J. Biochem, 2004, Vol. 136, No. 3 335-342
© 2004 The Japanese Biochemical Society
BIOCHEMISTRY |
Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin
1 Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and 2 Scientific Instrument Center, Toyama Medical and Pharmaceutical University, Toyama 930-0152
Cytolethal distending toxin (CDT) is one of the exotoxins produced by Actinobacillus actinomycetemcomitans, an agent of localized aggressive periodontitis. We constructed N-terminal deletion mutants of CdtA using an Escherichia coli expression system and found that A
19–47, with a deletion from Asn-19 to Pro-47, showed comparable CDT activity but no apparent heterogeneity of CdtA. The wild-type CDT (wtCDT) and the mutant CDT (A19–47CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified A19–47CDT showed strong CDT activity and a tripartite structure composed of CdtA (subunit A), 31 kDa CdtB (subunit B), and 18.5 kDa CdtC (subunit C) in nearly a 1:1:1 stoichiometry. Importantly, subunit A was identified as heterogeneous with three CdtA variants in wtCDT, but homogeneous in A19–47CDT. Purified CDTs also showed high stability that was absolutely dependent on the presence of sucrose in the buffer. In conclusion, the region from the Asn-19 to Pro-47 of CdtA contributes to the heterogeneous production of CdtA, but is dispensable for the toxin activity. Furthermore, this study describes an effective protocol for the purification of a native rather than reconstituted CDT, and clarifies the subunit composition of the active CDT holotoxin.
* To whom correspondence should be addressed: Tel: +81-3-3261-8763, Fax: +81-3-3264-8399, E-mail: keisaiki{at}tky.ndu.ac.jp
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  O. Matangkasombut, R. Wattanawaraporn, K. Tsuruda, M. Ohara, M. Sugai, and S. Mongkolsuk Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans Induces DNA Damage, S/G2 Cell Cycle Arrest, and Caspase- Independent Death in a Saccharomyces cerevisiae Model Infect. Immun., February 1, 2010; 78(2): 783 - 792. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. P. Rabin, J. G. Flitton, and D. R. Demuth Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Apoptosis in Nonproliferating Macrophages by a Phosphatase-Independent Mechanism Infect. Immun., August 1, 2009; 77(8): 3161 - 3169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Cao, A. Volgina, J. Korostoff, and J. M. DiRienzo Role of Intrachain Disulfides in the Activities of the CdtA and CdtC Subunits of the Cytolethal Distending Toxin of Actinobacillus actinomycetemcomitans Infect. Immun., September 1, 2006; 74(9): 4990 - 5002. [Abstract] [Full Text] [PDF]
|
|