Abstract
Src I is the first reported acidic actinoporin from sea anemone
Sagartia rosea with a p
I value of 4.8 and comprises 13.9% α-helix, 65.1% β-sheet, and 18.2% random coil. For structure–function studies, Src I was expressed in
Escherichia coli as a cleavable fusion protein. Recombinant Src I exhibited obviously hemolytic activity, but the fusion protein Trx-Src I almost lost its hemolytic activity, suggesting the importance of the N-terminal amphiphilic α-helix for its functional activity. The cytotoxic effects of Src I depending on the toxin concentration and incubation time were also observed on cultured cells. Among five cell lines: NIH/3T3, U251, NSCLC, BEL-7402, and BGC-823, NSCLC was the most sensitive cells with ID
50 2.8
μg/ml and BGC-823 was the least sensitive cells with ID
50 7.4
μg/ml. After incubated with lipid SUVs, such as SM-SUVs and SM/PC-SUVs, the hemolytic activity of Src I was inhibited to some extent. When incubated with calcein-entrapped lipid LUVs, such as SM-LUVs, SM/PC-LUVs, and SM/PG-LUVs, Src I induced release of entrapped calcein. According to the interaction with lipid vesicles, we proposed that it was the membrane matrix made up of phospholipids, not a particular phospholipid that facilitates Src I to react properly.
