Actinoporins from the Sea of Japan anemone Oulactis orientalis: Isolation and partial characterization

Abstract

2 Abbreviations: BLM, bilayer lipid membrane.tion is the absence of tryptophan and the presence of six Cys residues.The second group of cytolysins, which is the most thoroughly studied, are the highly basic polypeptides (  I 9 and higher) with the molecular masses of ca. 20 kDa. They are isolated from various species of the Actiniidae and Stichodactylidae families, which mainly inhabit tropical regions of the ocean [1][2][3][4][5]14]. They were shown to exhibit a powerful lytic action on the sphingomyelin-containing membranes of eukaryotes by forming in them cation-selective ion channels (pores), due to which they were named actinoporins [1,15,16]. The absence of cysteine is characteristic of their amino acid composition. The amino acid sequences of eight actinoporins have been elucidated up to date. A steadily increasing interest of researchers to studying the structure and function of recombinant forms of actinoporins prepared by the methods of genetic engineering is now noteworthy [3].The third group of cytolysins comprises the lethal cytolytic phospholipases Ä 2 from Stichodactyla helianthus [17] and Aiptasia pallida [18] with M 30-40 kDa and similar in physicochemical properties cytolysins devoid any enzymatic activity [3].The only representative of the fourth group of cytolysins is metridiolysin, isolated from the sea anemone Abstract -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 C 18 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.

Primary Structures of Actinoporins from Sea Anemone Oulactis orientalis

“…A comparison of the amino acid sequences of the Or-A and Or-G N -terminal fragments with the analogous amino acid sequences of other actinoporins revealed 25-75% homology between them [17]. The direct specific primers to the N -termini of Or-A (Dir1) and Or-G (Dir2) and the reverse primer (Rev) to the Cterminus, which is highly homologous in all the known actinoporins, were used for determination of complete amino acid sequences of the Oulactis actinoporins (table). The full-size cDNA library and the genomic DNA library of the O. orientalis sea anemone were used as a template for PCR with the Dir1 Dir2 and Rev primers.…”

“…As a result, two cytolytic polypeptides (Or-A and Or-G) were isolated. They have the physicochemical characteristics and action mechanism on the erythrocyte membrane and on the bilayer lipid membranes similar to those of the sphingomyelin-inhibited actinoporins from tropical species [17]. The values of hemolytic activity of the individual Or-A and Or-G (295.86 and 322.58 HU/mg, respectively) differ from those of the actinoporins from tropic species approximately by two orders of magnitide.…”

“…The values of hemolytic activity of the individual Or-A and Or-G (295.86 and 322.58 HU/mg, respectively) differ from those of the actinoporins from tropic species approximately by two orders of magnitide. Using the Edman degradation, we partially determined the amino acid sequences of N -terminal fragments of Or-A (ATFRVLAK-) and Or-G (GAIIAGAA-) [17].…”

The multigene families of actinoporins (part I): Isoforms and genetic structure

“…The development of similar isotoxins with small functional differences might be an evolutionary strategy occurring in anemones to successfully explore the variety of preys encountered in the marine environment (Fatehi et al, 1994;Anderluh and Ma cek, 2002). Examples of anemones with different isoforms of the same actinoporin are: Actinia equina (Ma cek and Lebez, 1988;Anderluh et al, 1999), Actinia tenebrosa Galletis and Norton, 1990), S. helianthus , H. crispa (also called Radianthus macrodactylus) (Monastyrnaya et al, 2002;Klyshko et al, 2004), H. magnifica (Wang et al, , 2008, Oulactis orientalis (Il'ina et al, 2005a(Il'ina et al, , 2005bMonastyrnaya et al, 2010), A. fragacea , Actineria villosa, and Phyllodiscus semoni (Uechi et al, 2010). It is likely that these organisms produce a higher number of isoforms that will be discovered when large scale methods, such as cDNA or transcriptome library sequencing, are performed.…”

“…The complete amino acid sequences of RTX-A and RTX-S2 (Fig. 3) from R. macrodactylus were deduced from cDNA nucleotide sequences (Il'ina et al, 2005a(Il'ina et al, , 2005b(Il'ina et al, , 2006Monastyrnaya et al, 2010) and their have similar hemolytic activity on rabbit erythrocytes (~3.5 Â 10 4 HU/mg) (Table 1).…”