Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin
Identifieur interne : 005051 ( Istex/Corpus ); précédent : 005050; suivant : 005052Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin
Auteurs : Keitarou Saiki ; Tomoharu Gomi ; Kiyoshi KonishiSource :
- Journal of Biochemistry [ 0021-924X ] ; 2004-09.
English descriptors
- KwdEn :
- ATM, Ataxia Telangiectasia–mutated protein; CDT, cytolethal distending toxin; LAP, localized aggressive periodontitis., Actinobacillus, Actinobacillus actinomycetemcomitans, Actinobacillus actinomycetemcomitans, cytolethal distending toxin, localized aggressive periodontitis, localized juvenile periodontitis, purification., Actinomycetemcomitans, Actinomycetemcomitans cdta, Becton dickinson, Cdta, Cdta deletion, Cdta proteins, Cdta variants, Cdtb, Cdtc, Cdts, Cell cycle arrest, Cell distending activity, Cell distention, Coli, Crude cell, Cytolethal, Cytolethal distending toxin, Deletion, Distending, Ducreyi, Haemophilus, Haemophilus ducreyi cytolethal distending toxin, Holotoxin, Immun, Last residue, Molar stoichiometry, Mutant, Phenylmethanesulfonyl fluoride, Plasmid, Protein bands, Purification protocol, Purification studies, Resultant plasmid, Stoichiometry, Subunit, Sucrose, Toxin, Ultrafiltration membrane, Wash buffer, Western blot analyses, Western blot analysis, Wtcdt.
- Teeft :
- Actinobacillus, Actinobacillus actinomycetemcomitans, Actinomycetemcomitans, Actinomycetemcomitans cdta, Becton dickinson, Cdta, Cdta deletion, Cdta proteins, Cdta variants, Cdtb, Cdtc, Cdts, Cell cycle arrest, Cell distending activity, Cell distention, Coli, Crude cell, Cytolethal, Cytolethal distending toxin, Deletion, Distending, Ducreyi, Haemophilus, Haemophilus ducreyi cytolethal distending toxin, Holotoxin, Immun, Last residue, Molar stoichiometry, Mutant, Phenylmethanesulfonyl fluoride, Plasmid, Protein bands, Purification protocol, Purification studies, Resultant plasmid, Stoichiometry, Subunit, Sucrose, Toxin, Ultrafiltration membrane, Wash buffer, Western blot analyses, Western blot analysis, Wtcdt.
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.
Url:
DOI: 10.1093/jb/mvh121
Links to Exploration step
ISTEX:A0D26CB4921AD66A339695BE7752437054F5FCCELe document en format XML
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and</mods:affiliation></affiliation></author><author><name sortKey="Gomi, Tomoharu" sort="Gomi, Tomoharu" uniqKey="Gomi T" first="Tomoharu" last="Gomi">Tomoharu Gomi</name><affiliation><mods:affiliation>Scientific Instrument Center, Toyama Medical and Pharmaceutical University, Toyama 930-0152</mods:affiliation></affiliation></author><author><name sortKey="Konishi, Kiyoshi" sort="Konishi, Kiyoshi" uniqKey="Konishi K" first="Kiyoshi" last="Konishi">Kiyoshi Konishi</name><affiliation><mods:affiliation>Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Biochemistry</title><title level="j" type="abbrev">J Biochem</title><idno type="ISSN">0021-924X</idno><imprint><publisher>Oxford University Press</publisher><date type="published" when="2004-09">2004-09</date><biblScope unit="volume">136</biblScope><biblScope unit="issue">3</biblScope><biblScope unit="page" from="335">335</biblScope><biblScope unit="page" to="342">342</biblScope></imprint><idno type="ISSN">0021-924X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0021-924X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>ATM, Ataxia Telangiectasia–mutated protein; 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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></front></TEI><istex><corpusName>oup</corpusName><keywords><teeft><json:string>cdta</json:string><json:string>subunit</json:string><json:string>distending</json:string><json:string>cdtb</json:string><json:string>wtcdt</json:string><json:string>deletion</json:string><json:string>actinomycetemcomitans</json:string><json:string>cdtc</json:string><json:string>cytolethal</json:string><json:string>toxin</json:string><json:string>sucrose</json:string><json:string>mutant</json:string><json:string>actinobacillus</json:string><json:string>cdts</json:string><json:string>stoichiometry</json:string><json:string>western blot analysis</json:string><json:string>actinobacillus actinomycetemcomitans</json:string><json:string>cytolethal distending toxin</json:string><json:string>holotoxin</json:string><json:string>immun</json:string><json:string>ducreyi</json:string><json:string>coli</json:string><json:string>plasmid</json:string><json:string>haemophilus</json:string><json:string>molar stoichiometry</json:string><json:string>cell distending activity</json:string><json:string>protein bands</json:string><json:string>actinomycetemcomitans