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.
Bourdenet
S
S
Laboratoire de Biochimie Microbienne, Université Claude Bernard Lyon I, Villeurbanne, France.
Vacheron
M J
MJ
Guinand
M
M
Michel
G
G
Arminjon
F
F
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D010450:Endopeptidases; D005098:Exotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009244:NAD+ Nucleosidase / Q000378:metabolism; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000302:isolation & purification; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000032:analysis* / Q000254:growth & development; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D010450:Endopeptidases; D005098:Exotoxins; D009244:NAD+ Nucleosidase; D010446:Peptide Fragments; D011065:Poly(ADP-ribose) Polymerases; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A