(Received for publication, March 25, 1997, and in revised form, June 11, 1997)
From the Glycobiology Unit and the ¶ Advanced
Technologies and Informatics Unit, GlaxoWellcome Medicines Research
Centre, Stevenage, Herts SG1 2NY, United Kingdom and the
Department of Medical Chemistry, Vrije Universiteit, Amsterdam,
The Netherlands
The lipopolysaccharide of certain strains of
Helicobacter pylori was recently shown to contain the Lewis
X (Lex) trisaccharide (Gal-1,4-(Fuc
(1,3))-GlcNAc).
Lex is an oncofetal antigen which appears on human gastric
epithelium, and its mimicry by carbohydrate structures on the surface
of H. pylori may play an important part in the interaction
of this pathogen with its host. Potential roles for bacterial
Lex in mucosal adhesion, immune evasion, and autoantibody
induction have been proposed (Moran, A. P., Prendergast, M. M., and Appelmelk, B. J. (1996) FEMS Immunol. Med.
Microbiol. 16, 105-115). In mammals, the final step of
Lex biosynthesis is the
(1,3)-fucosylation of GlcNAc in
a terminal Gal
(1
4)GlcNAc unit, and a corresponding
GDP-fucose:N-acetylglucosaminyl
(1,3) fucosyltransferase
(
(1,3)-Fuc-T) activity was recently discovered in H. pylori extracts. We used part of a human
(1,3)-Fuc-T amino
acid sequence to search an H. pylori genomic data base for related sequences. Using a probe based upon weakly matching data base
sequences, we retrieved clones from a plasmid library of H. pylori DNA. DNA sequence analysis of the library clones revealed a gene which we have named fucT, encoding a protein with
localized homology to the human
(1,3)-Fuc-Ts. We have demonstrated
that fucT encodes an active Fuc-T enzyme by expressing the
gene in Escherichia coli. The recombinant enzyme shows a
strong preference for type 2 (e.g. LacNAc) over type 1 (e.g. lacto-N-biose) acceptors in
vitro. Certain residues in a short segment of the H. pylori protein are completely conserved throughout the
(1,3)-Fuc-T family, defining an
(1,3)-Fuc-T motif which may be of
use in identifying new fucosyltransferase genes.
The Gram-negative bacterium Helicobacter pylori is a major cause of chronic gastritis and peptic and duodenal ulcers (1-5). It has also been implicated in gastric adenocarcinoma (6-9) and gastric lymphoma (10), leading to its classification as a type I human carcinogen (11). H. pylori is a chronic pathogen, and the means by which this organism is able to persist in the stomach and resist or evade destruction by the immune system is central to its involvement in disease. Some aspects of the host-pathogen interaction have been resolved, including the involvement of the Lewis b (Leb)1 epitope on epithelial cells in attachment of H. pylori (12), and characterization of a bacterial cytotoxin responsible for gastric epithelial damage (for a review see Ref. 13), but clearly much remains to be discovered.
Recent structural analysis of H. pylori lipopolysaccharides revealed that the O antigen contains fucosylated carbohydrate structures identical to the mammalian Lewis X (Lex) and Lewis Y (Ley) epitopes (14-17). It was further established that the bacterium contains endogenous galactosyltransferase (Gal-T) and fucosyltransferase (Fuc-T) activities necessary for biosynthesis of these structures (18) suggesting that they are synthesized de novo by H. pylori rather than scavenged from the surface of mammalian cells. Lex is an oncofetal antigen (19, 20) also expressed on adult human gastric mucosa (21), and its presence on H. pylori lipopolysaccharides may play a role in survival and pathogenesis. H. pylori infection is known to induce antibodies that cross-react with human gastric mucosa (22). In a recent report, Appelmelk et al. (23) demonstrated that the targets of this autoimmune response include Lex and/or Ley epitopes and provided evidence that anti-Lex/y antibodies may be involved in H. pylori-associated gastritis. Interestingly, molecular mimicry of Lex is also thought to be responsible for autoantibody production by Schistosoma mansoni (24, 25). In addition, surface carbohydrate antigens containing Lex structures may play a part in the immunopathology of H. pylori infection by promoting Th-1 to Th-2 switching as has been reported in schistosomal infections (26). Two recent reports (27, 28) that over 85% of H. pylori isolates from geographically widespread locations express Lex and/or Ley antigens would also seem to imply selective pressure for maintenance of these structures, given the considerable structural variability often shown by lipopolysaccharides from Gram-negative bacteria.
