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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 : 000167 ( Ncbi/Curation ); précédent : 000166; suivant : 000168

Utilizing 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.

RBID : PMC:2900885

Descripteurs français

KwdFr :
- Antigènes O (), Antigènes O (biosynthèse), Antigènes O (génétique), Conformation des glucides, Escherichia coli (métabolisme), Glycosyltransferase (génétique), Glycosyltransferase (métabolisme), Haemophilus influenzae (enzymologie), Haemophilus influenzae (génétique), Spécificité du substrat, Séquence glucidique, Test ELISA, Vecteurs génétiques (génétique).
MESH :
- biosynthèse : Antigènes O.
- enzymologie : Haemophilus influenzae.
- génétique : Antigènes O, Glycosyltransferase, Haemophilus influenzae, Vecteurs génétiques.
- métabolisme : Escherichia coli, Glycosyltransferase.
- Antigènes O, Conformation des glucides, Spécificité du substrat, Séquence glucidique, Test ELISA.

English descriptors

KwdEn :
- Carbohydrate Conformation, Carbohydrate Sequence, Enzyme-Linked Immunosorbent Assay, Escherichia coli (metabolism), Genetic Vectors (genetics), Glycosyltransferases (genetics), Glycosyltransferases (metabolism), Haemophilus influenzae (enzymology), Haemophilus influenzae (genetics), O Antigens (biosynthesis), O Antigens (chemistry), O Antigens (genetics), Substrate Specificity.
MESH :
- chemical , biosynthesis : O Antigens.
- chemical , chemistry : O Antigens.
- chemical , genetics : Glycosyltransferases, O Antigens.
- enzymology : Haemophilus influenzae.
- genetics : Genetic Vectors, Haemophilus influenzae.
- metabolism : Escherichia coli, Glycosyltransferases.
- Carbohydrate Conformation, Carbohydrate Sequence, Enzyme-Linked Immunosorbent Assay, Substrate Specificity.

Abstract

In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in Escherichia coli transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae. Analysis of these novel oligosaccharide-LPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the lsg 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 lsg locus, lsgC-F, were transformed into E. coli K12 (XL-1) which is defective in LPS O-antigen biosynthesis. This strategy relied on the inclusion of the H. influenzae gene product lsgG in every plasmid construct, which partially rescues the E. coli LPS biosynthesis defect by priming uridine diphosphate-undecaprenyl in the WecA-dependent O-antigen synthetic pathway with N-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 E. coli 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.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2900885

DOI: 10.1093/glycob/cwq033
PubMed: 20208062
PubMed Central: 2900885

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Le document en format XML

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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>
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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="Gibson, Bradford W" sort="Gibson, Bradford W" uniqKey="Gibson B" first="Bradford W." last="Gibson">Bradford W. Gibson</name>
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<series><title level="j">Glycobiology</title>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Carbohydrate Conformation</term>
<term>Carbohydrate Sequence</term>
<term>Enzyme-Linked Immunosorbent Assay</term>
<term>Escherichia coli (metabolism)</term>
<term>Genetic Vectors (genetics)</term>
<term>Glycosyltransferases (genetics)</term>
<term>Glycosyltransferases (metabolism)</term>
<term>Haemophilus influenzae (enzymology)</term>
<term>Haemophilus influenzae (genetics)</term>
<term>O Antigens (biosynthesis)</term>
<term>O Antigens (chemistry)</term>
<term>O Antigens (genetics)</term>
<term>Substrate Specificity</term>
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<keywords scheme="KwdFr" xml:lang="fr"><term>Antigènes O ()</term>
<term>Antigènes O (biosynthèse)</term>
<term>Antigènes O (génétique)</term>
<term>Conformation des glucides</term>
<term>Escherichia coli (métabolisme)</term>
<term>Glycosyltransferase (génétique)</term>
<term>Glycosyltransferase (métabolisme)</term>
<term>Haemophilus influenzae (enzymologie)</term>
<term>Haemophilus influenzae (génétique)</term>
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<term>Séquence glucidique</term>
<term>Test ELISA</term>
<term>Vecteurs génétiques (génétique)</term>
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<keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>O Antigens</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>O Antigens</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Glycosyltransferases</term>
<term>O Antigens</term>
</keywords>
<keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Antigènes O</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Haemophilus influenzae</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Haemophilus influenzae</term>
</keywords>
<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Genetic Vectors</term>
<term>Haemophilus influenzae</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Antigènes O</term>
<term>Glycosyltransferase</term>
<term>Haemophilus influenzae</term>
<term>Vecteurs génétiques</term>
</keywords>
<keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term>
<term>Glycosyltransferases</term>
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<term>Carbohydrate Sequence</term>
<term>Enzyme-Linked Immunosorbent Assay</term>
<term>Substrate Specificity</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>Antigènes O</term>
<term>Conformation des glucides</term>
<term>Spécificité du substrat</term>
<term>Séquence glucidique</term>
<term>Test ELISA</term>
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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>
</record>

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Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in Escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach

Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in Escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach

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Descripteurs français

English descriptors

Abstract

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