Cloning and expression of beta1,4-galactosyltransferase gene from Helicobacter pylori.
Helicobacter pylori, which is a human pathogen associated with gastric and duodenal ulcer, has been shown to express human oncofetal antigens Lewis X and Lewis Y. Although the mammalian glycosyltransferases that synthesize these structures are well characterized, little is known about the corresponding bacterial enzymes. We report that a novel beta1,4-galactosyltransferase gene (HpgalT) involved in the biosynthesis of lipopolysaccharides in H. pylori has been cloned and expressed in Escherichia coli. The deduced amino acid sequence of the protein (HpGal-T) encoded by HpgalT consists of 274 residues with the calculated molecular mass of 31,731 Da, which does not show significant similarity to those of beta1,4-galactosyltransferases from mammalian sources and Neisseria It was confirmed that HpGal-T catalyzed the introduction of galactose from UDP-Gal in a beta1,4 linkage to accepting N-acetylglucosamine (GlcNAc) residues by means of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). When the E.coli cells which overexpressed HpgalT was coupled with the UDP-Gal production system, which consisted of recombinant E.coli cells overexpressing its UDP-Gal biosynthetic genes and Corynebacterium ammoniagenes, N-acetyllactosamine, a core structure of lipopolysaccharide of H.pylori, was efficiently produced from orotic acid, galactose, and GlcNAc.
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194-8533, Japan.
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Ozaki
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D005700:Galactosyltransferases