In mammals, the defining step of Lex biosynthesis is
fucosylation of a type 2 core structure (Gal1
4GlcNAc). This
reaction is catalyzed in humans by one or more members of a family of
(1,3)-fucosyltransferases which employ GDP-fucose as an activated
sugar donor (29-38). Fuc-T and Gal-T activities have been detected in
H. pylori extracts (18), but although the order of sugar
transfer appears to follow the same course as in mammalian systems,
with galactosylation preceding fucosylation, little is known about the
bacterial Fuc-T and how it is related to the mammalian transferases.
If, as evidence is beginning to suggest, cell-surface Lex/y
epitopes play an important role in H. pylori persistence and pathogenesis (23, 39), the
(1,3)-Fuc-T may offer a nonbactericidal therapeutic target for eradication of H. pylori without
otherwise disturbing the balance of gut fauna.
Five members of the human (1,3)-Fuc-T gene family have been cloned
(Fuc-TIII-VII) (29-38). Homologs of some of these genes have also
been cloned from mouse (40-42), rat (43), and cow (44) cDNA. The
remarkable degree of sequence conservation between mammalian
(1,3)-Fuc-Ts and the recently cloned chicken
(1,3)-fucosyltransferase (CFT1) (45) suggests that other
nonmammalian
(1,3)-Fuc-Ts may also show significant homology to the
known members of this enzyme family. We describe here the
identification and cloning of a gene from H. pylori,
fucT, which encodes an active Fuc-T with localized sequence
similarity to the
(1,3)-Fuc-Ts.
GDP-fucose and N-acetyllactosamine
were from Sigma. AG 1-X8 mixed bed resin was from Bio-Rad.
GDP-[3H]fucose (2.22 TBq/mmol) was obtained from
Amersham. Oligosaccharides were obtained from Dextra and Oxford
Glycosystems. Xanthomonas manihotis (1,2)- and
(1,3/4)-fucosidases and Streptomyces plicatus N-acetyl-
-hexosaminidase were from New England Biolabs. Bovine testis
-galactosidase was from Böehringer Mannheim.
Bacteroides fragilis endo-
-galactosidase was supplied by
Oxford Glycosystems. Neutropak NH2 HPLC columns (5 µm,
150 × 4.6 mm) were from Capital Analytical. H. pylori
NCTC 11637 was obtained from the National Collection of Type Cultures
(NCTC). Buffer and media components were obtained from Sigma, Difco,
and Life Technologies, Inc. All chemicals were of the highest available
purity. Chemiluminescent detection film was from Kodak.
A 363-bp
sequence fragment from the H. pylori genomic data base
containing the Fuc-T homology region was amplified from H. pylori NCTC 11637 genomic DNA by PCR using the primers HPFT1
(5-CTT TGA AAA GAG GGT TTG CCA) and HPFT2 (5
-CAA GTA TCT CAC GTA ATC AAT). Amplified product was purified using the QiaQuick PCR
purification system (Qiagen) following the manufacturer's
instructions. Approximately 0.5 µg of the purified fragment was used
to prepare DIG-labeled probe using the DIG Hi-prime labeling kit
(Boehringer Mannheim). Probe was used without further purification.
Nylon membranes carrying plasmid DNA from an H. pylori plasmid library in high density gridded format were kindly supplied by Dr C. L. Clayton (Genomics Unit, GlaxoWellcome). Membranes were prehybridized for 4 h at 42 °C in 20 ml of 50% formamide (v/v), 1% SDS (w/v), 7.5% (w/v) dextran sulfate, 1 M sodium chloride, 1.5 × Denhardt's solution (46), 1.7 mM sodium pyrophosphate, 37.5 mM Tris·HCl, pH 7.5, containing 0.1 mg/ml denatured salmon sperm DNA. Probe was denatured by boiling for 5 min and added to the prehybridization buffer; hybridizations were carried out overnight at 42 °C. Membranes were washed twice for 30 min in 2 × SSC, 0.1% (w/v) SDS, room temperature; 1 × SSC, 0.1% (w/v) SDS, 45 °C; 0.1 × SSC, 0.1% (w/v) SDS, 68 °C. Detection was performed using the Boehringer Mannheim chemiluminescent DIG detection system with CSPD luminescent substrate. Membranes were exposed to film for 5-120 min at room temperature.
