This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jobling, M. G.
Right arrow Articles by Holmes, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jobling, M. G.
Right arrow Articles by Holmes, R. K.

 Previous Article  |  Next Article 

Journal of Bacteriology, July 2001, p. 4024-4032, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.4024-4032.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis

Michael G. Jobling and Randall K. Holmes*

Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80220

Received 11 December 2000/Accepted 27 March 2001

Cholera toxin (CT) is the prototype for the Vibrio cholerae-Escherichia coli family of heat-labile enterotoxins having an AB5 structure. By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties. Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin. Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68+V72). In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity. For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10- and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12- to 50-fold more than the reduction in toxicity. These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use.

* Corresponding author. Mailing address: Department of Microbiology, B175, University of Colorado Health Sciences Center, 4200 East 9th Ave., Denver, CO 80220. Phone: (303) 315-7903. Fax: (303) 315-6785. E-mail: Randall.Holmes{at}UCHSC.edu.

Journal of Bacteriology, July 2001, p. 4024-4032, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.4024-4032.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Wolf, A. A., Jobling, M. G., Saslowsky, D. E., Kern, E., Drake, K. R., Kenworthy, A. K., Holmes, R. K., Lencer, W. I. (2008). Attenuated Endocytosis and Toxicity of a Mutant Cholera Toxin with Decreased Ability To Cluster Ganglioside GM1 Molecules. Infect. Immun. 76: 1476-1484 [Abstract] [Full Text]  
  • Teter, K., Jobling, M. G., Sentz, D., Holmes, R. K. (2006). The Cholera Toxin A13 Subdomain Is Essential for Interaction with ADP-Ribosylation Factor 6 and Full Toxic Activity but Is Not Required for Translocation from the Endoplasmic Reticulum to the Cytosol. Infect. Immun. 74: 2259-2267 [Abstract] [Full Text]  
  • O'Neal, C. J., Jobling, M. G., Holmes, R. K., Hol, W. G. J. (2005). Structural Basis for the Activation of Cholera Toxin by Human ARF6-GTP. Science 309: 1093-1096 [Abstract] [Full Text]  
  • Teter, K., Jobling, M. G., Holmes, R. K. (2004). Vesicular Transport Is Not Required for the Cytoplasmic Pool of Cholera Toxin To Interact with the Stimulatory Alpha Subunit of the Heterotrimeric G Protein. Infect. Immun. 72: 6826-6835 [Abstract] [Full Text]  
  • Li, X., Erbe, J. L., Lockatell, C. V., Johnson, D. E., Jobling, M. G., Holmes, R. K., Mobley, H. L. T. (2004). Use of Translational Fusion of the MrpH Fimbrial Adhesin-Binding Domain with the Cholera Toxin A2 Domain, Coexpressed with the Cholera Toxin B Subunit, as an Intranasal Vaccine To Prevent Experimental Urinary Tract Infection by Proteus mirabilis. Infect. Immun. 72: 7306-7310 [Abstract] [Full Text]  
  • Massol, R. H., Larsen, J. E., Fujinaga, Y., Lencer, W. I., Kirchhausen, T. (2004). Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways. Mol. Biol. Cell 15: 3631-3641 [Abstract] [Full Text]  
  • Tinker, J. K., Erbe, J. L., Hol, W. G. J., Holmes, R. K. (2003). Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B5 Interface: Mutational Analysis and Development of an In Vitro Assembly System. Infect. Immun. 71: 4093-4101 [Abstract] [Full Text]  
  • Teter, K., Allyn, R. L., Jobling, M. G., Holmes, R. K. (2002). Transfer of the Cholera Toxin A1 Polypeptide from the Endoplasmic Reticulum to the Cytosol Is a Rapid Process Facilitated by the Endoplasmic Reticulum-Associated Degradation Pathway. Infect. Immun. 70: 6166-6171 [Abstract] [Full Text]  
  • Teter, K., Holmes, R. K. (2002). Inhibition of Endoplasmic Reticulum-Associated Degradation in CHO Cells Resistant to Cholera Toxin, Pseudomonas aeruginosa Exotoxin A, and Ricin. Infect. Immun. 70: 6172-6179 [Abstract] [Full Text]  
  • Sanchez, J., Wallerstrom, G., Fredriksson, M., Angstrom, J., Holmgren, J. (2002). Detoxification of Cholera Toxin without Removal of Its Immunoadjuvanticity by the Addition of (STa-related) Peptides to the Catalytic Subunit. A POTENTIAL NEW STRATEGY TO GENERATE IMMUNOSTIMULANTS FOR VACCINATION. J. Biol. Chem. 277: 33369-33377 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»