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Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2007 Aug 31; Vol. 282 (35), pp. 25314-21. Date of Electronic Publication: 2007 Jun 25. - Publication Year :
- 2007
-
Abstract
- The action of Clostridium difficile toxins A and B depends on processing and translocation of the catalytic glucosyltransferase domain into the cytosol of target cells where Rho GTPases are modified. Here we studied the processing of the toxins. Dithiothreitol and beta-mercaptoethanol induced auto-cleavage of purified native toxin A and toxin B into approximately 250/210- and approximately 63-kDa fragments. The 63-kDa fragment was identified by mass spectrometric analysis as the N-terminal glucosyltransferase domain. This cleavage was blocked by N-ethylmaleimide or iodoacetamide. Exchange of cysteine 698, histidine 653, or aspartate 587 of toxin B prevented cleavage of full-length recombinant toxin B and of an N-terminal fragment covering residues 1-955 and inhibited cytotoxicity of full-length toxin B. Dithiothreitol synergistically increased the effect of myo-inositol hexakisphosphate, which has been reported to facilitate auto-cleavage of toxin B (Reineke, J., Tenzer, S., Rupnik, M., Koschinski, A., Hasselmayer, O., Schrattenholz, A., Schild, H., and Von Eichel-Streiber, C. (2007) Nature 446, 415-419). N-Ethylmaleimide blocked auto-cleavage induced by the addition of myo-inositol hexakisphosphate, suggesting that cysteine residues are essential for the processing of clostridial glucosylating toxins. Our data indicate that clostridial glucosylating cytotoxins possess an inherent cysteine protease activity related to the cysteine protease of Vibrio cholerae RTX toxin, which is responsible for auto-cleavage of glucosylating toxins.
- Subjects :
- Bacterial Proteins isolation & purification
Bacterial Proteins metabolism
Bacterial Toxins isolation & purification
Bacterial Toxins metabolism
Dithiothreitol chemistry
Enterotoxins isolation & purification
Enterotoxins metabolism
Ethylmaleimide chemistry
Glucosyltransferases isolation & purification
Glucosyltransferases metabolism
Glycosylation
Humans
Inositol Phosphates chemistry
Iodoacetamide chemistry
Peptide Hydrolases isolation & purification
Peptide Hydrolases metabolism
Protein Structure, Tertiary
Recombinant Proteins chemistry
Recombinant Proteins isolation & purification
Recombinant Proteins metabolism
rho GTP-Binding Proteins metabolism
Bacterial Proteins chemistry
Bacterial Toxins chemistry
Clostridioides difficile enzymology
Enterotoxins chemistry
Glucosyltransferases chemistry
Peptide Hydrolases chemistry
Protein Processing, Post-Translational
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 282
- Issue :
- 35
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17591770
- Full Text :
- https://doi.org/10.1074/jbc.M703062200