Library Clone Retrieval and Sequence AnalysisClones which
hybridized strongly to the probe were retrieved from 384-well library
storage microtiter plates (stored at 80 °C) and grown overnight in
2 ml of L broth containing 100 µg/ml ampicillin. DNA was prepared by
the rapid alkaline lysis method (46). Larger quantities of plasmid DNA
were obtained from 1-200-ml cultures using the Qiagen plasmid (maxi)
system. DNA sequencing reactions performed using AmpliTaq FS with dye
terminators (Perkin-Elmer) and run on an Applied Biosystems ABI 373 automated sequencer. Sequence analysis was performed using GCG 8.1 (47)
and BLAST (48) software. Sequence alignments were created with Pileup (part of the GCG package) with a gap penalty of 5.0 and gap extension penalty of 0.5.
A
1.4-kb DNA fragment containing the fucT gene was amplified
from H. pylori NCTC11637 genomic DNA by PCR using the
primers HPFT3 (5-GAG TGT CTA ATG GGA TCC TTA TTT TTT AAC CCA CCT) and HPFT5 (5
-TAG CCC TAA TCA AGC CTT TG). PCR product was purified using
the QiaQuick PCR purification system (Qiagen), ligated to the A/T
cloning vector pCRTMII (Invitrogen) and introduced into
Escherichia coli XL-1 Blue (Stratagene) by electroporation.
Recombinant (white) clones were mapped with BssHII to
identify plasmids containing the cloned fragment in the desired
orientation (direction of transcription of fucT in the same
direction as lacZ). A 1.4-kb fragment containing the
fucT gene was excised from a suitable clone with
BamHI and ligated to pET-11a vector DNA (Novagen) which had
been linearized with BamHI and dephosphorylated using
shrimp alkaline phosphatase (Amersham, Little Chalfont, UK). Ligated
DNA was introduced into E. coli BL21(DE3), and transformants
were selected on L agar containing ampicillin (100 µg/ml).
Recombinant clones were identified by restriction mapping with
BssHII.
Recombinant clones were grown
overnight at 37 °C from single colony inocula in 5 ml of L broth
containing 100 µg/ml ampicillin. 20 ml of fresh L broth was
inoculated with 200 µl of the overnight culture and incubated at
37 °C. E. coli XL-1 Blue containing library plasmids were
grown for 5-8 h prior to harvesting. E. coli BL21(DE3) containing the expression plasmid pHPFT was incubated until an A600 of 0.4-0.6 was attained.
Isopropyl-1-thio--D-galactopyranoside was added to a
final concentration of 0.5 mM, and incubation continued for
an additional 3 h. Bacteria were harvested by centrifugation (4000 × g, 15 min, 4 °C) and washed in
phosphate-buffered saline. Pelleted bacteria were resuspended in 0.5 ml
of chilled solubilization buffer (0.1% (w/v) Triton X-100, 0.1 M NaCl, 25% (w/v) glycerol, 0.1 M NaCl, 2 mM dithiothreitol, 50 mM Tris-HCl, pH 7.0) and
sonicated on ice for 4 × 15 s bursts (MSE Soniprep 150),
with a 60-s cooling period on ice between bursts. Sonicate was cleared
by centrifugation (20,000 × g, 30 min, 4 °C) and
stored at
80 °C.
Fucosyltransferase activity was
measured by a modification (49) of the method of Prieels et
al. (50). Briefly, 12.5 µl of cell extract was incubated with 20 µM GDP-fucose, 100,000 cpm of
GDP-[3H]fucose, 5 mM acceptor, 5 mM MnCl2, 1 mM ATP, buffered to pH 7.2 with 50 mM HEPES-NaOH in a total volume of 50 µl for
1 h at 37 °C. Sensitivity of the H. pylori enzyme to
the inhibitor N-ethylmaleimide (NEM) was assessed by
including NEM at final concentrations of up to 15 mM in
Fuc-T assay reactions. Reactions were stopped by addition of 1 ml of
mixed bed resin slurry (AG 1-X8 (Cl form) 1:4 (w/v) in
water), vortexed briefly, and centrifuged for 5 min at 20,000 × g at room temperature. Radioactivity in 600 µl of
supernatant was measured by scintillation counting. Allowance was made
for nonspecific breakdown of GDP-fucose and fucose transfer to
endogenous acceptors by performing control reactions in the absence of
acceptor. Assay reactions were performed in duplicate.
Km for the acceptor N-acetyllactosamine (LacNAc) was determined by measuring reaction rates with 0-100 mM LacNAc and 200 µM GDP-fucose, while the
donor Km was obtained using 0-100 µM
GDP-fucose and 5 mM LacNAc.
