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J Biol Chem, Vol. 275, Issue 7, 4988-4994, February 18, 2000
From the The gastric pathogen
Helicobacter pylori can express the histo blood group
antigens, which are on the surface of many human cells. Most H. pylori strains express the type II carbohydrates, Lewis X and Y,
whereas a small population express the type I carbohydrates, Lewis A
and B. The expression of Lewis A and Lewis X, as in the case of
H. pylori strain UA948, requires the addition of fucose in
Lipopolysaccharide
(LPS)1 is an essential
structural and functional component of the Gram-negative bacterial cell
envelope. LPS is composed of lipid A, oligosaccharide core, and the
antigenic O-polysaccharide chain. Helicobacter pylori
express a range of Lewis antigens in their LPS O-chain. Most H. pylori strains express the type II glycoconjugate antigens, Lewis
X (Lex) and Lewis Y (Ley), whereas a small
proportion have the ability to express the type I glycoconjugates,
Lewis A (Lea) and Lewis B (Leb)
(1).2 The expression of the
Lewis antigens by H. pylori is thought to provide a way by
which the organism can avoid detection by the immune system because
these antigens are also expressed by the human gastric epithelium (2,
3). It has been suggested that the expression of Lewis antigens by
H. pylori may be the cause for an autoimmune response
leading to chronic type B gastritis and gastric as well as duodenal
ulcers (4), but the role of Lewis antigens in the disease process has
recently been questioned by two separate groups (5, 6). Presently, it
is unclear what role, if any, these antigens play in the disease
process because H. pylori isolates that do not express Lewis
antigens may still colonize and cause infection
(7).3
In the human body the expression and distribution of Lewis antigens is
tightly regulated by a series of glycosyltransferases that add
monosaccharides to precursor structures (8, 9). The synthesis of Lewis
antigens that contain two fucose moieties can occur in two ways, a
terminal fucosylation, by an Two highly homologous copies of the We have examined the FucTs from the H. pylori strain UA948
in an attempt to isolate the Bacterial Strains and Media--
H. pylori
strains were cultured by standard methods described by Taylor et
al. (31). Isolates from a frozen stock were thawed and plated out
on BHI-YE agar plates (3.7% brain heart infusion, 0.5% yeast extract,
15 µg/ml of both vancomycin and amphoterocin B, 5% of fetal bovine
serum, 1.2% agar). These plates were incubated at 37 °C under
microaerobic conditions for 2-4 days. Positive H. pylori
cultures were confirmed by urease test and microscopy. Transformants
containing the chloramphenicol acetyltransferase gene (32) inserted
into the fucT genes were isolated as described previously
(28) and cultured on BHI-YEA plates as described above containing 50 µg/ml chloramphenicol (Sigma-Aldrich). Escherichia coli
DH10 DNA Manipulation Techniques--
Standard DNA manipulation
techniques including the isolation, transformation, and restriction
enzyme digestion analysis of plasmid DNA were detailed by Sambrook
et al. (33). Both strands of the appropriate PCR fragments
were sequenced using the Thermosequenase sequencing kit according to
the manufacturer's instructions. Sequence analyses were performed with
the BLAST program from the National Center of Biotechnology Information
(Bethesda, MD). The Wisconsin Package (version 9.0) of the Genetics
Computer Group (Madison, WI) was used for the editing and alignment of sequences.
Cloning and Overexpression of the H. pylori fucT Genes--
The
primers used for amplification and cloning of the fucTs
are as follows. To clone fucTa DAVE55
5'-cgggatcccgGCGTGAATTACTACCTTTCTG -3' (positions 389088-389108)
and DAVE56 5'- cggaattccgCAAAACCCTCCTTTCTACTAATG (positions
390887-390868) were used. To clone fucTb DAVE53
5'-cgggatcccgAGCGACCAATCATTACAG-3' (position 698868-698852) and DAVE54
5'-cggaattccgACCTGGCAATTAGACAAC-3' (position 696838-696855) were
used. The capital letters denote sequences derived directly from the
published sequence from the strain 26695 (25), whereas the lowercase
letters of the primers denote the restriction endonuclease sites used
to facilitate cloning. PCR was performed as described previously (28)
producing fragments of 1769 and 1774 nucleotides for the
UA948fucTa and UA948fucTb fragments,
respectively. Restriction with EcoRI and BamHI
allowed cloning into a similarly digested pBluescript II
KS+. The respective clones containing the H. pylori
fucTa or fucTb were screened using the primers
described above. The proposed coding region of the
UA948fucTa and UA948fucTb were placed under the
control of the T7 promoter.
Recombinant plasmids pB948fucTa and pB948fucTb were introduced by
electroporation into E. coli K38 containing the plasmid pGP1-2 (which encodes a heat-inducible T7 RNA polymerase) (34). The
proteins encoded by the recombinant plasmids were expressed as follows.
