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

Eur J Biochem. 1990 Sep 11;192(2):379-85. doi: 10.1111/j.1432-1033.1990.tb19238.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • Pseudomonas aeruginosa Exotoxin A
  • Virulence Factors*

Substances

  • Bacterial Toxins
  • Exotoxins
  • Peptide Fragments
  • Virulence Factors
  • ADP Ribose Transferases
  • Poly(ADP-ribose) Polymerases
  • NAD+ Nucleosidase
  • Endopeptidases