0.5 ml of H. pylori Fuc-T (approximately 0.2 milliunit) was incubated with 5 mM acceptor (LacNAc or LNT), 3 mM GDP-fucose, 800,000 cpm of GDP-[3H] fucose, 0.1% (w/v) BSA, 2 µl (2 units) of shrimp alkaline phosphatase, 5 mM MnCl2, buffered to pH 7.2 with 50 mM HEPES-NaOH in 1 ml total volume for 40 h at 37 °C. Incubation mixtures were passed through a 2.5-ml Dowex AG 1-X8 ion exchange column, washed through with 7.5 ml of water. Column eluant and washings were pooled, evaporated to dryness, and redissolved in 0.5 ml of water. Samples from the Dowex column were applied to a Bio-Gel P2 gel filtration column (20 × 1 cm) eluted with water at 20 ml/h. 1-ml fractions were collected, and 5-µl aliquots were removed for liquid scintillation counting. Radioactive fractions were pooled and lyophilized, keeping discrete eluant peaks separate. To remove residual Triton X-100, products were dissolved in 500 µl of water and applied to disposable 100-mg Amprep C18 reverse phase columns (Amersham), washed through with 3 ml of water. Pooled eluant and washings were lyophilized and redissolved in 100 µl of water. Finally, fucosylated products were purified by HPLC on a Neutropak NH2 column in water/acetonitrile (25:75 by volume for products derived from LacNAc, 30:70 by volume for those produced from LNT). 0.5-ml fractions were collected, and radioactive fractions were pooled, lyophilized, and redissolved in 100 µl of water.
Glycosidase Treatment of Fucosylated Oligosaccharides20
µl of the purified LacNAc-derived product was incubated
overnight at 37 °C with 3 units (units as defined by the supplier) of (1,3/4)- or
(1,2)-fucosidase from X. manihotis in
30 µl of 50 mM sodium citrate, pH 6.0, containing 100 µg/ml BSA. 20 µl of the LNT-derived product was incubated overnight
at 37 °C with 6.25 milliunits of B. fragilis
endo-
-galactosidase in 50 µl of 50 mM sodium acetate,
pH 5.7, containing 250 µg/ml BSA; 10 milliunits of bovine
testis
-galactosidase in 50 µl of 50 mM sodium
citrate, pH 4.5; or 10 milliunits of bovine testis
-galactosidase and 10 milliunits of S. plicatus
N-acetyl-
-hexosaminidase in 50 µl of 50 mM sodium
citrate, pH 4.5.
Glycosidase reaction products were analyzed by HPLC on the Neutropak NH2 column in water/acetonitrile (25:75 by volume for products derived from LacNAc, 30:70 by volume for those generated from LNT). Elution profiles were generated by collecting 0.5-ml fractions for scintillation counting. Retention times for unlabeled oligosaccharide standards (LacNAc and for products derived from LacNAc using H. pylori Fuc-T, LNT, LacNAc, and Lex for those generated using LNT as acceptor) under corresponding chromatographic conditions were determined by monitoring absorbance at 205 nm.
Human (1,3)-fucosyltransferases (Fuc-TIII-VII) show a
high degree of sequence similarity at the amino acid level. To identify H. pylori sequences with homology to the human
fucosyltransferase enzymes, we performed a
TBLASTN2 search of a
GlaxoWellcome H. pylori genomic data set with part of the
catalytic domain (residues 152-303) of human Fuc-TVI, a strongly
conserved region among the human
(1,3)-Fuc-T family. A number of
H. pylori sequence fragments showed weak similarity to the
query (maximum BLAST score 0.0025), with matches localized to a short
region in each case (17 identities in 30 amino acids). Codon usage
plots indicated that this reading frame was likely to be protein coding
(data not shown). Closer examination of the sequence alignments
revealed that several of the matching residues from the H. pylori sequence are conserved across all five human
(1,3)-Fuc-Ts, suggesting that the data base sequence fragments may
be part of a related H. pylori gene. Since both
(1,3)-Fuc-T and
(1,4)-Gal-T Lex forming
activities have been reported in H. pylori (18), we carried
out a similar search with part of the catalytic domain of human
(1,4-Gal-T), but found no matching sequences.
Using primers derived from one of the matching data base sequences, we
amplified a short (approximately 400 bp) DNA fragment from H. pylori NCTC 11637 genomic DNA which was subsequently labeled with
digoxigenin and used to identify hybridizing clones in a plasmid
library of DNA from the same organism. Seven strongly hybridizing
clones were retrieved from the library for DNA sequence analysis, which
revealed considerable overlap between the cloned sequences in all seven
plasmids. DNA sequencing of all seven clones in both strands yielded a
total of approximately 2.7 kb of contiguous sequence (Fig.