E. coli K38(pGP1-2) harboring the plasmids containing the
recombinant UA948fucTa or UA948fucTb plasmids
were grown in 20 ml of liquid LB medium with appropriate antibiotics
(kanamycin and ampicillin) at 30 °C to an optical density of
0.5-0.7 at 600 nm. After collection by centrifugation, the cells were
washed once in M9 medium, and resuspended in 5 ml of supplemented M9 medium and further incubated for 1 h at 30 °C. To induce the
expression of the recombinant gene, the culture was shifted to 42 °C
by adding 5 ml of 55 °C supplemented M9 medium and incubated for a
further 15 min at 42 °C. Rifampicin was added to a final
concentration of 200 µg/ml, and incubation continued for 30 min. An
aliquot was removed and incubated a further 30 min with
[35S]methionine (50 nCi, Mandel Scientific Company Ltd.,
Guelph, ON, Canada), after which the cells were harvested by
centrifugation and resuspended in sample buffer and subjected to
SDS-polyacrylamide gel electrophoresis as described previously (28).
The remaining culture was prepared for enzyme assay by methods
previously described (28).
Fucosyltransferase Assay--
The FucT assays were
performed as described previously (35) with some modification.
Reactions were conducted at 37 °C for 20 min in a 20-µl volume
containing 1.8 mM acceptor, 50 µM GDP-fucose, 60,000 dpm GDP-[3H]fucose (American Radiolabeled
Chemicals Inc., St. Louis, MO), 20 mM Hepes buffer (pH
7.0), 20 mM MnCl2, 0.1 M NaCl, 35 mM MgCl2, 1 mM ATP, 5 mg/ml bovine
serum albumin, and 9.0 µl of the enzyme preparation. Acceptors
used in this study were: Type I ( Functional Inactivation of the UA948fucTa--
A chloramphenicol
cassette inserted at a unique XmnI site in the
fucT gene and the resultant construct (28) was used for transformation. Natural transformation of UA948 was accomplished by the
method of Ge and Taylor (36). Briefly, a 48-h culture from frozen stock
was restreaked and grown for 5 h on a BHI-YEA plate, 5 µg of DNA
containing the fucT::CAT was added to the growth. After a further incubation for 20 h at 37 °C, the culture was plated onto BHI-YEA plate containing 50 µg/ml of chloramphenicol. Cultures were grown from single colonies, and the genomic DNA was
extracted according to Ge and Taylor (36). Insertion of the CAT
cassette in the fucT gene on the chromosome was confirmed by
PCR with the previously described primers, which were specific to each
copy of the fucT gene. PCR products were subjected to electrophoresis on a 1% agarose gel and photographed under UV light.
Enzyme-linked Immunosorbent Assay for Lewis
Antigens--
The conditions for the enzyme-linked immunosorbent assay
were previously described (1, 37). The primary antibodies used were
anti-Lewis A (MAb BG-5, clone T174), anti-Lewis B (MAb BG-6, clone
T218), anti-Lewis X (MAb BG-7, clone P12), and anti-Lewis Y (MAb BG-8,
clone F3) from Signet Laboratories Inc. (Dedham, MA). These primary
antibodies were diluted 1:100, whereas the secondary antibody, goat
anti-mouse IgG + IgM conjugated to horseradish peroxidase (Biocan
number 115 035 068, Mississauga, ON, Canada), was diluted 1:2000.
Absorbance values were recorded at 405 nm using a Titretek Multiscan MC
microtitre plate reader. Absorbance values are an average of triplicate
wells with blanks subtracted. Values below 0.1 absorbance units were
considered negative.
Analysis of LPS by Acrylamide Gel Electrophoresis and
Immunoblot--
Whole cell extracts of the H. pylori
strains were treated with proteinase K, processed, and subjected to
electrophoresis as described previously (1). These gels were stained
with either zinc imidazole, according to the method of Hardy et
al. (38), or transferred to nitrocellulose membrane (Micron
Separations Inc., Westboro, MA; pore size, 0.22 µm) according to the
method described by Towbin et al. (39). Nitrocellulose
membranes, with transferred LPS, were probed with the primary and
secondary antibodies described above (anti-Lewis structure from Signet
Laboratories Inc.; antibodies 1:500 dilution and goat anti-mouse
conjugated to horseradish peroxidase diluted 1:2000, respectively).
Blots were developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech) according to the manufacturer's specifications, and
images were visualized on BioMax BM film (Eastman Kodak Co., Rochester, NY).
