(Received for publication, May 8, 1997, and in revised form, June 18, 1997)
From the Departments of Medical Microbiology and
Immunology, § Chemistry, and ¶ Biological Sciences,
University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Helicobacter pylori is an important
human pathogen which causes both gastric and duodenal ulcers and is
also associated with gastric cancer and lymphoma. This microorganism
has been shown to express cell surface glycoconjugates including Lewis
X (Lex) and Lewis Y. These bacterial oligosaccharides are
structurally similar to tumor-associated carbohydrate antigens found in
mammals. In this study, we report the cloning of a novel
1,3-fucosyltransferase gene (HpfucT) involved in the
biosynthesis of Lex within H. pylori. The
deduced amino acid sequence of HpfucT consists of 478 residues with the calculated molecular mass of 56,194 daltons, which is
approximately 100 amino acids longer than known mammalian
1,3/1,4-fucosyltransferases. The ~52-kDa protein encoded by
HpfucT was expressed in Escherichia coli CSRDE3
cells and gave rise to
1,3-fucosyltransferase activity but neither
1,4-fucosyltransferase nor
1,2-fucosyltransferase activity as
characterized by radiochemical assays and capillary zone
electrophoresis. Truncation of the C-terminal 100 amino acids of
HpFuc-T abolished the enzyme activity. An approximately 72-amino acid
region of HpFuc-T exhibits significant sequence identity (40-45%)
with the highly conserved C-terminal catalytic domain among known
mammalian and chicken
1,3-fucosyltransferases. These lines of
evidence indicate that the HpFuc-T represents the bacterial
1,3-fucosyltransferase. In addition, several structural features
unique to HpFuc-T, including 10 direct repeats of seven amino acids and
the lack of the transmembrane segment typical for known eukaryotic
1,3-fucosyltransferases, were revealed. Notably, the repeat region
contains a leucine zipper motif previously demonstrated to be
responsible for dimerization of various basic region-leucine zipper
proteins, suggesting that the HpFuc-T protein could form dimers.
Helicobacter pylori is a spiral, microaerophilic Gram-negative bacterium which has been recognized as an important human pathogen causing gastritis and both peptic and duodenal ulcers (1-3). This bacterium is also associated with an increased risk for the development of both gastric adenocarcinoma (4, 5) and primary gastric lymphoma (6). This pathogen is highly adapted to colonize human gastric mucosa and may remain in the stomach with or without causing symptoms for many years (7). Although H. pylori elicits local as well as systemic antibody responses (8), location in such a specific niche can permit it to escape elimination by the host immune response. Another mechanism by which H. pylori may protect itself from the action of the host immune response is the production of surface antigens mimicking those in the host.
Cell surface (1,3)- and
(1,2)-fucosylated oligosaccharides, that
is, Lewis X
(Lex)1 and Lewis
Y (Ley), are present on both eukaryotic and microbial cell
surfaces. In mammals, Lex is a stage-specific embryonic
antigen, and Lex, sLex, and Ley are
all regarded as tumor-associated markers (9-11). sLex
directly mediates cell to cell adhesion through the interaction with
selectins (12, 13). It has been proposed that Lex plays a
similar function during physiological and pathological processes (14).
