Differences Between Hemolytic Activity of the N-Terminal Region of the Sticholysins I e II
Pigossi, F.T.1; Crusca , E. J.1; Barbosa, S. C.1; Ros, U.3; Martinez, D.3; Lanio, M.E.3; Schreier, S.2; Alvarez, C.3; Cilli, E.M1.
UNESP – São Paulo State University, Instituto de Química, Araraquara1; Instituto de Química, USP, São Paulo2; Facultad de Biología, Universidad de La Habana; Cuba3.
Actinoporins consist of a family of cysteineless pore-forming proteins. Sticholysin I (StI) and Sticholysin II (StII) are highly hemolytic polypeptides isolated from the Caribbean Sea anemone Stichodactyla helianthus. Their high sequence homology (93%) indicates that they correspond to two isoforms of the same hemolysin. Spectroscopic measurements show a close similarity in the secondary structure content, conformation, and stability of both toxins. The largest difference between both toxins is the higher lytic activity of StII. The different pore forming capacity is probably related to the charge difference in the N-terminal fragment, that could considerably reduce StI insertion rate in the membrane, as well as the presence of an extra serine residue located at position 1 of the latter protein. In addition, our recent studies showed that peptides containing the first 30 residues of the N-terminal region of StII are hemolytic, but not as efficient as the native protein. In order to better understand the electrostatic effect on Sticholysin/membrane interaction and pore-forming mechanism, we have studied the role of different residues on the lower hemolytic activity of StI. For this purpose, N-terminal peptides of StI (+NH3-SELAGTIIDGASLTFEVLDKVLGELGKVSRK-COO-) and StII (+NH3-ALAGTIIAGASLTFQVLDKVLEELGKVSRK-COO-) were produced by SPFS using the Fmoc/tBut chemical approach. The peptides used in this study contained residues 12-31(StI [12-31]), 11-30 (StII [11-30]), 1-31 (StI [1-31]), and 1-30 (StII [1-30]). The hemolytic activity (HA) of the peptides was examined. These studies demonstrated that peptides StI [12-31] and StII [11-30] showed the same activity, but StI [1-31] was less hemolytic than StII [1-30]. Additionally, the absence of Ser in the peptide corresponding to StI yielded a compound with HA lower than that of StII [1-30], but higher than that of StI [1-31]. These results suggest that the presence of the polar residue Ser1 in StI, as well as the absence of the non polar residues Ala1 and Ala8 (present in StII) probably contribute to the lesser HA of StI when compared to StII. Supported by CNPQ, FAPESP and CAPES
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