Features of the
The protein translation of the sequenced PCR fragments revealed that
the UA948fucTa gene encodes an open reading frame (ORF) of
475 amino acids with a predicted molecular mass of 55.9 kDa, whereas
UA948fucTb does not encode an ORF containing a full-length FucT protein. An artifical increase or decrease in the cytosine and/or
adenosine tract of the UA948fucTb gene by any small number of nucleotides does not provide a full-length fucT ORF from
this gene, eliminating the possibility that the lack of functional protein is caused by slip strand mispairing. A similar modification was
discovered to be necessary for the production of full-length protein by
NCTC11637 (29) and J99FucTa (JHP1002) (26). An ORF with
homology to the identified FucTs does exist in the
UA948fucTb insert, but it is truncated.
A comparison of the UA948FucTa amino acid sequence with
previously identified H. pylori FucTs demonstrates a high
level of homology (Fig. 2). Overall, a
greater than 70% amino acid identity was noted between the identified
H. pylori FucTs. However, this homology is largely confined
to an internal approximately 270 amino acids, which demonstrates a
82.2% identity among all H. pylori FucTs. The highly
conserved internal 270 amino acids of H. pylori FucTs
contains the hypothetical
Among H. pylori FucTs the first 82 amino acids exhibit only
32.0% identity. Although it is difficult to calculate the degree of
identity over the carboxyl-terminal portion of these proteins (final
approximately 100 amino acids), as in the carboxyl terminus of all
identified Hp Protein Expression--
Both the UA948fucTa and
UA948fucTb were directionally cloned into pBluescript
KS+ under the control of the T7 promoter. Expression of the
protein was accomplished by utilization of the strain K38 containing a heat-inducible T7 polymerase on the plasmid pGP1-2 (34). Under expression conditions pBUA948fucTa-containing cells expressed a protein
of approximately 52 kDa, which is in close agreement with the molecular
mass of 55.9 kDa predicted from the sequence analysis (Fig.
3, lane 2). We infer that the
52-kDa protein is the active species as previous attempts to truncate
full-length FucT proteins have resulted in inactivation (28). As
predicted from the sequence analysis, the clone containing pBUA948fucTb did not show expression of full-length protein but did express some
lower molecular mass proteins, which may have arisen from alternate
start sites within the gene (Fig. 3, lane 1).
FucT Enzyme Activity--
By using a panel of acceptor
carbohydrate molecules the specificity and activity of the cloned
UA948FucT enzymes were determined. Extracts from the clone containing
the pBUA948fucTb did not have any measurable FucT enzyme activity on
the carbohydrate acceptors studied (data not shown), which is in
agreement with both the sequence analysis and protein expression data.
Significant FucT enzyme activity was detected in the extract from the
clone containing the pBUA948fucTa (Table
II). The UA948FucTa enzyme adds fucose to
type II carbohydrate acceptors (LacNAc and H type II) demonstrating
The addition of the fucose to type II carbohydrate chains appears to
occur irrespective of the fucosylation state of the terminal galactose.
There is no significant difference in the enzyme activity between the
transfer of fucose to the unfucosylated LacNAc as compared with the
transfer of fucose to the already terminally fucosylated H Type II
carbohydrate (Table II; 1.26 versus 1.47 milliunits/mg),
whereas the addition of fucose to type I chains is significantly
reduced (~70%) when the terminal galactose moiety is fucosylated
(Type I (0.22 milliunits/mg) versus H Type I (0.066 milliunits/mg)).
Effects of UA948fucTa Mutation--
The mutation of the
UA948fucT gene was accomplished by homologous recombination
of the fucT gene containing a chloramphenicol acetyltransferase gene (see "Experimental Procedures"). Many
chloramphenicol-resistant colonies were selected and screened on the
basis of the increase in size of the fucT PCR fragment,
using genomic DNA from transformants as the template, but all expressed
the same genotype. Using primers specific for each copy of the
fucT gene, the PCR fragments for UA948fucTa and
UA948fucTb are 1769 and 1774 base pairs, respectively. Using
the same primers it was demonstrated that the
UA948fucT
It has been previously demonstrated that UA948 expressed both Lewis A
and X simultaneously on the same LPS (1). The mutation of
UA948fucTa has a dramatic effect on the mobility of the LPS and the expression of Lewis antigens in the LPS (Fig.
5 and Table III). The LPS from
UA948fucTa The single functional H. pylori FucT enzyme identified
in this study is a novel enzyme in both activity and specificity when compared with the previously characterized H. pylori FucTs
(28, 29). Both previously identified FucTs almost exclusively
transferred fucose to type II carbohydrate acceptors (28, 29). Martin et al. (29) did note some enzyme activity with an elongated type I carbohydrate acceptor, but no activity was noted with the minimal type 1 disaccharide lacto-N-biose (Type I chain)
used in this study. The UA948FucTa enzyme identified in this work can add fucose to both type I and II carbohydrate acceptors (Table II),
representing the first It was also noted in the study by Martin et al. (29) that
the Although two homologous copies of the fucT gene exist within
the H. pylori genome of most H. pylori strains,
only one appears to be active in H. pylori UA948.