Bacterial Lex and Ley were first identified in
H. pylori by Aspinall and Monteiro (15) and Aspinall
et al. (16) who demonstrated that Lex and
Ley structures are present in the O-antigen regions of the
lipopolysaccharides purified from H. pylori cells using one-
and two-dimensional nuclear magnetic resonance spectroscopy. Expression
of Lex in H. pylori was further confirmed by
examination using immunoelectron microscopy and enzyme-linked
immunosorbent assay (17-19). Recently it was reported that production
of Lex and Ley is related to cagA+
H. pylori isolates which are associated with an increased
risk for the development of gastric cancer (20). The biological
functions of these bacterial oligosaccharide structures are not fully
understood. It has been suggested that such glycoconjugates produced by
H. pylori may mimic host cell antigens and could mask the
bacterium and thus reduce the immune response (19, 20). It is also
possible that these bacterial Lewis antigens could down-regulate the
host T-cell response (17). Therefore, production of such antigens may
contribute to colonization and long-term infection of the stomach by
H. pylori.
The final step in the synthesis of cell surface 1,3- and
1,4-fucosylated oligosaccharides in mammals is catalyzed by
1,3- or
1,3/1,4-fucosyltransferases (Fuc-Ts). The human FUT
genes encoding at least five distinct Fuc-Ts generating Lex
and related structures have been cloned and sequenced (21-29). Similar
FUTs were also identified in other animals, including bovine (30),
mouse (31), and chicken (32). The primary sequences of these eukaryotic
Fuc-Ts exhibit significant amino acid similarity (32, 33). It has been
demonstrated that the discrete peptide fragments of human Fuc-Ts III,
V, and VI contribute to discrimination of acceptor substrates (34,
35).
Chan et al. (18) demonstrated that the Lex
produced by H. pylori is synthesized by the addition of
galactose from UDP-Gal to GlcNAc and then fucose from GDP-Fuc to
Gal1-4GlcNAc (LacNAc), catalyzed by
1,4-galactosyltransferase
and Fuc-T, respectively. This pathway is identical to that found in
humans. However, the genetic basis for the bacterial Fuc-T was unclear.
Characterization of a gene encoding the
1,3-fucosyltransferase would
not only lead to elucidation of the pathogenic role of the
Lex determinant in the H. pylori infection but
would also provide new insights into the structure-function
relationship by comparison between eukaryotic and prokaryotic Fuc-Ts.
We now report the cloning and heterologous expression of the novel
bacterial Fuc-T gene, designated HpfucT, obtained from
H. pylori. The deduced amino acid sequence of
HpfucT is similar to the highly conserved catalytic domain
among known mammalian and chicken Fuc-Ts. Several sequence features
unique to this bacterial Fuc-T were also identified. In addition, the
HpFuc-T protein produced in Escherichia coli was
characterized biochemically.
Bacteria were used in this study, including
H. pylori strain NCTC11639 for cloning, E. coli
strain DH10B for production of recombinant plasmids, and E. coli CSRDE3 containing a Plac-controlled T7 RNA
polymerase (36) for overexpressing proteins of interest. H. pylori cells were cultured on BHI-YE agar or in BHI-YE broth under
microaerobic conditions as described previously (36). LB medium (37),
M9 medium (37), and a supplemented M9 medium (37) were used for growth
of E. coli cells. Either ampicillin (100 µg/ml) or
rifampicin (200 µg/ml) were added to the above media for growth of
plasmid-containing cells and for expression of plasmid-encoded
proteins. Plasmid vectors pBluescript II SK(+/) and
pCRTMII were purchased from Stratagene (La Jolla, CA) and
Invitrogen (San Diego, CA), respectively.
Gal1-4GlcNAc
-O-(CH2)8COOMe
(LacNAc-R),
Gal
1-3GlcNAc
-O-(CH2)8COOMe
(Gal
1-3GlcNAc-R), and
Gal
1-4GlcNAc
-O-(CH2)8CO-NHCH2CH2NH-TMR (LacNAc-TMR) were provided by Dr. O. Hindsgaul, Dept. of Chemistry, University of Alberta. GDP-[1-3H]fucose (5.1 Ci/mmol) was
obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO) and
phenyl-
-galactoside (Phenyl-Gal) as well as almond meal
-fucosidase from Sigma-Aldrich Canada, Ltd. (Mississauga, Ontario),
whereas GDP-fucose was synthesized by the method of Gokhale et
al. (38). Sep-Pak Plus C-18 reverse-phase cartridges were
purchased from Waters (Mississauga, Ontario). [
-32P]dCTP was from DuPont Canada Inc. (Mississauga,
Ontario). Protein concentrations were determined with the BCA protein
assay kit using bovine serum albumin as a standard according to the
supplier's instructions (Pierce). All restriction endonucleases and
other modified enzymes were obtained from Life Technologies, Inc.
unless specified.
Plasmid and H. pylori chromosomal DNA was prepared as described previously (36).
Standard molecular cloning procedures and Southern hybridization were
detailed by Sambrook et al. (37). Both strands of
recombinant plasmid DNA were sequenced using the Thermo sequenase
sequencing kit following the manufacturer's instructions (Amersham
Life Science, Inc.). [-32P] dCTP-labeled probes were
prepared using a random priming kit (Life Technologies, Inc.).