1A). The probe sequence occurs
within the only complete open reading frame in the sequence (designated
fucT), spanning 1002 bp and coding for a predicted 333-amino
acid polypeptide with localized sequence homology to the human
(1,3)-Fuc-Ts. A partial open reading frame occurs approximately 500 bp upstream of the fucT gene, running in the same direction.
The predicted translation of this part of the H. pylori
sequence shows homology to phosphoserine phosphatase (serB)
genes from Gram-negative bacteria, with greatest similarity to the
Haemophilus influenzae sequence (37% identity, 65 matching
residues over 173 amino acids).
Primary Structure of H. pylori fucT
The nucleotide sequence and predicted translation of H. pylori fucT are shown in Fig. 1. The GC content of the gene (36%) is typical for H. pylori coding sequences (3). The predicted amino acid sequence contains no recognizable signal peptide or transmembrane domain, a Kyte-Doolittle plot revealing that hydrophobic regions of the sequence are small and infrequent (Fig. 1B). A repetitive element occupies 49 amino acids of the C-terminal part of the protein. The repeat unit is imperfect, but leucine appears consistently at 7-amino acid intervals in a pattern reminiscent of the eukaryotic leucine zipper motif.
The similarity between the fucT gene product and other
(1,3)-Fuc-T is weak outside the short region originally identified by the data base search, spanning residues 101 to 129 of the H. pylori protein. Within this part of the sequence, however, 10 residues are completely conserved in all five human
(1,3)-Fuc-Ts and
also appear unchanged in bovine, murine, and avian
(1,3)-Fuc-T enzymes (Fig. 2). In addition, there are
a number of partially conserved positions (occupied by one of two amino
acids). Outside this region, similarity to other members of the
(1,3)-Fuc-T family diminishes very quickly, although a number
of isolated conserved residues can be identified. No significant
similarity to any enzyme class other than the
(1,3)-Fuc-T family
could be found for the H. pylori sequence.
Given the reported occurrence of Lex structures on the
H. pylori O antigen and detection by others (18) and
ourselves3 of corresponding
(1,3)-Fuc-T activity in cell extracts from this bacterium, we took
the exclusive, albeit localized, similarity between the deduced amino
acid sequence of H. pylori fucT and
(1,3)-Fuc-T enzymes
as an indication that it may encode an H. pylori
fucosyltransferase enzyme.
We assayed cell lysates
from the clones retrieved from the H. pylori plasmid library
for (1,3)-Fuc-T activity using N-acetyllactosamine (Gal
1
4GlcNAc) as an acceptor. All seven of the library clones tested showed measurable Fuc-T activity, while neither control clones
containing pUC18 nor untransformed E. coli possessed any activity (Table I), demonstrating that
cloned H. pylori sequences contained in the library plasmids
encode an active Fuc-T.
|
The
H. pylori library plasmids contain stretches of flanking
sequence on either side of fucT. To exclude the possibility
that coding sequences outside the identified fucT gene were
responsible for the observed Fuc-T activity, and in an effort to
increase levels of recombinant fucosyltransferase activity, we
subcloned H. pylori fucT into the E. coli
expression vector pET-11a. The resulting plasmid, pHPFT, contains
fucT as the sole H. pylori-derived coding
sequence under control of the T7lac promoter. E. coli BL21(DE3) containing pHPFT produced Fuc-T activity when
induced with isopropyl-1-thio--D-galactopyranoside, extracts typically showing a specific activity of 100-200 pmol/min/mg with N-acetyllactosamine as acceptor. Some activity could
also be detected in uninduced samples (10-20% of induced levels),
presumably as a result of "leaky" promoter repression. Maximal
activity levels produced from pHPFT were not, as we had hoped,
substantially higher than those in the library clones, nor was a highly
expressed protein of the expected molecular mass (approximately 40 kDa)
apparent from SDS-polyacrylamide gel electrophoresis analysis of
uncleared cell extracts (data not shown). The limited Fuc-T activity
produced by pHPFT thus appears to result from limited expression rather than accumulation of highly expressed but insoluble and inactive recombinant protein.
We measured the
activity of recombinant H. pylori FucT with a panel of
oligosaccharide acceptors, as shown in Table
II. The enzyme strongly preferred type 2 (Gal1
4GlcNAc) structures over type 1 (Gal
1
3GlcNAc)
acceptors. The type 2 tetrasaccharide (9) was a better
acceptor than LacNAc (1) suggesting that H. pylori FucT may prefer to fucosylate
-configured GlcNAc.
