Rational approach of the structure-function relationship using chimeric isoforms provides new insights into the functional differences of sticholysins

Sticholysin I and II are two isoforms of pore-forming proteins from Stichodactyla helianthus sea anemone, exhaustively characterized. Sticholysin I and II have 176 and 175 amino acid residues respectively, and only 12 amino acid substitutions. Consequently, these isoforms have 99% of similarity and 93% of identity of sequences with very similar three-dimensional structures. However, sticholysin II is hemolytically more active than sticholysin I on human red blood cells and guinea pig blood cells. The reasons for theses functional differences are not known but interestingly most of the amino acid changes are located in functional regions such as: N-terminal segment, loops associated with membrane binding site, and protein-protein interaction sites. To deepen into the hemolytic activity differences between sticholysin I and II we designed and obtained three chimeric sticholysins in Escherichia coli that exchange different segments of both isoforms. The analysis of the hemolytic activity of these chimeras and their homology modeling provided new evidences on the pore-forming activity of actinoporins, as well as on the amino acid residues that could be involved in the functional difference between sticholysins I and II, in the interaction of these proteins with membranes or in the oligomerization process.