Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 366, Asahi-machi, Machida-shi, Tokyo 1948533, Japan
Received on January 5, 2000; revised on March 1, 2000; accepted on March 3, 2000.
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Abstract |
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Key words: enzymatic synthesis/glycosyltransferase/oligosaccharide/ Helicobacter pylori
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Introduction |
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Here we describe the cloning and expression in E.coli of a novel ß1,4-galactosyltransferase gene from H.pylori. The ß1,4-galactosyltransferase used GlcNAc, GlcNAcß13Galß14Glc and GlcNAcß13Galß14GlcNAcß13Galß14Glc as acceptors. When E.coli cells that 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 (Koizumi et al., 1998; Endo et al., 1999
), LacNAc was efficiently produced from orotic acid, galactose, and GlcNAc.
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Results |
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Acceptor specificity of the HpGal-T
Since N-acetyllactosamine structure is the core structure of the LPS O-antigen in H.pylori, the natural acceptor for HpGal-T was predicted to be GlcNAc. As acceptor substrates of HpGal-T, GlcNAcß13Galß14Glc and GlcNAcß13Galß14GlcNAc-ß13Galß14Glc as well as GlcNAc could be utilized (Table I). When glucose was used as an acceptor, the very low activity of HpGal-T was detected (Table I). Other substrates such as lactose and lacto-N-neotetraose (Galß14GlcNAcß13Galß14Glc) were not utilized. These results suggested that the oligosaccharides containing GlcNAc at the non-reducing end could be acceptable for HpGal-T. When HpGal-T activity was measured using UDP-GlcNAc or UDP-GalNAc as the donors and GlcNAc or lactose as the acceptors, no products were formed. HpGal-T activity was not influenced by high concentrations of GlcNAc (up to 200 mM). Addition of -lactalbumin (0, 0.05, 0.2, 1, 5, 10 mg/ml) had no influence on LacNAc formation with GlcNAc (1 mM and 50 mM) and lactose formation with glucose (1 mM and 10 mM). These results indicated that HpGal-T is insensitive to
-lactalbumin.
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Discussion |
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Recently the ß 1,4-galactosyltransferase from bovine was crystallized and the structure of the catalytic domain were solved (Gastinel et al., 1999). HpGal-T catalyzes the same reaction as bovine ß1,4-galactosyltransferase, therefore comparison of the three-dimensional structure of the two enzymes should bring a deep insight about the reaction mechanism.
HpGal-T could be applied to a large-scale production of oligosaccharide containing galactose such as LacNAc, which had been established for the production of globotriose (Koizumi et al., 1998) and LacNAc (Endo et al., 1999
). HpGal-T was showed to utilize GlcNAc, GlcNAcß13Galß14Glc, and GlcNAcß13Galß14GlcNAcß13Galß14Glc as acceptors. On the other hand, glucose was not a good acceptor for HpGal-T in accordance with the observation that the H.pylori LPS lacked of lactose structure (Aspinal et al., 1994
). These results suggested that, by combining HpGal-T with UDP-Gal production system, various oligosaccharides, such as lacto-N-neotetraose and para lacto-N-neohexaose, could be also efficiently produced.
In conclusion, we have cloned the ß 1,4-galactosyltransferase gene from H.pylori. The availability of HpgalT was confirmed through the efficient production of LacNAc by the coupling with the UDP-Gal production system.
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Materials and methods |
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Cloning of the H.pylori galactosyltransferase gene (HpgalT)
The genomic library was prepared by introducing 35 kb fragments from a Sau3AI partial digest of the chromosomal DNA of H.pylori (NCTC 11637) into pUC118 as a vector. The library was plated on LB medium containing ampicillin, IPTG and X-gal and grown for 12 h at 37°C. Each 10 white colonies were picked up and incubated in an 800-µl of LB medium with ampicillin and were grown for 12 h at 37°C. The cells were collected by centrifugation and measured for the galactosyltransferase activity as described below. The positive pools were plated, and then colonies were cultivated and analyzed again for the activity. The DNA sequence was determined by the dideoxy sequencing using the 373A DNA sequencer (Applied Biosystems, Foster City, CA). Hydropathy was calculated by the method of Kyte and Doolittle (Kyte and Doolittle, 1982). DNA manipulations were performed according to the standard methods (Sambrook et al., 1989
).
