others (Sket et al., 1974, Norton et al., 1990, Galettis and Norton, 1990, Turk, 1991, Maček et al., 1994, Malavašič et al., 1996, Tejica et al., 1996, Tejica et al., 1999, Poklar et al., 1997, Pungerčar et al., 1997, De los Rios et al., 1998). It has been assumed that biological role of these toxins consist in the capture and killing of prey, digestion, repelling of predators and intraspecific spatial competition (Maček, 1992). They act on natural and model lipid membranes by forming cation-selective channels (or pores) of ∼1 nm in diameter (Varanda and Finkelstein, 1980, Rudnev et al., 1984, Brezhestovsky et al., 1988, Maček et al., 1994, De los Rios et al., 1998, Maček et al., 1995). Besides, cytolysins were applied as important tools in the cell membrane research (Brezhestovsky et al., 1988, Chanturiya, 1990, Shnirov et al., 1992, Zhadan et al., 1994).
Until recently beside actinoporins (hemolysins with ‘direct’ lytic action), hemolysins with phospholipase A2 activity (‘undirect’ lytic action) have been isolated from two sea anemones, Aptasia pallida (Hessinger and Lenhoff, 1976) and S. helianthus (Gomes et al., 1987).
Besides toxins, a good deal of proteinase inhibitors of polypeptide nature (molecular weight 5000–6000) were isolated from several sea anemones such as Anemonia sulcata (Fritz et al., 1972), Stoichactis sp. (Wunderer et al., 1976), Rhodactis rhodostoma, and Radianthus koseirensis (Mebs et al., 1983), A. equina (Shiomi et al., 1989), S. helianthus (Delfin et al., 1994), and Radianthus macrodactylus (Zykova et al., 1985b). Biological role of proteinase inhibitors has not been completely understood.
Among tropical anemones from the Indo-Pacific coral reefs, the species R. macrodactylus (genus Heteractis, the former name Radianthus) are widely distributed and more completely studied (Zykova et al., 1985a, Zykova and Kozlovskaya, 1989a, Zykova and Kozlovskaya, 1989b, Zykova et al., 1985b, Zykova et al., 1988a, Zykova et al., 1988b, Kozlovskaya et al., 1984, Rudnev et al., 1984, Ivanov et al., 1987, Ivanov et al., 1988). The earlier publications were devoted to the isolation and investigation of physicochemical properties of some biologically active polypeptides from the anemones of the genus Heteractis, such as cytolysin and proteinase inhibitor from R. koseirensis (Mebs et al., 1983), neurotoxins from Radianthus paumotensis (Schweitz et al., 1985), and Radianthus crispus (Shiomi et al., 1996), three cytolysins from H. magnifica (Khoo et al., 1993, Samejima et al., 2000), five neurotoxins and a trypsin inhibitor from R. macrodactylus (Zykova et al., 1988b, Zykova et al., 1985b). At present, only the primary structures of one neurotoxin from R. paumotensis (Schweitz et al., 1985), five neurotoxins and one trypsin inhibitor from R. macrodactylus (Zykova et al., 1985b, Zykova et al., 1988b), three cytolysins from H. magnifica (Khoo et al., 1993, Wang et al., 2000), and polypeptide toxin from R. crispus (Shiomi et al., 1996) have been identified.
In this paper, we describe the isolation and partial characterization of three high molecular weight and two low molecular weight cytolysins, as well as one trypsin inhibitor from a water extract of R. macrodactylus.
Section snippets
Materials
Akrilex P-4 was from Reanal (Hungary); polytetrafluoroethylene-1 (polychrom-1) was from Olaine (Latwiya); CM-32 cellulose was from Whatman (England); Sephadex G-15, Sepharose 4B, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) molecular weight markers, isoelectric point (pI) markers of pH range 3.5–11 were purchased from Pharmacia-LKB Biotechnology (Uppsala, Sweden); N-bensoil-d,l-arginin p-nitroanilid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,
Acetone precipitation
Almost all polypeptides with the hemolytic and inhibitory activities extracted from R. macrodactylus with water were shown to precipitated with 80% acetone. For the further purification we applied 50–80% acetone powder. A scheme of the separation of high and low molecular weight cytolysins and protein inhibitors is shown in Table 1.
Isolation of biologically active polypeptides
Results of the desalting of water solution of acetone powder from R. macrodactylus by gel filtration with Akrilex P-4 are shown in Fig. 1. The peaks labelled as I,
Discussion
In the last three decades the biologically active polypeptides from sea anemones have been presented as objects of numerous publications and reviews (Fritz et al., 1972, Kem, 1988, Harvey, 1990, Norton et al., 1990, Bernheimer, 1990, Turk, 1991, Maček, 1992, Maček et al., 1994; Shiomi et al., 1994; Bunc et al., 1999). The unique structure and variety of pharmacological properties of these substances designate great interest to them. In this paper we described the purification and
Acknowledgements
The authors thank Dr N. Prokof'eva (Laboratory of Bioassays, the Pacific Institute of Bioorganic Chemistry, the Russian Academy of Sciences, Vladivostok) for the determination of toxicity and cytotoxicity; Dr I. Nasimov (Institute of Bioorganic Chemistry, the Russian Academy of Sciences, Moscow) for automated N-terminal amino asid sequence analyse of RTX-A; Mrs N. Shepetova for fruitful discussion and translation of this article into English.
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