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

Abstract

SummaryClostridium 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 × 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.

Cleavage of a C-terminal Peptide Is Essential for Heptamerization of Clostridium perfringens ε-Toxin in the Synaptosomal Membrane

“…Another likely chaperonic function of the C-terminal peptide is to prevent the active ⑀-toxin from aggregating in solution. The 5.1-kDa Cterminal propeptide of C. septicum ␣-toxin has been shown to be associated with the toxin even after proteolytic activation, preventing the toxin from aggregating (8). Further study, of the association/dissociation of the C-terminal peptide with the active ⑀-toxin, and its effect on unfolding/refolding of the toxin, should be undertaken to elucidate the roles of the C-terminal peptide in the structure and function of ⑀-toxin.…”

“…It has been suggested to be a pore-forming toxin based on the following observations. (i) ⑀-Toxin can form a large complex in the membrane of MDCK 1 cells, and it permeabilizes them (5, 6); (ii) the large complex formed by ⑀-toxin is not dissociated by SDS treatment (6), which is a common feature of pore-forming toxins such as aerolysin (7), Clostridium septicum ␣-toxin (8), and Pseudomonas aeruginosa cytotoxin (9); and (iii) the CD spectrum of ⑀-toxin shows it mainly consists of ␤-sheets (10), as is characteristically observed for pore-forming ␤-barrel toxins.…”

Crystal structure of the precursor of galactose oxidase: An unusual self-processing enzyme

“…The best-studied examples are proteases where the pro-sequence is relatively long, comprising some 70 to more than 200 aa (29)(30)(31)(32)(33). Processing, commonly by protease activity, leads to removal of the prosequence and activation of the enzyme.…”

Cholesterol-Dependent Cytolysins, a Family of Versatile Pore-Forming Toxins

“…One common mechanism to control oligomerization is the proteolytic cleavage of a propeptide of the toxin by a membrane-restricted protease. The small pore-forming toxins such as aerolysin from Aeromonas hydrophila, C. septicum alpha-toxin, and anthraxprotective antigen all require the proteolytic cleavage of a propeptide to initiate oligomerization (2,16,24,48,84). Typically, a membrane-restricted protease, such as furin (25), cleaves at an amino-or carboxy-terminal site of the membranebound toxin that removes the covalent attachment of the propeptide from the main structure of the toxin.…”