cdta</json:string><json:string>cdta deletion</json:string><json:string>purification studies</json:string><json:string>wash buffer</json:string><json:string>crude cell</json:string><json:string>cdta proteins</json:string><json:string>haemophilus ducreyi cytolethal distending toxin</json:string><json:string>ultrafiltration membrane</json:string><json:string>cell cycle arrest</json:string><json:string>phenylmethanesulfonyl fluoride</json:string><json:string>resultant plasmid</json:string><json:string>purification protocol</json:string><json:string>western blot analyses</json:string><json:string>last residue</json:string><json:string>cell distention</json:string><json:string>cdta variants</json:string><json:string>becton dickinson</json:string></teeft></keywords><author><json:item><name>Keitarou Saiki</name><affiliations><json:string>Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and</json:string></affiliations></json:item><json:item><name>Tomoharu Gomi</name><affiliations><json:string>Scientific Instrument Center, Toyama Medical and Pharmaceutical University, Toyama 930-0152</json:string></affiliations></json:item><json:item><name>Kiyoshi Konishi</name><affiliations><json:string>Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Actinobacillus actinomycetemcomitans, cytolethal distending toxin, localized aggressive periodontitis, localized juvenile periodontitis, purification.</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ATM, Ataxia Telangiectasia–mutated protein; CDT, cytolethal distending toxin; LAP, localized aggressive periodontitis.</value></json:item></subject><arkIstex>ark:/67375/HXZ-VP3ZD1SR-F</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>other</json:string></originalGenre><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. 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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.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>KWD</head><item><term>Actinobacillus actinomycetemcomitans, cytolethal distending toxin, localized aggressive periodontitis, localized juvenile periodontitis, purification.</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>ABR</head><item><term>ATM, Ataxia Telangiectasia–mutated protein; CDT, cytolethal distending toxin; LAP, localized aggressive periodontitis.</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2004-09">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/A0D26CB4921AD66A339695BE7752437054F5FCCE/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="corpus oup, element #text not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="US-ASCII"</istex:xmlDeclaration><istex:docType PUBLIC="-//NLM//DTD Journal Publishing DTD v2.3 20070202//EN" URI="journalpublishing.dtd" name="istex:docType"></istex:docType><istex:document><article xml:lang="en" article-type="other"><front><journal-meta><journal-id journal-id-type="hwp">jbiochem</journal-id><journal-id journal-id-type="nlm-ta">J Biochem</journal-id>
<journal-id journal-id-type="publisher-id">jbchem</journal-id><journal-title>Journal of Biochemistry</journal-title><abbrev-journal-title abbrev-type="publisher">J Biochem</abbrev-journal-title><issn pub-type="ppub">0021-924X</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="other">mvh121</article-id><article-id pub-id-type="doi">10.1093/jb/mvh121</article-id><article-categories><subj-group subj-group-type="heading"><subject>BIOCHEMISTRY</subject></subj-group></article-categories><title-group><article-title>Deletion and Purification Studies to Elucidate the Structure of the <italic>Actinobacillus actinomycetemcomitans</italic> Cytolethal Distending Toxin</article-title></title-group><contrib-group>
<contrib contrib-type="author"><name><surname>Saiki</surname><given-names>Keitarou</given-names></name>
<xref rid="CHDFCBEE">1</xref>
<xref rid="FN1">*</xref>
</contrib><contrib contrib-type="author"><name><surname>Gomi</surname><given-names>Tomoharu</given-names></name>
<xref rid="CHDIFAHG">2</xref>
</contrib><contrib contrib-type="author"><name><surname>Konishi</surname><given-names>Kiyoshi</given-names></name>
<xref rid="CHDFCBEE">1</xref>
</contrib><aff> <target target-type="aff" id="CHDFCBEE"></target><label>1</label>Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and <target target-type="aff" id="CHDIFAHG"></target><label>2</label>Scientific Instrument Center, Toyama Medical and Pharmaceutical University, Toyama 930-0152</aff></contrib-group><pub-date pub-type="ppub"><month>09</month><year>2004</year></pub-date><volume>136</volume><issue>3</issue><fpage>335</fpage><lpage>342</lpage><permissions><copyright-year>2004</copyright-year></permissions><abstract xml:lang="en">
