Departments of Medical Microbiology and Immunology1 and Chemistry2, University of Alberta, Edmonton, AB, Canada T6G 2H7
Author for correspondence: Diane E. Taylor. Tel: +1 780 492 4777. Fax: +1 780 492 7521. e-mail: diane.taylor{at}ualberta.ca
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ABSTRACT |
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Keywords: Helicobacter pylori, 1,2-fucosyltransferase, Lewis antigens
Abbreviations: FucT, fucosyltransferase; Hp, H. pylori; LacNAc, N-acetyllactosamine; Lea, Lewis a; Leb, Lewis b; LeX, Lewis X; LeY, Lewis Y; TMR, tetramethylrhodamine
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INTRODUCTION |
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In mammalian cells, the synthesis of Lewis antigens is regulated by several glycosyltransferases that add monosaccharides to a precursor molecule in a sequential fashion (Fig. 1b) (for reviews see Avent, 1997
; Herry et al., 1995
; Kleene & Berger, 1993
; Watkins, 1995
). Lewis a (Lea) is synthesized from the type 1 precursor by an
1,3/4-fucosyltransferase (FucT) encoded by fut-3 (Le gene), and the same enzyme is responsible for the synthesis of Lewis b (Leb) from H type 1. At least five different human FucT genes (fut-3, fut-4, fut-5, fut-6 and fut-7) have been identified that encode enzymes involved in the synthesis of LeX and Lea structures (Herry et al., 1995
).
1,2-FucT transfers fucose to the terminal ßGal unit of precursor chains (type 1 or LacNAc) to form H antigens. At least two distinct
1,2-FucTs are present in human tissues. The
1,2-FucT encoded by the H gene (fut-1) is active mainly on erythrocyte membranes, while the
1,2-FucT encoded by the Se gene (fut-2) catalyses the synthesis of H antigen mainly in epithelial cells and in body fluids such as saliva (Avent, 1997
). Classical models assume that difucosylated structures (Leb or LeY; see Fig. 1a
) are synthesized through sequential action of the
1,2- and
1,3/4-FucTs through H determinants (Avent, 1997
; Watkins, 1995
). However, unusual
1,2-FucT activity that synthesizes Leb from Lea or LeY from LeX has been found in some human cancer cells or tissues (Blaszczyk-Thurin et al., 1988
; Yazawa et al., 1993
), and recently such an unusual (the third type)
1,2-FucT was also found in the normal cells of rabbit (Hitoshi et al., 1996
).
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METHODS |
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DNA manipulation techniques.
Standard DNA manipulation techniques, including the isolation, transformation and restriction enzyme digestion analysis of plasmid DNA, as well as partial DNA sequencing, were as detailed by Sambrook et al. (1989) .
Overexpression of the Hp FucT in E. coli.
In a typical experiment, E. coli CLM4(pGP1-2) habouring a plasmid carrying an Hp fucT gene (pBKHp763fucT39, pGEMH2, pGEMI6 or pGEMB3) was grown in 25 ml liquid LB medium with appropriate antibiotics (kanamycin and ampicillin) at 30 °C to an OD600 of 0·50·7. After being collected, the cells were washed once with M9 medium, resuspended in 5 ml supplemented M9 medium, and further incubated at 30 °C for 1 h. To induce the expression of the fucT gene, the culture was shifted to 42 °C by adding 5 ml prewarmed (55 °C) supplemented M9 medium. After incubation at 42 °C for 15 min, rifampicin was added to a final concentration of 200 µg ml-1, and cell growth was continued at 42 °C for 20 min.
For analysis of the protein by SDS-PAGE, a small aliquot (0·5 ml) of the cell culture was taken, and 2·5 µl [35S]methionine (4·35x1013 Bq mmol-1, 3·7x108 Bq ml-1, New England Nuclear) was added. After further growth at 30 °C for 30 min, the cells were harvested, resuspended in 100 µl sample buffer (50 mM Tris/HCl, pH 6·8; 1%, w/v, SDS; 20 mM EDTA; 1%, v/v, mercaptoethanol; 10%, v/v, glycerol), and boiled for 3 min before loading on to the gel. For the preparation of the sample for the enzyme assay, the remaining part (major aliquot, 9·5 ml) of the cell culture after induction was further incubated at 30 °C for 30 min, then harvested. The cells were washed with 1·5 ml 20 mM HEPES (pH 7·0), and resuspended in 1·5 ml of this buffer supplemented with 0·5 mM PMSF.