Similar preferences have been reported for human Fuc-TIV and to a
lesser extent for Fuc-TV with these two acceptors (51). With sialylated LacNAc acceptors the H. pylori FucT most closely resembled
human Fuc-Ts V and VII in that 3
-sialyl-LacNAc (6) was a
substrate, while 6
-sialyl-LacNAc (7) was not (51, 52). No
activity was observed with the type 1 disaccharide
lacto-N-biose (2), although
lacto-N-tetraose (LNT) (10) was an efficient
acceptor. Fucosylation of the terminal galactose of LNT in the
2-position (11) or GlcNAc in the 4-position (12)
significantly reduced incorporation rates, while fucosylation on both
GlcNAc and glucose (15) abolished fucose incorporation
altogether. This suggests that H. pylori FucT may may be
capable of fucosylating predominantly the glucose residue of LNT-based
acceptors, as has been demonstrated for Fuc-TV (51). 6
-Sialylation of
GlcNAc also blocked fucosylation of type 2 structures with the
recombinant H. pylori enzyme (18) whereas
3
-sialylation of the terminal galactose residue (17) caused
only a minor reduction in relative activity. Unlike Fuc-TV (but like
Fuc-TVI and Fuc-TVII), however, the H. pylori Fuc-T showed
no activity toward 2
-fucosyllactose (5), implying that in
the synthesis of Ley by H. pylori
2-fucosylation of Gal may occur after
3-fucosylation of GlcNAc. Taken together, these results suggest that the
Helicobacter enzyme has little
(1,2)- or
(1,4)-Fuc-T
activity, but efficiently
(1,3)-fucosylates neutral and
(2,3)-sialylated type 2 acceptors. To further define the catalytic
properties of H. pylori Fuc-T, Km values
were determined for LacNAc (0.5 mM) and GDP-fucose (9 µM). Sensitivity to the inhibitor NEM was measured by
performing assays in the presence of NEM at concentrations up to 15 mM. The enzyme showed very limited NEM sensitivity, with
Fuc-T activity reduced by only 34% in the presence of 15 mM NEM. For comparison, FucT-III (expressed in COS cells)
is inhibited approximately 85% at the same NEM concentration, while
extracts from Schistosoma mansoni and COS cells expressing
Fuc-TIV retain about 50% of their Fuc-T activity (53).
|
Some of the inferences drawn from the acceptor preferences of H. pylori Fuc-T were investigated further by examining the sensitivity of fucosylated products to glycosidase treatment. Radiolabeled oligosaccharide products were generated by incubating the acceptors LacNAc and LNT with H. pylori Fuc-T in the presence of GDP-[3H]fucose. Following removal of excess sugar nucleotide, free fucose and residual Triton X-100 from the reaction mixture by successive ion exchange, size exclusion, and reverse phase chromatographic steps, oligosaccharide products were purified by HPLC on a Neutropak NH2 column.
Incubation of LacNAc with H. pylori Fuc-T yielded a single
radiolabeled product with a retention time on HPLC corresponding to
that of Lex (Fig.
3A). The product was
unaffected by treatment with a selective (1,2)-fucosidase, while
treatment with
(1,3/4)-fucosidase resulted in complete conversion to
a new product which eluted from the Neutropak NH2 column
more rapidly than LacNAc. These observations support the conclusion
that the major fucosylated product generated by H. pylori
Fuc-T with LacNAc as acceptor is the Lex trisaccharide.
Accordingly, the product is resistant to
(1,2)-fucosidase, but
sensitive to
(1,3/4)-fucosidase, which liberates fucose as the sole
radiolabeled product.
To investigate the fucosylation of the type 1 acceptor LNT
(10) by H. pylori Fuc-T, labeled product was
treated with endo--galactosidase. The action of this glycosidase is
inhibited by fucosylation of residues flanking the
-galactoside
linkage (54, 55) and thus if, as substrate preferences seem to suggest, H. pylori Fuc-T fucosylates the glucose residue
of LNT, the product should be resistant to endo-
-galactosidase
cleavage. Since endo-
-galactosidase activity may also be hampered by
fucosylation at more distant sites (for example, on GlcNAc or the
distal Gal residue of LNT) (55), the product was also treated with
bovine testis
-galactosidase, alone or in combination with
-N-acetylhexosaminidase, to examine this possibility.
Analysis of the labeled oligosaccharides produced by glycosidase
digestion of H. pylori Fuc-T-generated fucosyl-LNT is shown
in Fig. 3B. As expected, endo-
-galactosidase had no effect on the fucosylated oligosaccharide, while LNT itself was readily
cleaved under similar conditions (data not shown). Digestion with
bovine testis exo-
-galactosidase resulted in substantial (>50%)
conversion to a product with a retention time close to that of LNT.
