Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N‐terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365–404). As a result, the polypeptides contained a large portion (13–34 amino acids) of domain Ib linked to the adjacent C‐terminal domain III (amino acids 405–613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S‐fragment; residues 392–613; 24201 Da; pI 4.7) possessed the same level of ADP‐ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37‐fold higher NAD‐glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP‐ribosyltransferase activity of both exotoxin A and the S‐fragment, but not the NAD‐glycohydrolase activity of the S‐fragment. Antibodies against the S‐fragment neutralized the ADP‐ribosyltransferase activity of exotoxin A. These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392–404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF‐2) and show that antibodies recognize the EF‐2‐binding site but not the NAD + ‐binding site.