Interestingly, even though the UA948fucTb gene is inactive,
with respect to FucT activity, no insertions of the CAT cassette into
this gene could be obtained. We have also observed this phenomenon in
other H. pylori isolates while attempting to make a double
mutant in which both of the fucT genes are insertionally
inactivated.4 This suggests that UA948fucTb may
encode an unknown essential gene product or the genes flanking
UA948fucTb are essential and may be transcriptionally or
translationally linked. Upstream of UA948fucTb is the gene
encoding a cytochrome C biogenesis protein, which terminates only 13 nucleotides upstream of the proposed start codon of the
fucTb ORF in both 26695 (25) and J99 (26). It is possible
that these genes are coordinately transcribed and that insertion of the
CAT cassette into the fucTb gene affects the synthesis of
the cytochrome C biogenesis protein, which would be deleterious to the
cell, whereas there are amino acid biosynthetic genes in both the
upstream and downstream positions of UA948fucTa. Probably
the inactivation of these genes do not pose a strong selective pressure
on the H. pylori, considering the richness of the media used
for culture.
The LPS of UA948fucTa It has been noted in the identification of the key amino acids involved
in the specificity of the human FucTs that single amino acid changes
can change or eliminate the expression of the FucT activity (12,
17-22). Because of the low level homology between the prokaryotic and
eukaryotic Obvious differences were observed when the UA948FucTa was compared with
the previously identified H. pylori FucTs. Firstly, there
was a low level of homology (~30%) in the amino-terminal 80 amino
acids of H. pylori FucTs, which in the human FucTs is the
location of the transmembrane and hypervariable region (Fig. 2B). The hypervariable region is the location of essential
amino acids responsible for the determination of the enzyme acceptor specificity (11, 12). All of the H. pylori FucTs examined by
our group5 appear to have differing rates for transfer of
fucose to identical acceptors, which may be a direct reflection of the
variability exhibited in the primary sequence in the amino terminus of
the proteins (Fig. 2A).
Significant differences are also observed in the carboxyl-terminal
portions among the different H. pylori FucTs. This region contains the heptad repeats, that is the proposed leucine zipper region, UA948FucTa exhibits significant amino acid sequence variability when compared with previously identified H. pylori We have demonstrated the existence of an We thank P. Boulton and Nora Chan for
assistance with the FucT assays.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by A Doctoral Research Award from the Medical Research
Council of Canada and an Alberta Heritage Foundation for Medical
Research Studentship and Incentive Award.
¶
Supported by a Postdoctoral Fellowship from the Canadian
Association of Gastroenterology and Astra Canada in association with an
Medical Research Council-Pharmaceutical Manufacturer's Association of
Canada award as well as a fellowship from the Alberta Heritage Foundation for Medical Research.
**
Supported by the Natural Sciences and Engineering Research Council
of Canada.
2
Monteiro, M. A., Appelmelk, B. J., Rasko, D. A.,
Hynes, S. O., McLean, L. L., Chan, K. H., St. Michael, F., Logan, S. M., O'Rourke, J., Lee, A., Moran, A. P., Taylor, D. E., and Perry, M. B. (2000) Eur. J. Biochem. 267, in press.
3
Rasko, D. A., Wilson, T. J. M., Zopf, D., and
Taylor, D. E. (2000) J. Infect. Dis., in press.
4
D. A. Rasko, unpublished data.
5
D. A. Rasko, G. Wang, M. M. Palcic,
and D. E. Taylor, unpublished data.
The abbreviations used are:
LPS, lipopolysaccharide;
PCR, polymerase chain reaction;
ORF, open reading
frame;
CAT, chloramphenicol acetyltransferase.
Cloning and Characterization of the 
(1,3/4)
Fucosyltransferase of Helicobacter pylori*
§,¶,
**, and
Department of Medical Microbiology and
Immunology and the Department of Chemistry, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4 and 1,3 linkages to type I or type II carbohydrate backbones, respectively. This work describes the cloning and characterization of a
single H. pylori fucosyltransferase (FucT) enzyme, which has the ability to transfer fucose to both of the aforementioned linkages in a manner similar to the human fucosyltransferase V (Fuc-TV). Two homologous copies of the fucT gene have been
identified in each of the genomes sequenced. The characteristic
adenosine and cytosine tracts in the amino terminus and repeated
regions in the carboxyl terminus are present in the DNA encoding the
two UA948fucT genes, but these genes also contain
differences when compared with previously identified H. pylori
fucTs. The UA948fucTa gene encodes an approximately
52-kDa protein containing 475 amino acids, whereas
UA948fucTb does not encode a full-length FucT protein. In vitro, UA948FucTa appears to add fucose with a greater
than 5-fold preference for type II chains but still retains significant activity using type I acceptors. The addition of the fucose to the type
II carbohydrate acceptors, by UA948FucTa, does not appear to be
affected by fucosylation at other sites on the carbohydrate acceptor,
but the rate of fucose transfer is affected by terminal fucosylation of
type I acceptors. Through mutational analysis we demonstrate that only
FucTa is active in this H. pylori isolate and that
inactivation of this enzyme eliminates expression of all Lewis antigens.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,2 fucosyltransferase (FucT), followed
by subterminal fucosylation, by an 1,3/4 FucT, or subterminal
fucosylation followed by terminal fucosylation (see Fig. 1).
