RESUMO
A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10-27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins.
Assuntos
Venenos de Cnidários/química , Citotoxinas/química , Anêmonas-do-Mar/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Venenos de Cnidários/genética , Venenos de Cnidários/isolamento & purificação , Citotoxinas/genética , Citotoxinas/isolamento & purificação , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Anêmonas-do-Mar/genética , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Two new serine proteinase inhibitors (RmIn I and RmIn II) from the tropical sea anemone Radianthus macrodactylus have been isolated and characterized. The purification procedure includes polychrome-1 hydrophobic chromatography, Superdex Peptide 10/30 FPLC, and Nucleosil C(18) reverse-phase HPLC. The molecular masses of RmIn I, RmIn II, and the complexes RmIn II/trypsin and RmIn I,II/alpha-chymotrypsin have been determined. The K(i) values of RmIn I and RmIn II for trypsin and alpha-chymotrypsin have been determined. The polypeptides RmIn I and RmIn II are shown to be nontoxic and to exhibit antihistamine activity. The N-terminal amino acid sequences of RmIn I (GICSEPIVVGPCKAG-) and RmIn II (GSTCLEPKVVGPCKA-) have been determined. A high homology of the amino acid sequences is demonstrated for the proteinase inhibitors produced by such evolutionarily distant species as coelenterates, reptiles, and mammals.
