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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in <italic>Escherichia coli</italic> to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach</title><author><name sortKey="Johansen, Eric B" sort="Johansen, Eric B" uniqKey="Johansen E" first="Eric B." last="Johansen">Eric B. Johansen</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Pharmaceutical Chemistry and Pharmaceutical Sciences</addr-line>,<institution>University of California</institution>,<addr-line>San Francisco, CA 94143</addr-line>,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Szoka, Francis C" sort="Szoka, Francis C" uniqKey="Szoka F" first="Francis C." last="Szoka">Francis C. Szoka</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Pharmaceutical Chemistry and Pharmaceutical Sciences</addr-line>,<institution>University of California</institution>,<addr-line>San Francisco, CA 94143</addr-line>,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Zaleski, Anthony" sort="Zaleski, Anthony" uniqKey="Zaleski A" first="Anthony" last="Zaleski">Anthony Zaleski</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology</addr-line>,<institution>University of Iowa</institution>,<addr-line>Iowa City, IA 52242</addr-line>,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Apicella, Michael A" sort="Apicella, Michael A" uniqKey="Apicella M" first="Michael A." last="Apicella">Michael A. Apicella</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology</addr-line>,<institution>University of Iowa</institution>,<addr-line>Iowa City, IA 52242</addr-line>,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Gibson, Bradford W" sort="Gibson, Bradford W" uniqKey="Gibson B" first="Bradford W." last="Gibson">Bradford W. Gibson</name><affiliation><nlm:aff id="aff3"><institution>Buck Institute for Age Research</institution>,<addr-line>8001 Redwood Blvd., Novato, CA 94945</addr-line>,<country>USA</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">20208062</idno><idno type="pmc">2900885</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2900885</idno><idno type="RBID">PMC:2900885</idno><idno type="doi">10.1093/glycob/cwq033</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">000126</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000126</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in <italic>Escherichia coli</italic> to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach</title><author><name sortKey="Johansen, Eric B" sort="Johansen, Eric B" uniqKey="Johansen E" first="Eric B." last="Johansen">Eric B. Johansen</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Pharmaceutical Chemistry and Pharmaceutical Sciences</addr-line>,<institution>University of California</institution>,<addr-line>San Francisco, CA 94143</addr-line>,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Szoka, Francis C" sort="Szoka, Francis C" uniqKey="Szoka F" first="Francis C." last="Szoka">Francis C. Szoka</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Pharmaceutical Chemistry and Pharmaceutical Sciences</addr-line>,<institution>University of California</institution>,<addr-line>San Francisco, CA 94143</addr-line>,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Zaleski, Anthony" sort="Zaleski, Anthony" uniqKey="Zaleski A" first="Anthony" last="Zaleski">Anthony Zaleski</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology</addr-line>,<institution>University of Iowa</institution>,<addr-line>Iowa City, IA 52242</addr-line>,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Apicella, Michael A" sort="Apicella, Michael A" uniqKey="Apicella M" first="Michael A." last="Apicella">Michael A. Apicella</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology</addr-line>,<institution>University of Iowa</institution>,<addr-line>Iowa City, IA 52242</addr-line>,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Gibson, Bradford W" sort="Gibson, Bradford W" uniqKey="Gibson B" first="Bradford W." last="Gibson">Bradford W. Gibson</name><affiliation><nlm:aff id="aff3"><institution>Buck Institute for Age Research</institution>,<addr-line>8001 Redwood Blvd., Novato, CA 94945</addr-line>,<country>USA</country></nlm:aff></affiliation></author></analytic><series><title level="j">Glycobiology</title><idno type="ISSN">0959-6658</idno><idno type="eISSN">1460-2423</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in <italic>Escherichia coli</italic> transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (<italic>lsg</italic>) from <italic>Haemophilus influenzae.</italic> Analysis of these novel oligosaccharide-LPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the <italic>lsg</italic> locus. Here, we adapted this strategy to construct a modular approach to study the synthetic properties of individual glycosyltransferases expressed alone and in combinations. To this end, a set of expression vectors containing one to four putative glycosyltransferase genes from the <italic>lsg</italic> locus, <italic>lsgC-F</italic>, were transformed into <italic>E. coli</italic> K12 (XL-1) which is defective in LPS O-antigen biosynthesis<italic>.</italic> This strategy relied on the inclusion of the <italic>H. influenzae</italic> gene product <italic>lsgG</italic> in every plasmid construct, which partially rescues the <italic>E. coli</italic> LPS biosynthesis defect by priming uridine diphosphate-undecaprenyl in the WecA-dependent O-antigen synthetic pathway with <italic>N</italic>-acetyl-glucosamine (GlcNAc). This GlcNAc-undecaprenyl then served as an acceptor substrate for further carbohydrate extension by transformed glycosyltransferases. The resultant LPS-linked chimeric glycans were isolated from their <italic>E. coli</italic> constructs and characterized by mass spectrometry, methylation analysis and enzyme-linked immunosorbent assays. These structural data allowed the specificity of various glycosyltransferases to be unambiguously assigned to individual genes. LsgF was found to transfer a galactose (Gal) to terminal GlcNAc. LsgE was found to transfer GlcNAc to Gal-GlcNAc, and both LsgF and LsgD were found to transfer Gal to GlcNAc-Gal-GlcNAc but with differing linkage specificities. This method can be generalized and readily adapted to study the substrate specificity of other putative or uncharacterized glycosyltransferases.