Preparation of cell lysates or cell extracts for the fucosyltransferase assay.
The E. coli cells containing overproduced Hp FucT proteins, which were in HEPES buffer with PMSF as described above, were disrupted with a French press at 7000 p.s.i. (48 kPa) at 4 °C. The cell lysates were used directly for enzyme assays. For determining the location of the enzyme activities, the cytoplasmic and membrane fractions were separated as follows. The cell lysates were centrifuged at 13000 g at 4 °C for 10 min. The cell debris were discarded and the supernatant was subjected to ultracentrifugation at 128000 g (Beckman TL100/rotor 100.2) at 4 °C for 1 h. The supernatant was collected as the cytoplasmic fraction. The membrane pellets were resuspended in a small volume of the same buffer and treated with 1 M NaCl.
For determining the enzyme activity from H. pylori cells, cells grown for 3 d in 25 ml BHI-YE broth were harvested and washed with 5 ml 20 mM HEPES buffer (pH 7·0). Finally the cells were resuspended in 2 ml of the same HEPES buffer plus 0·5 mM PMSF. The H. pylori cells were disrupted with a French press as described above for E. coli cells, and the cell lysates were directly used for enzyme assays.
Fucosyltransferase assay.
Assays of Hp 1,2- and
1,3-FucT activities were carried out according to the method described by Chan et al. (1995)
with some modifications. Reactions were conducted at 37 °C for 20 min in a volume of 20 µl containing 1·8 mM acceptor, 50 µM GDP-fucose, 60000 d.p.m. GDP-[3H]fucose, 20 mM HEPES buffer (pH 7·0), 20 mM MnCl2, 0·1 M NaCl, 35 mM MgCl2, 1 mM ATP, 5 mg BSA ml-1, and 6·2 µl of the enzyme preparation. The acceptors used in this study were: LacNAc [ßGal1-4ßGlcNAc], LeX [ßGal1-4 (
Fuc1-3)ßGlcNAc], type 1 [ßGal1-3ßGlcNAc] and Lea [ßGal1-3(
Fuc1-4)ßGlcNAc]. These acceptors were provided by Dr O. Hindsgaul, Department of Chemistry, University of Alberta. GDP-[3H]fucose (1·9x1011 Bq ml-1 mmol-1) was obtained from American Radiolabelled Chemicals. Sep-Pak Plus C-18 reverse-phase cartridges were purchased from Waters. For calculation of the specific activity of the enzyme [micro-units (µU) per mg protein], protein concentrations of the cell extracts were determined with a BCA protein assay kit (Pierce) using BSA as a standard according to the suppliers instructions.
Capillary electrophoresis assays.
These assays were performed to identify the products synthesized by the protein preparation of E. coli cells overexpressing UA802 1,2-FucT. For a negative control, the protein preparation of E. coli cells containing pGEM vector was used. The reaction mixture, in a volume of 20 µl, contained 8 µl of the protein preparation, 1·8 mM acceptor labelled with tetramethylrhodamine (TMR), 1·8 mM GDP-fucose, 20 mM HEPES buffer (pH 7·0), 20 mM MnCl2, 0·1 M NaCl, 35 mM MgCl2, 1 mM ATP and 5 mg BSA ml-1. The reaction was carried out at 37 °C for 20 min. The sample was applied to a conditioned Sep-Pak C-18 Cartridge (Palcic et al., 1988
), washed with 20 ml water, and the TMR-labelled oligosaccharides were eluted with 3 ml HPLC-grade methanol. Subsequently, the sample was prepared and analysed by capillary electrophoresis by injecting 12 pl onto a column (60 cm long x 10 µm i.d.) at 1 kV for 5 s as described previously (Chan et al., 1995
). The electrophoretic separations were performed at a running voltage of 400 V cm-1.