Inactivation or lack of expression of one of these FucTs leads to the
expression of monofucosylated Lewis antigens. The Lea
structure is synthesized by addition of fucose by an (1,3/4) FucT
encoded by the FUT-3 gene to a Type I acceptor
(lacto-N-biose (Gal
1-3GlcNAc; see Fig. 1A) (10). Six
separate human FucT enzymes have been identified that have the capacity
to catalyze the transfer of fucose to the Type II precursor, LacNAc, to
produce Lex: Fuc-TIII, Fuc-IV, Fuc-V, Fuc-VI, Fuc-VII, and
Fuc-TIX (11, 12) (see Fig. 1B). Among them, Fuc-TIII and Fuc-TV have
demonstrated both (1,3) and (1,4) FucT activity and thus have the
potential to produce Lea and Lex (10, 13),
although the (1,3) activity of Fuc-TIII has been disputed (14-16).
It has been demonstrated in Fuc-TIII that a single amino acid change
resulting from a single nucleotide change in the gene is responsible
for enzyme inactivation (17-20), whereas other single amino acid
changes are found to be responsible for alteration of enzyme substrate
specificity (differential fucose transfer to type I or type II
carbohydrate acceptors) (12, 21, 22). A consensus amino acid sequence
for the (1,3) FucT enzymes (11, 23, 24) has also been identified.
(1,3) fucT have been
identified in both of the H. pylori genomes sequenced to
date (25, 26). It is thought that the fucT genes are
controlled by a slip strand repair mechanism at tracts of cytosines and
adenosines in the 5' end of the gene (27). In previous studies a
fucT gene from NCTC11637 and a fucT gene from
NCTC11639 have been cloned, and in vitro characterization of
the FucT enzymes did not demonstrate (1,4) FucT activity (28, 29).
This is consistent with the observation that the lipopolysaccharide of
these strains does not contain any type I Lewis antigens
(30).4 We have previously
identified an H. pylori strain, UA948 that expresses both
type I (Lea) and type II (Lex) carbohydrate
structures simultaneously (1). For H. pylori to produce the
Lewis structures (Lea and Lex) in the LPS
O-chain, as for the production of Lewis antigens by human cells, there
is a requirement for the addition of fucose to both (1,3) and
(1,4) linkages. Thus it is expected that UA948 contains both
(1,3) and (1,4) FucT activities (Fig.
1).
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Fig. 1.
Lewis antigens used and/or detected in this
study. A demonstrates how the mono- and difucosylated
Lewis antigens can be produced from the hypothetical starting Type I
carbohydrate chain of Lewis C. B shows how Type II
carbohydrates can be synthesized from the starting carbohydrate
backbone, LacNAc. The only difference between Type I and Type II
carbohydrate chains is the linkage of the sugars in the backbone being
1-3 and 1-4, respectively.
(1,4) FucT activity. In this study we
demonstrate that a single H. pylori FucT enzyme from UA948 contains both (1,3) and (1,4) FucT activity responsible for the
production of both Lea and Lex in the LPS
O-side chain, while the other copy of the fucT gene does not
encode a functional FucT enzyme. Comparisons of the nucleotide and
amino acid sequences of the newly identified H. pylori
(1,3/4) FucT with the previously identified H. pylori
(1,3) FucTs allowed us to predict some of the domains of the enzyme
that are potentially responsible for broadening the range of acceptors
used by this enzyme.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were grown on Lauria broth agar plates containing 100 µg/ml
ampicillin and/or 50 µg/ml chloramphenicol or 50 µg/ml kanamycin depending on the resistance markers present on the plasmids within the cells.
Gal 1-3 GlcNAc-O-(CH2)8CO2CH3),
H Type I (Fuc1-2 Gal 1-3 GlcNAc-O-(CH2)8CO2CH3), LacNAc (Gal 1-4
GlcNAc-O-(CH2)8CO2CH3),
and H Type II (Fuc 1-2 Gal 1-4 GlcNAc-O-(CH2)8CO2CH3).