To
clone the fucosyltransferase gene from H. pylori NCTC11639,
degenerate primers were generated from the several regions conserved by
three mammalian 1-3 fucosyltransferases including human Fuc-TVI
(29), bovine Fuc-TIII (33), and mouse Fuc-TIV (34). Primer FUTF3
(5
TT(T/C)TA(T/C)CT(T/C/A/G)GC(G/A/T/C)TT(T/C)GA(A/G)AA3
) corresponds
to residues 242-248 of human Fuc-TVI, whereas primer FUCTR2
(5
AA(A/G)TC(A/G)TC(G/ATC)AC(A/G)TG(G/A/T/C)AG(A/G)AA3
) is
complementary to the sequence deduced from its residues 289-295. An expected DNA fragment of ~170 nucleotides was PCR-amplified from
chromosomal DNA of H. pylori NCTC11639 with the primer pair of FUCTF3 and FUCTR2 under a thermocycling program of 40 cycles: for
the first two cycles, 1 min at 94 °C, 30 s at 40 °C, and
40 s at 72 °C; for the remaining cycles, 1 min at 94 °C,
30 s at 50 °C, and 40 s at 72 °C, followed by extension
at 72 °C for 10 min. The PCR products were cloned into vector
pCRTMII according to the supplier's instructions.
Subsequently, the inserts in recombinant plasmids were sequenced with
Thermo sequenase, and their nucleotide sequences and deduced amino acid
sequences were used in the search for related proteins in data bases
with the software of Blast included in the GCG package (version 8.0, Genetic Computing Group, Inc., Madison, WI). A clone, designated pCRHpfucT3, was demonstrated to contain the insert encoding the amino
acid sequence homologous to known mammalian
1,3-fucosyltransferases.
To clone a putative intact fucT gene from H. pylori, chromosomal DNA from H. pylori NCTC11639 was
digested with restriction endonucleases, including BglII,
EcoRI, BamHI, BglII-EcoRI,
EcoRI-BamHI, and
BglII-BamHI and then separated in a 1% agarose
gel. DNA fragments containing the putative fucT gene were
demonstrated by Southern hybridization with a
[-32P]dCTP-labeled probe made from pCRHpfucT3 DNA. The
2.2-kb EcoRl-BglII and 4.5-kb
EcoRI-BamHI fragments were cloned into vector
pBluescript II KS
which was digested with EcoRI and
BamHI. Two clones, pBKHpfucT8 carrying a 2.2-kb
EcoRl-BglII fragment and pBKHpfucT31 carrying a
4.5-kb, EcoRI-BamHI fragment, were selected for
further characterization.
To construct recombinant plasmids containing an intact or
partial H. pylori fucT gene, three primers were generated
from the nucleotide sequence in Fig. 1: ZGE37 corresponding to
nucleotides 1-19; ZGE38 and ZGE39 complementary to nucleotides
1215-1233 and 1692-1710, respectively. ZGE37 contained a
BamHI site, whereas ZGE38 and ZGE39 contained an
EcoRI site. PCR products were amplified from pBKHpfucT31
with a primer pair of either ZGE37/ZGE38 or ZGE37/ZGE39. These
PCR-amplified DNA fragments were digested with EcoRl and BamHl and then cloned into pBluescript II KS. The
respective clones containing the H. pylori fucT gene of
interest were screened by PCR with the corresponding pair of the above
primers. Two clones, designated pBKHp763fucT38 and pBKHp763fucT39,
contained a partial and an intact H. pylori fucT gene,
respectively. The coding region of the H. pylori fucT gene
was controlled under the T7 promoter. The sequence of the PCR-amplified
DNA fragments in pBKHp763fucT38 and pBKHp763fucT39 was demonstrated by
sequencing to be identical to that of the native template.
Recombinant plasmids pBKHp763fucT38 and pBKHp763fucT39 were introduced into E. coli CSRDE3 cells by electroporation. The proteins encoded by the recombinant plasmids were overexpressed in the presence of either [35S]methionine or cold methionine as described previously (36). The cells expressing [35S]methionine-labeled proteins were boiled for 5 min in 1 × sample buffer and separated in a 13.5% SDS-polyacrylamide gel (36).