Plasmid construction and expression of the HpgalT gene in E.coli
The plasmid pPAC31, which contains the replication origin and ampicillin resistance gene from pBR322, PL promoter, and temperature-sensitive cI857 repressor from phage lambda, was used for the construction of the expression plasmids (Koizumi et al., 1998). The HpgalT was amplified by PCR using the primers: 5' primer, 5'-AACATCGATGGGAGTCTAACCTATGCGTGTTTTTATC-3' (ClaI site shown in bold italics), and 3' primer, 5'-AGCGGATCCTAAAAAGTCTTAGT-3' (BamHI site shown in bold italics). Conditions for PCR cycling included denaturation at 94°C for 1 min, annealing at 37°C for 2 min and extension at 72°C for 3 min (30 cycles). The 0.9 kb PCR product was digested with ClaI and BamHI and cloned into the ClaIBamHI sites in pPAC31 to form pPT6.
Measurement of galactosyltransferase activity
The galactosyltransferase activity from E.coli genomic library was measured with Nymeen-treated cells prepared as described previously (Endo et al., 1999). Reaction was conducted at 37°C for 12 h in 100 µl of 50 mM citrate acid buffer (pH 7.0), 5 mM MnCl2, 0.2 mM FCHASE-labeled GlcNAcß1,3Galß1,4Glc, 0.2 mM UDP-Gal, and Nymeen-treated cells. The reaction mixtures were analyzed by thin layer chromatography on silica-60 TLC plates (Merck, Darmstadt, Germany) (Wakarchuk et al., 1996
).
Substrate specificity of galactosyltransferase
Determination of acceptor specificity was performed with crude extract of E.coli harboring pPT6. The reactions were carried out at 37°C in 100 µl of 50 mM citrate acid buffer (pH 7.0), 5 mM MnCl2, 1.0 mM acceptor, 1.0 mM UDP-Gal, and various amount of enzyme. The reactions were terminated by boiling for 2 min, and analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Dionex DX-500 system (Lee, 1990). One unit of the enzyme activity is defined as an amount of enzyme that catalyzes the formation of 1 µmol of product per min.
LacNAc production
The production of LacNAc was carried out in a 200-mL vessel containing 30 ml of the reaction mixture, i.e., 150 g/l (wet weight) of C.ammoniagenes DN510 cells, 50 g/l (wet weight) of NM522/pNT25/pNT32 cells, 50 g/l (wet weight) of NM522/pPT6 cells, 50 g/l of fructose, 50 g/l of galactose, 50 g/l of GlcNAc, 25 g/l of KH2PO4, 5 g/l of MgSO4-7H2O, 10 g/l of orotic acid (potassium salt), 4 g/l of Nymeen S-215 (Nippon Oil and Fats, Tokyo, Japan), and 10 ml/l of xylene. The reaction was carried out at 32°C with agitation (900 rpm), and the pH was kept at 7.2 with 4 N NaOH.
Analyses
UDP-Gal was measured by HPLC (Langnas and Diez-Masa, 1994). LacNAc and other saccharides were analyzed by means of HPAEC-PAD using a Dionex DX-500 system equipped with a Carbopac PA10 column (Dionex, Sunnyvale, CA) (Lee, 1990
). Nucleotides were measured according to the method described before (Fujio and Maruyama, 1997
). Inorganic phosphate was determined with Determiner IP-S Kit (Kyowa Medex, Tokyo, Japan).
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Acknowledgments |
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Footnotes |
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2 Present address: Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 11 Kyowa-cho, Hofu-shi, Yamaguchi 7478522, Japan
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