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Glycobiology</journal-id><journal-id journal-id-type="publisher-id">glycob</journal-id><journal-id journal-id-type="hwp">glycob</journal-id><journal-title-group><journal-title>Glycobiology</journal-title></journal-title-group><issn pub-type="ppub">0959-6658</issn><issn pub-type="epub">1460-2423</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">20208062</article-id><article-id pub-id-type="pmc">2900885</article-id><article-id pub-id-type="doi">10.1093/glycob/cwq033</article-id><article-id pub-id-type="publisher-id">cwq033</article-id><article-categories><subj-group subj-group-type="heading"><subject>Orignal Article</subject></subj-group></article-categories><title-group><article-title>Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in <italic>Escherichia coli</italic> to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach</article-title><alt-title>E B Johansen</alt-title><alt-title alt-title-type="right-running">Combinatorial approach to assess glycosyltransferase function</alt-title><alt-title alt-title-type="left-running">EB Johansen et al.</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Johansen</surname><given-names>Eric B.</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Szoka</surname><given-names>Francis C.</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Zaleski</surname><given-names>Anthony</given-names></name><xref ref-type="aff" rid="aff2">3</xref></contrib><contrib contrib-type="author"><name><surname>Apicella</surname><given-names>Michael A.</given-names></name><xref ref-type="aff" rid="aff2">3</xref></contrib><contrib contrib-type="author"><name><surname>Gibson</surname><given-names>Bradford W.</given-names></name><xref rid="cor1" ref-type="corresp">1</xref><xref ref-type="aff" rid="aff3">4</xref></contrib><aff id="aff1"><label>2</label><addr-line>Department of Pharmaceutical Chemistry and Pharmaceutical Sciences</addr-line>,<institution>University of California</institution>,<addr-line>San Francisco, CA 94143</addr-line>,<country>USA</country></aff><aff id="aff2"><label>3</label><addr-line>Department of Microbiology</addr-line>,<institution>University of Iowa</institution>,<addr-line>Iowa City, IA 52242</addr-line>,<country>USA</country></aff><aff id="aff3"><label>4</label><institution>Buck Institute for Age Research</institution>,<addr-line>8001 Redwood Blvd., Novato, CA 94945</addr-line>,<country>USA</country></aff></contrib-group><author-notes><corresp id="cor1"><label>1</label>To whom correspondence should be addressed: Tel: 415-209-2032; Fax: 415 209-2231; e-mail:
<email>bgibson@buckinstitute.org</email></corresp></author-notes><pub-date pub-type="ppub"><month>6</month><year>2010</year></pub-date><pub-date pub-type="epub"><day>5</day><month>3</month><year>2010</year></pub-date><volume>20</volume><issue>6</issue><fpage>763</fpage><lpage>774</lpage><history><date date-type="received"><day>7</day><month>8</month><year>2009</year></date><date date-type="rev-recd"><day>26</day><month>2</month><year>2010</year></date><date date-type="accepted"><day>27</day><month>2</month><year>2010</year></date></history><permissions><copyright-statement>© The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org</copyright-statement><copyright-year>2010</copyright-year><copyright-holder>Oxford University Press</copyright-holder></permissions><abstract><p>In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in <italic>Escherichia coli</italic> transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (<italic>lsg</italic>) from <italic>Haemophilus influenzae.</italic> Analysis of these novel oligosaccharide-LPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the <italic>lsg</italic> locus. Here, we adapted this strategy to construct a modular approach to study the synthetic properties of individual glycosyltransferases expressed alone and in combinations. To this end, a set of expression vectors containing one to four putative glycosyltransferase genes from the <italic>lsg</italic> locus, <italic>lsgC-F</italic>, were transformed into <italic>E. coli</italic> K12 (XL-1) which is defective in LPS O-antigen biosynthesis<italic>.</italic> This strategy relied on the inclusion of the <italic>H. influenzae</italic> gene product <italic>lsgG</italic> in every plasmid construct, which partially rescues the <italic>E. coli</italic> LPS biosynthesis defect by priming uridine diphosphate-undecaprenyl in the WecA-dependent O-antigen synthetic pathway with <italic>N</italic>-acetyl-glucosamine (GlcNAc). This GlcNAc-undecaprenyl then served as an acceptor substrate for further carbohydrate extension by transformed glycosyltransferases. The resultant LPS-linked chimeric glycans were isolated from their <italic>E. coli</italic> constructs and characterized by mass spectrometry, methylation analysis and enzyme-linked immunosorbent assays. These structural data allowed the specificity of various glycosyltransferases to be unambiguously assigned to individual genes. LsgF was found to transfer a galactose (Gal) to terminal GlcNAc. LsgE was found to transfer GlcNAc to Gal-GlcNAc, and both LsgF and LsgD were found to transfer Gal to GlcNAc-Gal-GlcNAc but with differing linkage specificities. This method can be generalized and readily adapted to study the substrate specificity of other putative or uncharacterized glycosyltransferases.</p></abstract><kwd-group><title>Keywords</title><kwd>assay</kwd><kwd>chimera</kwd><kwd>E. coli</kwd><kwd>glycosyltransferase</kwd><kwd>mass spectrometry</kwd></kwd-group></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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