Isolation, properties and partial amino acid sequence of a new actinoporin from the sea anemone Radianthus macrodactylus

• Published in: Toxicon (Oxford) Vol. 44; no. 3; pp. 315 - 324
• Main Authors: Klyshko, Elena V, Issaeva, Marina P, Monastyrnaya, Margarita M, Il'yna, Anna P, Guzev, Konstantin V, Vakorina, Tatyana I, Dmitrenok, Pavel S, Zykova, Tatyana A, Kozlovskaya, Emma P
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.09.2004; Elsevier Science
• Subjects: Actinoporins; Amino Acid Sequence; Animals; Base Pairing; Biological Assay; Chromatography, High Pressure Liquid; Cytolysins; Cytotoxins - chemistry; Cytotoxins - genetics; Cytotoxins - toxicity; DNA Primers; Electrophoresis, Polyacrylamide Gel; Hemolysis - drug effects; Hemolytic activity; Heteractis magnifica; Lethal Dose 50; Marine; Mass Spectrometry; Mice; Molecular Sequence Data; Primary and secondary structures; Radianthus macrodactylus; Sea Anemones; Sequence Alignment; Sequence Analysis, Protein; Spectrophotometry, Ultraviolet; Sphingomyelin; Sphingomyelin; Actinoporins; Sea anemones; Hemolytic activity; Primary and secondary structures; Cytolysins
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2004.06.006

A new cytolytic toxin, actinoporin RTX-S II, was isolated from the sea anemone Radianthus macrodactylus with a high degree of purity by a combination of gel filtration, ion-exchange and reverse-phase chromatography. RTX-S II has molecular mass of 19,280 Da and isoelectric point of 10.0. The hemolytic activity of RTX-S II is inhibited by sphingomyelin. RTX-S II had an LD 50 of 70 mg/kg, and is lacking in phospholipase activity. The amino acid composition of this protein contains a high amount of basic and non-polar amino acids and no cysteine. The N-terminal sequence of RTX-S II was determined. The partial amino acid sequence (141 aa) of RTX-S II was deduced based on the cDNA sequence obtained with two oligonucleotides encoding the N-terminal portion of RTX-S II and the internal conserved cytolysin peptide by PCR. A comparison of the RTX-S II cDNA sequence and the rtx-s II gene obtained with the same PCR primers indicates that they are 100% identical at the nucleotide level. It shows that no introns are present in the corresponding region of the rtx-s II gene. Multiple alignments of RTX-S II with known sequences of actinoporins show that RTX-S II is highly homologous to magnificalysin II from Heteractis magnifica. The predicted secondary structure of RTX-S II is predominantly anti-parallel β-structure, which is in good agreement with experimental data obtained from other sea anemones–actinoporins.