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RESULTS |
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In mammalian cells, the same 1,2-FucT enzyme (H or Se, tissue-specific) is normally responsible for the synthesis of both H type 1 and H type 2 structures (Sarnesto et al., 1990
, 1992
). To determine whether the Hp
1,2-FucT is also involved in the synthesis of Leb, we measured its activity with type 1 oligosaccharide acceptors (Table 1
, B). Even though UA802 does not express type 1 Lewis antigen, its
1,2-FucT enzyme can transfer fucose to type 1 and Lea acceptors. Compared to LeX, type 1 and Lea are even more efficient substrates for Hp
1,2-FucT (twofold more active). Thus, Hp
1,2-FucT can also synthesize H type 1 and Leb.
Analysis of the reaction products of Hp 1,2-FucT by capillary electrophoresis
The reaction products synthesized from different acceptors by the Hp 1,2-FucT were further characterized by capillary electrophoresis with laser-induced fluorescence detection. The reaction mixture contained the overproduced UA802
1,2-FucT protein (from the pGEMI6 clone), GDP-fucose, and different acceptors labelled with TMR. The results (Fig. 3
) confirmed the data from the enzyme assay using radioactive labelled GDP-fucose (Table 1
, B) by identifying the products of the reactions.
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Hp 1,2-FucT is a soluble protein
DNA sequence analysis predicted the Hp 1,2-FucT to be a hydrophilic protein (Wang et al., 1999
), and the same is true for Hp
1,3-FucT (Ge et al., 1997
). However, the determination of Hp
1,3-FucT activity from the overexpressed proteins demonstrated that the majority of the activity was present in the membrane fraction (Ge et al., 1997
). To delineate the cellular location of the Hp
1,2-FucT activity, cytoplasmic and membrane fractions of E. coli cells overproducing Hp
1,2-FucT proteins were prepared as described in Methods. The activity in both fractions was determined using LeX or type 1 as acceptors (Table 2
). There was no detectable activity in the membrane fraction when using LeX as an acceptor. By using type 1 as an acceptor, a very low amount of activity (negligible) was detected in the membrane fraction, which accounted for less than 3% of the total activity. These results indicated that Hp
1,2-FucT is a soluble cytoplasmic protein. Compared to the data shown in Table 1
, which were obtained from measurement of immediate cell lysates, the specific activities [µU (mg protein)-1] obtained here are much lower (three- to fourfold). Most probably, many enzyme activities were lost in the procedure for separating cytoplasmic and membrane fractions.
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Lower-level expression of the 1,2-FucT from H. pylori strain 26695
The fucT2 gene in H. pylori 26695 is a variant because it is split into two potential smaller ORFs due to frameshift mutation at the centre of the gene (Fig. 2a, pGEMB3). In vitro expression of this gene has demonstrated that the full-length protein (equivalent to that of prototype UA802
1,2-FucT) can be produced from this gene, most probably by a mechanism of translational frameshifting (the frequency was around 50%) (Wang et al., 1999
). In this study, the plasmid pGEMB3 carrying the 26695 fucT2 gene was transferred into E. coli CLM4(pGP1-2), and the gene was expressed in the same way as described above. In contrast to UA802 fucT2 gene (pGEMI6; Fig. 2
, lane 5), the expression of the 26695 fucT2 gene produces a much lower amount of the full-length protein (Fig. 2
, lane 6). Concomitantly, an additional faint band at 17 kDa, representing the half-length
1,2-FucT, was observed. This suggested that the expression of the gene in vitro may be very different from that in vivo.
In agreement with the low amount of the expressed protein, a low level of enzyme activity was detected for 26695 1,2-FucT (Table 1
, D). Using LacNAc or LeX as acceptor, the activity was undetectable. Using type 1 or Lea as acceptor, a low activity of about 20 µU (mg protein)-1 was detected, which is only about 7% of the activity of UA802
1,2-FucT. Considering that UA802
1,2-FucT has a lower activity on LeX than on type 1 or Lea, it is not surprising that the activity of 26695
1,2-FucT on LeX is too low to be detected.