These acceptors were kindly provided by Dr. O. Hindsgaul. For
calculation of the specific activity of the enzyme (milli-units (which
indicates the amount of enzyme/milliliter needed to convert 1 nmol of
acceptor to product within the defined time period) per milligram of
protein), protein concentrations of the cell extracts were determined
with a BCA protein assay kit (Pierce) using bovine serum albumin as a
standard according to the supplier's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1,3/4) fucT Gene--
PCR and subsequent
sequence analysis of the inserts of the pB948fucTa and pB948fucTb
demonstrated a significant difference in the size of the
UA948fucTb gene compared with the predicted size from strain
26695. The insert in pB948fucTb contained 1774 base pairs, whereas the
predicted size is 2030 base pairs. The main reason for the difference
in size is the absence of a number of repeated sequences in
UA948fucTb that exist in the 3' region of
26695fucTb. On the other hand, the PCR product of
UA948fucTa is 1769 base pairs long, which is similar to the
predicted size of 1799 base pairs. The nucleotide sequence of
UA948fucTa is 85.5% identical to the fucTa of
26695, whereas the UA948fucTb is 87.6% identical when
compared with the fucTb gene in 26695. Like in other
H. pylori fucT genes identified (25, 26, 28, 29), characteristic cytosine and adenosine tracts exist in the 5' end of the
UA948fucT genes (Table I).
Comparison of nucleotide and amino acid repeats of fucosyltransferases
(1,3) catalytic domains identified in
eukaryotic FucTs by sequence alignments (23, 24) (Fig. 2A,
domains I and II). All H. pylori FucTs
contain a conserved domain I with a low level identity with only 2 of
19 amino acids being conserved, whereas a much greater level of
identity is present in domain II, with 12 of 23 amino acids being
conserved (Fig. 2A). The human FucTs (HU-FucT) have a
conserved carboxyl terminus containing the catalytic domain and an
amino terminus containing the transmembrane and variable regions,
whereas the H. pylori FucT (Hp-FucT) have an internally
conserved region containing the catalytic domain, and both the amino
and carboxyl termini are variable (Fig. 2B).
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Fig. 2.
A, comparison of the amino acid
sequences of the known H. pylori FucTs. J99A, JHP1002
(AAD06573); J99B, JHP0596 (AAD06169); 26695A,
HP0379 (AAD07447); 26695B, HP0651 (AAD07710);
NCTC11639, the one copy identified (AAB81031); NCTC11637, the one copy
identified (AAB93985); UA948A, FucTa of H. pylori UA948
(AF194963). Numbers in parentheses above refer to the
GenBankTM accession number. Asterisks indicate
amino acid identity, double dots indicate a conserved amino
acid substitution, and a blank space indicates a
nonconservative amino acid substitution. The conserved catalytic
domains are designated by a line on top of the
sequence, and the cross-species conserved amino acids within this
region are in bold type. Amino acid sequences were aligned
by CLUSTAL alignment. B, a diagrammatic alignment of human
FucTs with H. pylori FucTs. In both cases N
denotes the amino terminus and C is the carboxyl terminus,
whereas CAT represents the catalytic domains of the FucT enzymes.
HV is the hypervariable region identified in the human
FucTs, which contain mutations responsible for the alteration of enzyme
characteristics. TM is the transmembrane region present only
in the human FucTs. V is the variable regions identified in
the H. pylori FucTs.
(1,3) FucTs (25, 26, 28, 29) there exists a variable
number and sequence of a 7-amino acid repeat (heptad repeat) (Table I
and Fig. 2). These heptad repeats are thought to function as a leucine
zipper in dimerization, which may be essential for function (28, 29).
In most other H. pylori (1,3) FucTs identified the heptad
repeat consists of the amino acids DDLRVNY. UA948FucTa contains five
internal repeats that are of this consensus heptad sequence, whereas
the remaining three repeats show divergence. The two heptads that
border the repeat region contain the amino acid sequence DDLRRDH,
whereas the second last heptad contains the amino acid sequence DDLRRDR
(Table I and Fig. 2A). Following the heptad repeats there is
also a 15-amino acid addition at the carboxyl terminus of the
UA948FucTa protein (Fig. 2) that does not show homology with any
protein or motif presently in the data bases.
View larger version (51K):
[in a new window]
Fig. 3.
Heterologous protein expression in E. coli K38(pGP1-2) grown under induction conditions described
in experimental procedures. Lane 1 contains extract of
K38(pGP1-2) containing plasmid pBUA948fucTb; lane 2 contains
cell extract from the host containing plasmid pBUA948fucTa; and
lane 3 contains sample from the host strain with pBluescript
KS+. The arrow indicates a 52-kDa protein that
is expressed by the clone containing the full-length active FucT. The
clone containing the pBUA948fucTb does produce protein under these
conditions, but none would code for a full-length FucT as is
demonstrated by the lack of FucT activity (see text).