<p>Cytolethal distending toxin (CDT) is one of the exotoxins produced by <italic>Actinobacillus actinomycetemcomitans</italic>, an agent of localized aggressive periodontitis. We constructed N-terminal deletion mutants of CdtA using an <italic>Escherichia coli</italic> expression system and found that AΔ<sub>19–47</sub>, 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 (A<sub>Δ</sub><sub>19–47</sub>CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified A<sub>Δ</sub><sub>19–47</sub>CDT 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 A<sub>Δ</sub><sub>19–47</sub>CDT. 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.</p>
</abstract><kwd-group kwd-group-type="KWD" xml:lang="en">
<kwd><italic>Actinobacillus actinomycetemcomitans</italic>, cytolethal distending toxin, localized aggressive periodontitis, localized juvenile periodontitis, purification.</kwd>
</kwd-group><kwd-group kwd-group-type="ABR" xml:lang="en">
<kwd> ATM, Ataxia Telangiectasia–mutated protein; CDT, cytolethal distending toxin; LAP, localized aggressive periodontitis.</kwd>
</kwd-group><custom-meta-wrap><custom-meta><meta-name>hwp-legacy-fpage</meta-name><meta-value>335</meta-value></custom-meta><custom-meta><meta-name>hwp-legacy-dochead</meta-name><meta-value>BIOCHEMISTRY</meta-value></custom-meta></custom-meta-wrap></article-meta><notes><p content-type="arthw-misc">Received March 26, 2004; accepted June 10, 2004</p></notes></front>
</article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Deletion and Purification Studies to Elucidate the Structure of the Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin</title></titleInfo><name type="personal"><namePart type="given">Keitarou</namePart><namePart type="family">Saiki</namePart><affiliation>Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Tomoharu</namePart><namePart type="family">Gomi</namePart><affiliation>Scientific Instrument Center, Toyama Medical and Pharmaceutical University, Toyama 930-0152</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Kiyoshi</namePart><namePart type="family">Konishi</namePart><affiliation>Department of Microbiology, Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo 102-8159; and</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="other" displayLabel="other" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-7474895G-0">other</genre><originInfo><publisher>Oxford University Press</publisher><dateIssued encoding="w3cdtf">2004-09</dateIssued><copyrightDate encoding="w3cdtf">2004</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract 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.</abstract><note>Received March 26, 2004; accepted June 10, 2004</note><subject lang="en"><genre>KWD</genre><topic>Actinobacillus actinomycetemcomitans, cytolethal distending toxin, localized aggressive periodontitis, localized juvenile periodontitis, purification.</topic></subject><subject lang="en"><genre>ABR</genre><topic>ATM, Ataxia Telangiectasia–mutated protein; CDT, cytolethal distending toxin; LAP, localized aggressive periodontitis.</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Biochemistry</title></titleInfo><titleInfo type="abbreviated"><title>J Biochem</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">0021-924X</identifier><identifier type="PublisherID">jbchem</identifier><identifier type="PublisherID-hwp">jbiochem</identifier><identifier type="PublisherID-nlm-ta">J Biochem</identifier><part><date>2004</date><detail type="volume"><caption>vol.</caption><number>136</number></detail><detail type="issue"><caption>no.</caption><number>3</number></detail><extent unit="pages"><start>335</start><end>342</end></extent></part></relatedItem><identifier type="istex">A0D26CB4921AD66A339695BE7752437054F5FCCE</identifier><identifier type="DOI">10.1093/jb/mvh121</identifier><identifier type="local">mvh121</identifier><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-GTWS0RDP-M">oup</recordContentSource></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/document/A0D26CB4921AD66A339695BE7752437054F5FCCE/metadata/json</uri></json:item></metadata><annexes><json:item><extension>jpeg</extension><original>true</original><mimetype>image/jpeg</mimetype><uri>https://api.istex.fr/document/A0D26CB4921AD66A339695BE7752437054F5FCCE/annexes/jpeg</uri></json:item><json:item><extension>gif</extension><original>true</original><mimetype>image/gif</mimetype><uri>https://api.istex.fr/document/A0D26CB4921AD66A339695BE7752437054F5FCCE/annexes/gif</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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