The propeptide of Clostridium septicum alpha toxin functions as an intramolecular chaperone and is a potent inhibitor of alpha toxin-dependent cytolysis.

Clostridium septicum alpha toxin is activated by a proteolytic cleavage at Arg-398 in its carboxy terminus, which yields a 41.3-kDa cytolytically active toxin and a 5.1-kDa propeptide. Studies were performed to determine when the propeptide dissociated from the toxin after proteolytic activation of the protoxin (AT(pro)) and to demonstrate the chaperone activity of the propeptide. The propeptide was found to remain associated with the toxin after activation with trypsin (AT(act)) when analysed by gel filtration or affinity chromatography of a polyhistidine-tagged derivative that contained the polyhistidine tag on the propeptide. The affinity of the propeptide for the toxin was decreased significantly when a mutation was introduced in which Val-400 was converted to a cysteine residue. This mutation destabilized the interaction of the propeptide with the toxin and the propeptide was found to dissociate from the toxin under the same gel-filtration conditions used for the wild-type toxin. The separation of the propeptide in the V400C mutant did not affect the cytolytic activity of the toxin and therefore the propeptide was not necessary for cytolytic activity. These data suggested that the propeptide did not dissociate from the main body of the toxin after proteolysis. Further analysis demonstrated that purified propeptide was a potent inhibitor of alpha toxin activity, which inhibited the oligomerization of alpha toxin into a functional pore. These data suggest that the propeptide does not participate in the final oligomerized complex and that oligomerization appears to displace the propeptide from AT(act). The importance of the propeptide to the solution stability of alpha toxin was also demonstrated. When AT(pro) was activated in solution with trypsin a significant level (approximately 50%) of inactive aggregate formed. This aggregate, which could be removed by centrifugation at 14,000 x g, was made up of both SDS-sensitive and -resistant aggregates, suggesting that a variety of inactive aggregates formed when the monomers interacted in solution. Significantly higher levels of haemolytic activity (approximately 16-fold) were observed when alpha toxin was proteolytically activated after membrane binding instead of in solution. These results support the role of the propeptide as an intramolecular chaperone that stabilizes the monomeric AT(pro) and shuttles it to the membrane where it is activated by protease, oligomerizes into a pre-pore complex and forms a pore. The data suggest that oligomerization of the toxin displaces the propeptide from the monomer form of alpha toxin and that the propeptide does not participate in, and is not necessary to, the final cytolytic complex.