Detection of 1,2-FucT activity directly from H. pylori cell extracts
After the characterization of the Hp 1,2-FucT protein overproduced in E. coli, we attempted to detect the enzyme activity directly from H. pylori cells, which would be useful for screening high
1,2-FucT-producing strains. Two major difficulties hinder the achievement of this goal: (i) the expression level of the enzyme in natural H. pylori cells is very low; (ii) other fucosyltransferases (mainly
1,3-FucT), which co-exist in H. pylori cell extracts and have much higher activity than
1,2-FucT, interfere with the enzyme assay when using some acceptors such as LacNAc that are not specific for
1,2-FucT. Therefore, we analysed the enzyme activity from different strains and using different acceptors. Finally, we succeeded in detecting very low levels of
1,2-FucT activities from some LeY-producing H. pylori strains (Table 3
).
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DISCUSSION |
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Recently, we have shown that H. pylori mutants carrying a disrupted fucT2 gene (encoding 1,2-FucT) express more LeX than the wild-type cells (Wang et al., 1999
). This phenomenon may suggest that LeX is the direct substrate for LeY synthesis, but the mutagenesis experiment itself cannot exclude the other possible pathway of LeY synthesis (via H type 2), because disruption of
1,2-FucT might lead to accumulation of LacNAc, providing more substrates for
1,3-FucT to synthesize LeX (Fig. 4a
). Therefore, determination of activities of the fucosyltransferases responsible will be direct proof to distinguish between the two possible pathways. The observation in this study that LeX but not LacNAc is the substrate for the Hp
1,2-FucT clearly indicated that H. pylori prefers to use the LeX pathway to synthesize LeY (Fig. 4a
). Other supporting evidence came from the enzyme assay for Hp
1,3-FucT: (i) LacNAc is an excellent substrate for Hp
1,3-FucT (Ge et al., 1997
; Martin et al., 1997
; this study, Table 1
, A); and (ii) Martin et al. (1997)
found that H type 2 was not the substrate of an Hp
1,3-FucT. It should be noted, however, that the fucosyltransferases from different H. pylori strains may have different acceptor specificity. Further studies on combined analysis of the
1,3- and
1,2-FucTs from various H. pylori strains are needed to elucidate whether this novel pathway for the synthesis of LeY is general in H. pylori or is strain-specific.
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Aberrant glycosylation seems to be crucial in human tumour progression (Hakomori, 1989 ). In addition to that of sialyl Lea and sialyl LeX, the role of Leb and LeY as ligands for E-selectin and in adhesion to tumour necrosis factor
-activated endothelial cells has also been demonstrated (Kannagi, 1997
; Miyake & Hakamori, 1991
; Sakamoto et al., 1986
).
1,2-FucT, the key enzyme regulating the biosynthesis of these structures, has become a marker of tumour progression (Sun et al., 1995
). Here, we show that H. pylori
1,2-FucT is functional in the synthesis of both Leb and LeY, and the synthetic pathways (Fig. 4
) are similar to those found in some human cancer cells or tissues (Blaszczyk-Thurin et al., 1988
; Yazawa et al., 1993
) (Fig. 1b
, unusual pathway). We have shown that the expression of the
1,2-FucT-encoding gene in H. pylori is regulated at multiple levels including replication, transcription and translation (Wang et al., 1999
), and the expression of this gene in H. pylori cells is at a very low level (Table 3
). Whether elevated expression of this gene/enzyme in vivo, when H. pylori cells are attached to human gastric epithelial cells, is related to H. pylori-associated development of human gastric cancer is an important issue which needs to be addressed. To our knowledge, H. pylori
1,2-FucT is the first bacterial
1,2-fucosyltransferase that has been characterized. In addition to the biological advantages that H. pylori might gain with altered specificity of its
1,2-FucT compared to the counterpart of its host, the novel substrate specificity is of great potential pharmaceutical interest for enzymic synthesis of oligosaccharides.
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ACKNOWLEDGEMENTS |
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Received 8 April 1999;
revised 12 July 1999;
accepted 22 July 1999.