(1,3) FucT activity. Transfer of fucose to type I carbohydrate acceptors (Type I chain and H Type I) was also observed. This demonstrates (1,4) FucT activity, although it is 5-20-fold lower than the (1,3) FucT activity (0.22 and 0.066 milliunits/mg
versus 1.26 and 1.47 milliunits/mg; Table II).
Transfer of fucose by the heterologously expressed UA948FucTa enzyme
transformants all contained CAT
insertions into UA948fucTa but not into
UA948fucTb (Fig. 4). Because
the nucleotide sequences of the fucT genes are highly
homologous, it was expected that the fucT::CAT
fragment would have a similar chance to recombine into both genes.
View larger version (63K):
[in a new window]
Fig. 4.
Gel of PCR products after fucT
inactivation. Lanes 1 and 2 contain
the PCR products for the fucTb (1774 base pairs) and
fucTa (1769 base pairs), respectively, from the wild type
UA948. Lanes 3 and 4 are the PCR products from
the UA948fucT using the fucTb and
fucTa specific primers described under "Experimental
Procedures." The UA948fucTa is the only gene that appears
to have the chloramphenicol acetyltransferase gene insertion as is
demonstrated by the increase in size of the PCR product (approximately
700 base pairs).
migrates faster through the
SDS-polyacrylamide gel electrophoresis gel, as indicated by the
arrow in Fig. 5A, when compared with the wild
type LPS. The band is more diffuse and thus less intense than the wild
type LPS. The expression of the Lewis antigens have also been
eliminated in UA948fucTa (Fig. 5, B
and C). The results were confirmed by quantitative determination of the Lewis antigens expression by enzyme-linked immunosorbent assay (Table III). This indicates that the fucose cannot
be transferred to the growing LPS O side chain to produce the Lewis
antigenic structures in UA948fucTa, indicating
that the only functional FucT in H. pylori UA948 is
FucTa.
View larger version (33K):
[in a new window]
Fig. 5.
LPS and immunoblot analysis of the UA948 and
UA948fucTa . In all panels
lane 1 shows a proteinase K-treated sample of UA948, and
lane 2 shows a similarly treated sample of
UA948fucTa. A is a
zinc-imidazole-stained polyacrylamide gel showing the change in
mobility of the LPS from the UA948fucTa
isolate as is indicated by the arrow. B and
C are immunoblots probed with anti-Lewis A and anti-Lewis X,
respectively. Both panels demonstrate that the
UA948fucTa isolate no longer expresses Lewis
antigens.
Enzyme-linked immunosorbent assay values of UA948 wild type versus
UA948fucTa
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1,3/4) FucT to be identified in H. pylori. There is a greater than 5-fold preference for the type II
carbohydrate acceptors over type I carbohydrate acceptors, which is
more similar to the enzyme characteristics exhibited by the human
Fuc-TV than any other enzyme (40). The human Fuc-TV enzyme shows a
slight preference for type II acceptors but still retains significant
activity on type I carbohydrate receptors.
(1,3)FucT isolated could not use the type II carbohydrate, H
Type II, as an acceptor to produce Lewis Y, thus leading the authors to
believe that the production of the difucosylated Lewis antigens,
Leb and Ley, may be routed through a
subterminal monofucosylation by an (1,3) or (1,3/4) FucT followed
by the terminal fucosylation by an (1,2)FucT (41) (Fig. 1,
A and B, right-hand pathway). It is
apparent from the data presented in this study that this is not true
for all H. pylori (1,3)FucTs, because UA948FucTa has the
ability to add fucose in a subterminal position on the GlcNAc of H Type
II very efficiently, as well as to H Type I with reduced efficiency produce a difucosylated antigens (Fig. 1, A and
B, left-hand pathway). Our recent
observations5 show that the
(1,2) FucT of some H. pylori isolates can use both the
subterminally fucosylated (Lewis A or Lewis X) as well as the
unfucosylated carbohydrate chains (Type I and LacNAc) as acceptors.
More work with both the (1,2) FucT and the (1,3/4) FucT is
necessary to definitively identify the pathway for the synthesis of
difucosylated antigens by H. pylori.
no longer contains any
of the complex, fucose-containing, Lewis antigens that are present in
the wild type UA948 strain, even though only a single fucT
gene has been inactivated (Fig. 5 and Table III). This further
supports the conclusion that only one copy of the fucT gene
is active in this H. pylori isolate. The inactivation of the
only fucT gene in this H. pylori isolate provides
an opportunity to determine what role, if any, the formation of
complexed fucose containing carbohydrates in the LPS plays in the
infectious process of H. pylori.
