Glycobiology, 2000, Vol. 10, No. 8 809-813
© 2000 Oxford University Press
Cloning and expression of ß1,4-galactosyltransferase gene from Helicobacter pylori
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3–6–6, Asahi-machi, Machida-shi, Tokyo 194–8533, 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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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 ß1,4-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 ß1,4-galactosyltransferases from mammalian sources and Neisseria. It was confirmed that HpGal-T catalyzed the introduction of galactose from UDP-Gal in a ß1,4 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.
Key words: enzymatic synthesis/glycosyltransferase/oligosaccharide/ Helicobacter pylori
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Helicobacter pylori is a spiral, microaerophilic Gram-negative bacterium which is involved in the pathogenesis of gastritis, gastric glandular atrophy, and both peptic and duodenal ulcers (Graham, 1991
; Peterson, 1991; Warren and Marshall, 1983). It is reported that O-antigen regions of the lipopolysaccharide (LPS) of H.pylori commonly expresses human oncofetal antigens Lewis X [Galß1,4(Fuc
1,3)GlcNAcß] and Lewis Y [(Fuc1,2) Galß1,4(Fuc1,3)GlcNAcß] (Aspinal et al., 1994; Chan et al., 1995; Sherburne and Taylor, 1995; Applemelk et al., 1996). Although the precise role of these oligosaccharide structures is not known, it is presumed to be a form of molecular mimicry that aids in the evasion of the host immune response (Applemelk et al., 1996; Wirth et al., 1997). Lewis X structure in H.pylori is synthesized by the addition of galactose to GlcNAc and then fucose to N-acetyllactosamine (LacNAc), catalyzed by the ß1,4-galactosyltransferase and the 1,3-fucosyltransferase, respectively (Chan et al., 1995; Wang et al., 1999). Although mammalian glycosyltransferases that synthesize Lewis X structure are well characterized (Schachter, 1991; Kleene and Berger, 1993), little is known about corresponding bacterial glycosyltransferases. Recently two genes encoding the 1,3-fucosyltransferase of H.pylori were cloned and characterized (Ge et al., 1997; Martin et al., 1997). However, the gene encoding the ß1,4-galactosyltransferase of H.pylori has not been cloned, although the whole genome sequences have been determined (Tomb et al., 1997). On the other hand, the gene encoding the ß1,4-galactosyltransferase was cloned and characterized from bacteria belonging to Neisseria (Gotschlich, 1998).
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, GlcNAcß1–3Galß1–4Glc and GlcNAcß1–3Galß1–4GlcNAcß1–3Galß1–4Glc 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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Cloning and sequencing of a H.pylori galactosyltransferase gene
To clone the gene coding for the ß 1,4-galactosyltransferase of H.pylori, the galactosyltransferase activity was measured using gene products of the library of H.pylori (NCTC11637). One E.coli strain harboring a plasmid (pPT1) containing the intact HpgalT was obtained. Nucleotide sequences of 2.0 kb in pPT1 were determined in both strands (Figure 1, DDBJ/EMBL/GenBank accession no. AB035971). An open reading frame (ORF) of 822 bp, starting at nucleotide 216 and ending at nucleotide 1037, which could code 274 amino acid residues with the molecular mass of 31,731 Da, was found in this region. The deduced amino acid sequence of this ORF with known sequences in GenBank using the BLAST search showed significant similarity (96% identity) to LPS 5G8 epitope biosynthesis-associated protein (Lex2B, HP0826) of H.pylori, indicating HP0826 was the structural gene for ß 1,4-galactosyltransferase. The initiation codon of HpgalT was decided TTG in accordance with HP0826 annotated by means of GenMark and GenSmith that is generally used in the genome analysis (Tomb et al., 1997
). In addition the sequence showed similarity to HP0805 of H.pylori (43%) and Lex2B of Haemophilus influenzae (31%), both of which were involved in the biosynthesis of LPS. However, the sequence of HpGal-T did not show significant homology to the ß1,4-galactosyltransferase (LgtB) of Neisseria gonorrhoeae (15%) (Gotschlich, 1998; Figure 2). The mammalian ß1,4-galactosyltransferase, such as ß4Gal-T1, did not show significant homology either (17%) (Schwientek et al., 1998). Analysis of the sequence of the downstream region of HpGal-T revealed two open reading frames, which showed homology to the bacterial DNA binding protein (HP0827) and ATP synthase (HP0828). A hydropathy plot of the HpGal-T indicated that the sequence did not contain a potential hydrophobic transmembrane segment (data not shown).
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Expression of HpgalT
To characterize the HpGal-T more precisely, HpgalT was overexpressed by inserting into the expression vector pPAC31 to form pPT6. HpgalT in pPT6 was controlled by PL promoter. pPT6 gave rise to a specific product of 31kDa which was close to the predicted molecular mass of HpGal-T (data not shown). The product was not produced in cells cultivating at 28°C, and was induced after cultivating at 40°C for 3 h. In addition, the protein was not produced in cells containing pPAC31 without the insert (data not shown). These results suggested that the product was HpGal-T overexpressed in E.coli.