This indicates that the distal Gal residue of the major product is not
fucosylated, as this would otherwise block exo-
-galactosidase
action. Combined treatment with
-galactosidase and
N-acetyl-
-hexosaminidase yielded a more rapidly eluting
product, consistent with removal of distal Gal and GlcNAc residues to
leave a fucose-containing trisaccharide. Further
-galactosidase
action is presumably blocked by fucose branching at the Glc residue of the remaining galactoside. These findings suggest that H. pylori Fuc-T fucosylates LNT predominantly at the glucose residue.
Resistance of part of the product material to bovine testis
-galactosidase may reflect some degree of
(1,2)- or
(1,4)-fucosylation, although the substrate preferences of this Fuc-T
indicate that incomplete galactosidase digestion is perhaps a more
likely explanation.
By searching an H. pylori genomic data set with part of
the catalytic domain sequence of a human (1,3)-Fuc-T and sequencing corresponding clones from a plasmid library of H. pylori DNA
we were able to identify a gene (fucT) with highly localized
similarity to known
(1,3)-Fuc-T enzymes. Cell extracts from library
clones containing the H. pylori gene possessed Fuc-T
activity, and by subcloning fucT into an E. coli
expression vector we were able to confirm that it encodes an active
(1,3)-Fuc-T. H. pylori fucT is the first Fuc-T gene to be
cloned from an invertebrate, although enzyme activity has been detected
in the freshwater snail Lymnea stagnalis (56) and in the
parasite S. mansoni (53).
Sequence similarity between the mammalian Fuc-Ts and chick
fucosyltransferase (CFT1) provided evidence for evolutionary
conservation of (1,3)-Fuc-T sequences (45). Conservation between
H. pylori FucT and the mammalian enzymes, although limited
and highly localized, suggests that aspects of the
(1,3)-Fuc-T
sequence have survived unchanged through evolution from bacteria to
higher mammals and man. The lack of overall sequence
similarity to human
(1,3)-Fuc-Ts would seem to preclude the idea
that H. pylori acquired the Fuc-T gene from a mammalian
source. The base composition of the gene (35% GC) is also much closer
to the average for H. pylori (36%) than to mammalian and
avian
(1,3)-Fuc-T genes, which are typically GC-rich (e.g.
CFT-1, 69% GC).
Unlike eukaryotic Fuc-Ts which have a hydrophobic transmembrane domain near their N terminus and share a common type II membrane protein topology, the H. pylori enzyme contains no recognizable membrane insertion elements. Aligned on the basis of the short, highly conserved region of homology (Fig. 2), the bacterial enzyme appears to lack a region corresponding to the transmembrane and stem domains of other Fuc-Ts. Most of the "hypervariable region" previously implicated in acceptor binding specificity in human Fuc-TIII and -V (residues 34 to 161 in Fuc-TIII) (57) is also absent, suggesting that the architecture of the H. pylori protein is substantially different from the rest of the enzyme family. The alignment also reveals that the C terminus of the bacterial sequence extends for approximately 100 amino acids beyond that of the other Fuc-Ts, half of this C-terminal extension being taken up by a periodic 7-amino acid leucine zipper-like motif. The function of this region, which has no counterpart in mammalian or avian Fuc-T sequences, is unknown. One possibility is that it mediates homo- or heteromultimer formation through coiled-coil type interactions, but at present the subunit structure of the H. pylori protein is unknown and further work will be necessary to establish the role of the zipper-like region.
Recombinant H. pylori Fuc-T has a strong preference for type
2 acceptors, and analysis of oligosaccharides generated by fucosidase digestion of the product generated by this Fuc-T with LacNAc indicates that H. pylori Fuc-T is indeed capable of synthesizing the
Lex epitope. Some type 1 structures are also fucosylated,
but our studies suggest that with these acceptors fucose may be
transferred predominantly to glucose rather than GlcNAc, implying that
the enzyme has little (1,2)- or
(1,4)-Fuc-T activity, as has been reported for human Fuc-TV (51). Biosynthesis of the Ley
epitope found on the surface of many H. pylori isolates is
therefore likely to involve a separate
(1,2)-Fuc-T activity.
Overall, the acceptor specificity of H. pylori Fuc-T does
not match that reported for any of the human enzymes or indeed that of
S. mansoni
(1,3)-Fuc-T (53). Like the schistosome enzyme
and human Fuc-Ts IV and VII, however, H. pylori Fuc-T shows
only slight sensitivity to NEM inhibition. Interestingly,
3
-sialyl-LacNAc is an efficient acceptor (although 6
-sialyl-LacNAc is
not), implying that H. pylori Fuc-T may be capable of
synthesizing the sialyl-Lex (sLex) structure
which was recently detected in a small number of H. pylori
isolates by Wirth et al. (27). The absence of
sLex from the majority of H. pylori isolates may
therefore reflect a lack of sialyltransferase activity in these
strains.