(1,3) FucTs, it is difficult to use the amino acid
sequence of the human FucTs to predict which amino acid changes may be
responsible for the alteration of acceptor specificity of H. pylori FucTs, but the human system may provide clues as to the
identification of the region responsible for this activity in the
H. pylori FucTs. Breton et al. (23, 24)
identified two conserved motifs within the catalytic domain that is
identical in all of the human (1,3) FucT enzymes regardless of the
acceptor specificity, and these domains are present in the (1,3)
FucT of other species (11). By sequence analysis these two motifs have
been localized in the H. pylori (1,3) FucTs (Fig.
2A, domains I and II), and they are
also present in the (1,3/4) FucT of H. pylori. The
H. pylori FucTs have a high level of amino acid identity (~82%) in the internal 270 amino acids of the FucT proteins
corresponding to the proposed catalytic domain. In the human FucTs
mutations in this highly conserved region generally correspond to the
inactivation of the FucT activity of the protein (18-20, 42).
(1,3)
FucTs (Fig. 2). The first four amino acids of the UA948FucTa heptad repeat are conserved, DDLR, but the final three amino acids are divergent. The UA948FucTa heptad repeats still contain the leucine moieties in the appropriate spacing to continue acting as a leucine zipper. It is clear that although variable, there is some conservation of physical attributes of the variable amino acids in the repeat region
because the net charge of these three amino acids is relatively conserved. It is unclear at this time whether the nonhomologous flanking heptad repeats are involved in the broadening of the acceptor
range to include the type I carbohydrate acceptors or whether these
regions are responsible only for the hypothesized function of
dimerization (28, 29). Finally, there is also a carboxyl-terminal
15-amino acid addition in UA948FucTa, which is not present in the other
H. pylori (1,3) FucTs and does not share any homology
with any identified sequence. It was noted in Ge et al. (28)
that a carboxyl-terminal 115-amino acid truncation eliminated all FucT
enzyme activity, proving that this region is essential for enzyme activity.
(1,3/4)FucT from H. pylori that is responsible for the production of both
Lea and Lex (Fig. 1). This enzyme exhibits
significant sequence divergence at both the nucleotide and amino acid
level from previously identified H. pylori FucTs. The
regions of variability will need further investigation to determine
their role, if any, in the activity and expanded acceptor range of
UA948FucTa. It will require a careful molecular study, identification
of more H. pylori (1,3/4) FucTs, and domain swapping
experiments similar to those performed with the human FucTs to
determine exactly which of the changes are responsible for the broader
acceptor range of UA948FucTa.
ACKNOWLEDGEMENTS
FOOTNOTES
Alberta Heritage Foundation for Medical Research Scientist.
Supported from Canadian Bacterial Diseases Network as well as the
National Cancer Institute of Canada with funds from the Terry Fox Run.
To whom correspondence should be addressed: University of Alberta,
Dept. of Medical Microbiology and Immunology, 1-28 Medical Sciences
Bldg., Edmonton, AB T6G 2H7, Canada. Tel.: 780-492-4777; Fax:
780-492-7521; E-mail: diane.taylor@ualberta.ca.
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B. Ma, G. Wang, M. M. Palcic, B. Hazes, and D. E. Taylor C-terminal Amino Acids of Helicobacter pylori{alpha}1,3/4 Fucosyltransferases Determine Type I and Type II Transfer J. Biol. Chem., June 6, 2003; 278(24): 21893 - 21900. [Abstract] [Full Text] [PDF]
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A. Lozniewski, X. Haristoy, D. A. Rasko, R. Hatier, F. Plenat, D. E. Taylor, and K. Angioi-Duprez Influence of Lewis Antigen Expression by Helicobacter pylori on Bacterial Internalization by Gastric Epithelial Cells Infect. Immun., May 1, 2003; 71(5): 2902 - 2906. [Abstract] [Full Text] [PDF]
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A. L. Sherwood, D. A. Upchurch, M. R. Stroud, W. C. Davis, and E. H. Holmes A highly conserved His-His motif present in {alpha}1->3/4fucosyltransferases is required for optimal activity and functions in acceptor binding Glycobiology, October 1, 2002; 12(10): 599 - 606. [Abstract] [Full Text] [PDF]
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B. J. Appelmelk, M. C. Martino, E. Veenhof, M. A. Monteiro, J. J. Maaskant, R. Negrini, F. Lindh, M. Perry, G. Del Giudice, and C. M. J. E. Vandenbroucke-Grauls Phase Variation in H Type I and Lewis a Epitopes of Helicobacter pylori Lipopolysaccharide Infect. Immun., October 1, 2000; 68(10): 5928 - 5932. [Abstract] [Full Text] [PDF]
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K.-A. Karlsson The human gastric colonizer Helicobacter pylori: a challenge for host-parasite glycobiology Glycobiology, August 1, 2000; 10(8): 761 - 771. [Abstract] [Full Text] [PDF]
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