Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in Escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach
Identifieur interne : 000088 ( Pmc/Checkpoint ); précédent : 000087; suivant : 000089Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in Escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach
Auteurs : Eric B. Johansen [États-Unis] ; Francis C. Szoka [États-Unis] ; Anthony Zaleski [États-Unis] ; Michael A. Apicella [États-Unis] ; Bradford W. Gibson [États-Unis]Source :
- Glycobiology [ 0959-6658 ] ; 2010.
Abstract
In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in
Url:
DOI: 10.1093/glycob/cwq033
PubMed: 20208062
PubMed Central: 2900885
Affiliations:
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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>
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,<institution>University of California</institution>
,<addr-line>San Francisco, CA 94143</addr-line>
,<country>USA</country>
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<country xml:lang="fr">États-Unis</country>
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<author><name sortKey="Szoka, Francis C" sort="Szoka, Francis C" uniqKey="Szoka F" first="Francis C." last="Szoka">Francis C. Szoka</name>
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<author><name sortKey="Zaleski, Anthony" sort="Zaleski, Anthony" uniqKey="Zaleski A" first="Anthony" last="Zaleski">Anthony Zaleski</name>
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<author><name sortKey="Apicella, Michael A" sort="Apicella, Michael A" uniqKey="Apicella M" first="Michael A." last="Apicella">Michael A. Apicella</name>
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<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 wicri:level="1"><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>
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<country xml:lang="fr">États-Unis</country>
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<author><name sortKey="Szoka, Francis C" sort="Szoka, Francis C" uniqKey="Szoka F" first="Francis C." last="Szoka">Francis C. Szoka</name>
<affiliation wicri:level="1"><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>
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<country xml:lang="fr">États-Unis</country>
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<author><name sortKey="Zaleski, Anthony" sort="Zaleski, Anthony" uniqKey="Zaleski A" first="Anthony" last="Zaleski">Anthony Zaleski</name>
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,<institution>University of Iowa</institution>
,<addr-line>Iowa City, IA 52242</addr-line>
,<country>USA</country>
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<country xml:lang="fr">États-Unis</country>
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<author><name sortKey="Apicella, Michael A" sort="Apicella, Michael A" uniqKey="Apicella M" first="Michael A." last="Apicella">Michael A. Apicella</name>
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,<addr-line>Iowa City, IA 52242</addr-line>
,<country>USA</country>
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<country xml:lang="fr">États-Unis</country>
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<country xml:lang="fr">États-Unis</country>
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<series><title level="j">Glycobiology</title>
<idno type="ISSN">0959-6658</idno>
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<imprint><date when="2010">2010</date>
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<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>
<affiliations><list><country><li>États-Unis</li>
</country>
</list>
<tree><country name="États-Unis"><noRegion><name sortKey="Johansen, Eric B" sort="Johansen, Eric B" uniqKey="Johansen E" first="Eric B." last="Johansen">Eric B. Johansen</name>
</noRegion>
<name sortKey="Apicella, Michael A" sort="Apicella, Michael A" uniqKey="Apicella M" first="Michael A." last="Apicella">Michael A. Apicella</name>
<name sortKey="Gibson, Bradford W" sort="Gibson, Bradford W" uniqKey="Gibson B" first="Bradford W." last="Gibson">Bradford W. Gibson</name>
<name sortKey="Szoka, Francis C" sort="Szoka, Francis C" uniqKey="Szoka F" first="Francis C." last="Szoka">Francis C. Szoka</name>
<name sortKey="Zaleski, Anthony" sort="Zaleski, Anthony" uniqKey="Zaleski A" first="Anthony" last="Zaleski">Anthony Zaleski</name>
</country>
</tree>
</affiliations>
</record>
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