Mammalian (1,3)-Fuc-Ts are a closely-related family of enzymes,
making it difficult to identify residues of potential structural and/or
catalytic importance from sequence alignments. The recently cloned
avian
(1,3)-Fuc-T, CFT-1 (45), also shows a high level of
sequence similarity to the corresponding mammalian proteins, with
46.3% sequence identity to human Fuc-TIV. This is not the case with
the H. pylori enzyme, which shows significant homology to
the other
(1,3)-Fuc-Ts only in one short region. A consensus motif
derived from this local area of homology (Fig. 2B) is unique to members of the
(1,3)-Fuc-T family, including the H. pylori enzyme and an open reading frame from a
Caenorhabditis elegans cosmid4 (58). This highly
conserved
(1,3)-Fuc-T motif may be useful in identifying novel
(1,3)-Fuc-T genes in genomic and expressed sequence tag sequence
data, since its appearance seems to be a reliable predictor of
membership of this enzyme family. It may also provide a tool for
cloning
(1,3)-Fuc-Ts in a manner similar to the demonstrated utility
of the L- and S-sialyl motifs in cloning novel
sialyltransferases (59).
The functional significance of the (1,3)-Fuc-T motif is at present
unclear. Marked differences in acceptor preferences among members of
the
(1,3)-Fuc-T family would seem to argue against a role in
acceptor binding. Human Fuc-TIV and -VII for example both contain the
(1,3)-Fuc-T motif, but while Fuc-TVII uses 2,3-sialylated acceptors
almost exclusively, Fuc-TIV strongly prefers neutral type 2 substrates
(41) in in vitro assays. The behavior of Fuc-TIV in
vivo is apparently more complex (60). The
(1,3)-Fuc-T motif lies outside sequence regions implicated by efforts to define acceptor-discriminating residues in
(1,3)-Fuc-Ts (51, 57, 61). Given
that the enzymes transfer fucose from a common sugar nucleotide donor,
it seems more likely that the
(1,3)-Fuc-T motif is involved in
binding GDP-fucose or Mn2+. The motif lies some
considerable distance from a cysteine residue implicated in GDP-fucose
protectable inhibition by NEM (62), although it may be much closer in
space within the folded protein than the primary sequence suggests. The
corresponding position in the H. pylori Fuc-T is occupied by
tyrosine (Fig. 2A), in keeping with observations that
enzymes with Cys at this location are inhibited by NEM while those with
other amino acids (Fuc-TIV has Ser, Fuc-TVII has Thr) are resistant to
NEM inhibition (62).5
Interestingly, the conserved motif contains a lysine residue (Fig.
2A), possibly a candidate for the so far unidentified
GDP-fucose-protected lysine residue identified by pyridoxal phosphate
labeling of a human fucosyltransferase (63). Further work is clearly
needed to test these speculations, but in this respect the lack of
overall similarity between the H. pylori and mammalian
transferase sequences may be advantageous. The relatively small number
of conserved residues inside and outside the
(1,3)-Fuc-T motif may
provide a useful focus for mutagenesis experiments to probe structural and mechanistic aspects of the
(1,3)-fucosyltransferases. The dissimilarity of H. pylori and human Fuc-T enzymes would
also seem to auger well for the design of selective inhibitors of the bacterial enzyme.
The H. pylori enzyme, which lacks a transmembrane domain and is, presumably, nonglycosylated, is devoid of some of the features which make eukaryotic Fuc-Ts difficult to work with. The possibility of bacterial expression also makes this enzyme a promising candidate for chemoenzymatic glycoconjugate synthesis. The same features may simplify the task of structural determination. Given the conserved motif, it seems reasonable to assume that this enzyme shares at least some structural features in common with its mammalian counterparts which have so far resisted structural elucidation.
Mounting evidence appears to point to a role for Lewis antigen mimicry in H. pylori pathogenesis. Identification and cloning of a Fuc-T gene from this organism will allow us to probe the biosynthesis of Lex by H. pylori in vivo via disruption of fucT and may make it possible to test the role of Lex directly in models of H. pylori pathogenesis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF006039.
We thank Dr. Chris Clayton for providing the H. pylori plasmid library and Valerie Kelly and Dr. Christopher Britten for assistance with fucosyltransferase assays. We would also like to thank Dr. Hans Mulder and Dr. William McDowell for helpful discussions.