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Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

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Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

Auteurs : Keitarou Saiki ; Tomoharu Gomi ; Kiyoshi Konishi

Source :

Journal of Biochemistry [ 0021-924X ] ; 2004-09.

RBID : ISTEX:A0D26CB4921AD66A339695BE7752437054F5FCCE

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- topic : Saccharose.

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Abstract

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 AΔ19–47, 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 (AΔ19–47CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified AΔ19–47CDT 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 AΔ19–47CDT. 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.

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https://api.istex.fr/document/A0D26CB4921AD66A339695BE7752437054F5FCCE/fulltext/pdf

DOI: 10.1093/jb/mvh121

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Keitarou Saiki

<affiliation><mods:affiliation>Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and</mods:affiliation>
<wicri:noCountry code="subField">and</wicri:noCountry>
</affiliation>

Tomoharu Gomi

<affiliation><mods:affiliation>Scientific Instrument Center, Toyama Medical and Pharmaceutical University, Toyama 930-0152</mods:affiliation>
<wicri:noCountry code="subField">930-0152</wicri:noCountry>
</affiliation>

Kiyoshi Konishi

<affiliation><mods:affiliation>Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and</mods:affiliation>
<wicri:noCountry code="subField">and</wicri:noCountry>
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Le document en format XML

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<term>Actinobacillus</term>
<term>Actinobacillus actinomycetemcomitans</term>
<term>Actinobacillus actinomycetemcomitans, cytolethal distending toxin, localized aggressive periodontitis, localized juvenile periodontitis, purification.</term>
<term>Actinomycetemcomitans</term>
<term>Actinomycetemcomitans cdta</term>
<term>Becton dickinson</term>
<term>Cdta</term>
<term>Cdta deletion</term>
<term>Cdta proteins</term>
<term>Cdta variants</term>
<term>Cdtb</term>
<term>Cdtc</term>
<term>Cdts</term>
<term>Cell cycle arrest</term>
<term>Cell distending activity</term>
<term>Cell distention</term>
<term>Coli</term>
<term>Crude cell</term>
<term>Cytolethal</term>
<term>Cytolethal distending toxin</term>
<term>Deletion</term>
<term>Distending</term>
<term>Ducreyi</term>
<term>Haemophilus</term>
<term>Haemophilus ducreyi cytolethal distending toxin</term>
<term>Holotoxin</term>
<term>Immun</term>
<term>Last residue</term>
<term>Molar stoichiometry</term>
<term>Mutant</term>
<term>Phenylmethanesulfonyl fluoride</term>
<term>Plasmid</term>
<term>Protein bands</term>
<term>Purification protocol</term>
<term>Purification studies</term>
<term>Resultant plasmid</term>
<term>Stoichiometry</term>
<term>Subunit</term>
<term>Sucrose</term>
<term>Toxin</term>
<term>Ultrafiltration membrane</term>
<term>Wash buffer</term>
<term>Western blot analyses</term>
<term>Western blot analysis</term>
<term>Wtcdt</term>
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<term>Actinobacillus actinomycetemcomitans</term>
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<term>Cdta proteins</term>
<term>Cdta variants</term>
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<term>Cdtc</term>
<term>Cdts</term>
<term>Cell cycle arrest</term>
<term>Cell distending activity</term>
<term>Cell distention</term>
<term>Coli</term>
<term>Crude cell</term>
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<term>Cytolethal distending toxin</term>
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<term>Haemophilus</term>
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<term>Last residue</term>
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<term>Purification protocol</term>
<term>Purification studies</term>
<term>Resultant plasmid</term>
<term>Stoichiometry</term>
<term>Subunit</term>
<term>Sucrose</term>
<term>Toxin</term>
<term>Ultrafiltration membrane</term>
<term>Wash buffer</term>
<term>Western blot analyses</term>
<term>Western blot analysis</term>
<term>Wtcdt</term>
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<front><div type="abstract" xml:lang="en">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 AΔ19–47, 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 (AΔ19–47CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified AΔ19–47CDT 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 AΔ19–47CDT. 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.</div>
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Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

Source :

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English descriptors

Abstract

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