PMID:2170123

Citation	Bourdenet, S, Vacheron, MJ, Guinand, M, Michel, G and Arminjon, F (1990) Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa. Eur. J. Biochem. 192:379-85
Abstract	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.
Links	PubMed
Keywords	ADP Ribose Transferases; Amino Acid Sequence; Bacterial Toxins; Endopeptidases; Exotoxins/chemistry; Exotoxins/isolation & purification; Exotoxins/metabolism; Kinetics; Molecular Sequence Data; Molecular Weight; NAD+ Nucleosidase/metabolism; Neutralization Tests; Peptide Fragments/isolation & purification; Poly(ADP-ribose) Polymerases/metabolism; Pseudomonas aeruginosa/analysis; Pseudomonas aeruginosa/growth & development; Virulence Factors
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PSEAE:TOXA		GO:0047286: NAD+-diphthamide ADP-ribosyltransferase activity	ECO:0000314:		F		Figure 3: ADP-ribosylation activity of exotoxin A with NAD+ after 30 minutes.	complete CACAO 4212
PSEAE:TOXA	enables	GO:0047286: NAD+-diphthamide ADP-ribosyltransferase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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