Fucosyltransferase AssaysE. coli CSRDE3 cells
expressing nonradioactively labeled proteins encoded by pBKHp763fucT38
and pBKHp763fucT39 were harvested and suspended in Hepes buffer (20 mM Hepes, pH 7.0) supplemented with 0.5 mM
phenylmethylsulfonyl fluoride (a proteinase inhibitor). Subsequently,
membrane and soluble fractions of the cells were prepared after
disruption with a French press as described previously (36). The
membrane pellets were resuspended in the same Hepes buffer, frozen in
liquid nitrogen, and stored at 70 °C prior to use.
Assays of H. pylori 1,3- and
1,4-fucosyltransferase
activities were carried out according to the method described by Chan et al. (18) with some modifications. Reactions were
conducted at 37 °C for 20 min in a volume of 20 µl containing
either 720 µM LacNAc-R for
1,3-fucosyltransferase
activity, Gal
1-3GlcNAc-R for
1,4-fucosyltransferase activity, or
5.33 mM Phenyl-Gal for
1,2-fucosyltransferase activity,
50 µM GDP-fucose, 100,000 dpm GDP-[3H]fucose, 20 mM Hepes buffer (pH 7.0),
20 mM MnCl2, 0.2% bovine serum albumin, and
8.5 µl of the enzyme fraction.
The incubation mixtures
contained 16 µl of the membrane fraction containing the intact
HpFuc-T protein, 100 µM LacNAc-TMR, 100 µM
GDP-fucose in a total volume of 20 µl of 20 mM Hepes (pH 7.0) containing 20 mM MnCl2 and 0.2% bovine
serum albumin. Incubation was done at 37 °C for 30 min.
Subsequently, the sample was prepared and analyzed by capillary
electrophoresis by injecting 12 pl onto an electrophoresis column (60 cm long) at 1 kV for 5 s as described previously (18). The
electrophoretic separations were performed at a running voltage of 400 V/cm. -Fucosidase treatment was done by incubating the sample (10 µM total TMR) with 4 microunits of almond meal
-fucosidase in a total volume of 40 µl of 50 mM sodium citrate buffer, pH 5.0, at 37 °C for 90 h. Products were
isolated and analyzed by capillary electrophoresis as described
above.
Three recombinant plasmids, pCRHpfucT3, pBKHpfucT8, and
pBKHpfucT31 containing the intact or partial sequence of the
fucT gene from H. pylori NCTC11639 were obtained
as described under "Experimental Procedures" (Fig.
1). The nucleotide sequences of these
recombinant clones were sequenced from both strands using nested
primers. Fig. 2A gave rise to
the nucleotide sequence of 1710 base pairs derived from clone
pBKHpfucT31. A major open reading frame (ORF), starting at nucleotide
145 and ending at nucleotide 1578, was predicted in this region. An
unusual sequence feature of this ORF was 10 direct repeats of 21 nucleotides (Fig. 2A). An AA to GG transition at positions
12 and 13 of this repeat has occurred in repeat copies III, V, and
VIII. An SD sequence, a ribosomal binding site in prokaryotes (39),
precedes the predicted translation initiation codon AUG. In addition,
the sequence "ACCATGT," which is similar to the Kozak's consensus
context "ACCATGG" (a common ribosomal binding site in eukaryotes)
(40), is also present at the beginning of the ORF. Putative
transcription elements including 10 and
35 regions immediately
upstream of the ORF and a stem-loop structure following the stop codon
of the ORF, which probably act as a transcription promoter and
-independent transcription terminator (41), were identified (Fig.
2A). An asymmetric inverted repeat sequence was found,
encompassing 18 nucleotides and containing the putative
10 region
(Fig. 2A). Another ORF downstream from the major ORF, in the
opposite orientation as indicated in Fig. 2A, encodes the
amino acid sequence similar to the corresponding region of the
glutamate dehydrogenase identified in Corynebacterium glutamicum (nucleotide sequence accession number S32227).
Features of the Deduced Amino Acid Sequence of the H. pylori fucT
A protein consisting of 478 amino acids with a calculated
molecular mass of 56,194 daltons was predicted from this ORF. A hydropathy profile calculated by the method of Kyte and Doolittle (42)
indicates that the deduced amino acid sequence is primarily hydrophilic
and does not contain a potential transmembrane segment (Fig.