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, GlcNAcß1–3Galß1–4Glc and GlcNAcß1–3Galß1–4GlcNAc-ß1–3Galß1–4Glc 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 (Galß1–4GlcNAcß1–3Galß1–4Glc) 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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Production of LacNAc
E.coli cells that overexpressed HpgalT were coupled with the UDP-Gal production system, which consisted of recombinant E.coli cells overexpressing the UDP-Gal biosynthetic genes, galT, galK, galU, and ppa, and C.ammoniagenes which contributes the production of UTP from orotic acid (Figure 3). The reaction was carried out on a 30 ml scale in a 200 ml beaker. GlcNAc, galactose (Gal), and orotic acid were also added to the reaction mixture, as well as polyoxyethylene octadecylamine (Nymeen S-215) and xylene to permeabilize the cells. Fructose was added as an energy source. After 20 h, 157 mM (60 g/l) of LacNAc was produced from 278 mM Gal and 226 mM GlcNAc (Figure 4). Almost no other peaks than fructose, GlcNAc, and LacNAc were observed after 20 h as analyzed by HPAEC-PAD (data not shown). These results indicated that the coupling of UDP-Gal production system and recombinant E.coli which overexpressed ß1,4-galactosyltransferase gene from H.pylori worked well. After the reaction, LacNAc was isolated through several steps of purification including activated charcoal chromatography from the reaction mixture. In order to obtain a pure sample for instrumental analyses, the compound was further purified by gel-filtration and freeze-dried. The structure of the purified compound was identified as LacNAc by means of 1H NMR and 13C NMR spectroscopy (data not shown).
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Discussion |
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In this study, we have cloned and sequenced a novel ß 1,4-galactosyltransferase gene from H.pylori (HpgalT). HpgalT was located upstream of two genes of a DNA binding protein(HP0827) and ATP synthase(HP0828) unrelated to LPS synthesis, apart from the
1,3-fucosyltransferase genes (HP0379, HP0651; Tomb et al., 1997). It was indicated that the genes involved in the biosynthesis of LPS in H.pylori were scattered on the chromosome unlike those of Neisseria (Gotschlich, 1998). Sequence analysis and homology search revealed that HpgalT showed little homology to ß1,4-galactosyltransferases from mammalian source and Neisseria (Figure 2). However, the sequence was similar to the lex2B of H.influenzae (Gregory and Eric, 1994) and HP0805 of H.pylori (Tomb et al., 1997), both of which were involved in LPS 5G8 epitope biosynthesis. Considering the LPS O-antigen structures of H.pylori, HP0805 might be a glycosyltransferase, such as ß1,3-N-acetylglucosaminyltransferase, although any glycosyltransferase activities of HP0805 have not been detected using oligosaccharide as acceptors. HP805 might have rigid acceptor specificity and require oligosaccharide linked to a hydrophobic group, such as the case of the gene product of cps14I of Streptococcus pneumoniae (Kolkman et al., 1997).
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, GlcNAcß1–3Galß1–4Glc, and GlcNAcß1–3Galß1–4GlcNAcß1–3Galß1–4Glc 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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Materials
H.pylori NCTC 11637 was a kind gift from Dr. Takeshi Ito (Tokyo Metropolitan Research Laboratory of Public Health, Japan). E.coli strain NM522 was purchased from Stratagene (La Jolla, CA). LB medium (Sambrook et al., 1989
) was used for growth of E.coli, and ampicillin (100 µg/ml), isopropyl-1-thio-ß-D-galactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) were added if necessary. E.coli cells were cultivated in a 5 l jar fermenter according to the method described previously (Koizumi et al., 1998). C.ammoniagenes DN510 cells were cultivated in a 5 l jar fermenter according to the method described before (Fujio and Maruyama, 1997). Cells were collected by centrifugation and stored at –20°C until used. Plasmid vector pUC118 that was treated with BamHI and alkaline phosphatase was purchased from Takara Shuzo (Kyoto, Japan). Aminophenyllactoside and para lacto-N-neohexaose were purchased from Sigma-Aldrich (St. Louis, MO), LacNAc and lacto-N-neotetraose were purchased from Oxford GlycoSystems (Rosedale, NY) and 6 (5-fluorescein-carboxamide)-hexsanoic acid succimidylester (FCHASE) was purchased from Funakoshi (Tokyo, Japan). FCHASE-amminophenyl- GlcNAcß1–3Galß1–4Glc was synthesized from FCHASE-amminophenyl- Galß1–4Glc and UDP-GlcNAc, with the activity of recombinant E.coli overexpressing lgtA gene from N.gonorrhoeae (Wakarchuk et al., 1996; Gotschlich, 1998). BioGel P-2 was purchased from Bio-Rad Laboratories (Hercules, CA). Orotic acid was the product of Kyowa Hakko Kogyo (Tokyo, Japan). All other chemicals used were commercially available and of analytical grade.
Cloning of the H.pylori galactosyltransferase gene (HpgalT)
The genomic library was prepared by introducing 3–5 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 ClaI–BamHI 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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We thank Natsumi Kudo for technical assistance.
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1 To whom correspondence should be addressed
2 Present address: Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1–1 Kyowa-cho, Hofu-shi, Yamaguchi 747–8522, Japan
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