2B). The predicted protein carries 10 direct repeats of seven amino acid residues proximal to the C terminus. There is a
conservative replacement of valine with isoleucine at position 5 found
in repeats III, V, and VIII, which results from the corresponding AA to
GG mutations as mentioned above. Five putative N-linked glycosylation sites were predicted, two of which are proximal to the N
terminus similar to those identified in mammalian Fuc-Ts. However, the
remaining three such sites are close to the C terminus. This latter
feature is similar to the sites identified in rabbit and human
1,2-fucosyltransferases (43). Comparison of this polypeptide
sequence with other proteins in the data bases using the Blast search
revealed significant sequence similarity (40-45% identity) to
1-3
and 1-3/1-4 fucosyltransferases from mammalian sources including
human Fuc-Ts III to VII (21-29), bovine Fuc-TIII (30), mouse Fuc-TIV
(31), and CFT1 from chicken (32) within an approximately 72-amino acid
stretch. As denoted in Fig.
3A, this region is located in
the proposed C-terminal catalytic domains of Fuc-Ts (44, 45).
Therefore, we designated this gene as HpfucT.
The remaining sequences beyond this conserved region between HpFuc-T and eukaryotic Fuc-Ts are relatively divergent. HpFuc-T appears to lack a transmembrane segment which is common to eukaryotic Fuc-Ts and which is usually located in their N-terminal region. On the other hand, these eukaryotic Fuc-Ts do not contain the ~100-amino acid region encompassing ten "DDLR(V/I)NY" repeats. Sequence similarity searches revealed that this region is significantly similar to the domain potentially forming a leucine zipper structure within several homeobox-leucine zipper proteins (HD-Zip protein) including ATHB-1, ATHB-5 to -7 from Arabidoposis thaliana (46-48), and tomato (nucleotide sequence accession number X94947) (Fig. 3B). The conserved leucine residues are also colinear to those present in the leucine zipper motif of the group of the basic region-leucine zipper (bZip) proteins found in eukaryotes including yeast, higher plant, animals, and recently in a bacterium (49-52).
Characterization of the HpfucT Gene Product in E. coli CellsTo investigate whether or not the predicted
HpfucT gene represents a complete locus, a modified maxicell
system using CSRDE3 (36) was applied for the HpfucT
expression. Two recombinant plasmids, pBKHp763fucT38 carrying the
partial HpfucT gene and pBKHp763fucT39 carrying the intact
HpfucT gene, were constructed (Fig. 1). The
HpfucT genes in these two plasmids were controlled by a T7
promoter. The expression results were shown in Fig.
4. pBKHp763fucT39 gave rise to a specific
product of ~52 kDa (Fig. 4, lane 2) which is close to the
predicted molecular mass of 56 kDa. In addition, a protein of ~41 kDa
was produced from pBKHp763fucT38-containing cells (lane 1).
The size of this product is consistent with the predicted 42 kDa of the
truncated HpFuc-T in which the C-terminal 115 amino acids of HpFuc-T
was removed. In contrast, these HpfucT-encoded proteins were
not produced in cells containing either a vector without the insert or
with no plasmid (Fig. 4, lanes 3 and 4). Two
strong protein bands of ~35 kDa and 29 kDa, respectively, were
present in all the samples, indicating that they were encoded by host
genes. Therefore, the above evidence demonstrates that the cloned
HpfucT represents a complete locus.
Biochemical Assay of the Overproduced HpFuc-T Protein
The
partial sequence of this bacterial Fuc-T is homologous to the catalytic
domain of mammalian Fuc-Ts, suggesting that HpFuc-T is a
fucosyltransferase. The nature of this enzyme activity was investigated. To delineate the cellular location of the enzyme activity, membrane and cytoplasmic fractions of E. coli
cells producing the HpFuc-T proteins were prepared. The 1,3-HpFuc-T activity was quantitated using LacNAc-R as an acceptor and GDP-fucose as the donor. Approximately 85% of the total enzyme activity was associated with the membrane fraction containing the intact HpFuc-T protein expressed from pBKHp763fucT39, whereas the remaining portion was present in the cytoplasmic fraction (Table
I). There was no detectable activity of
either
1,2-Fuc-T or
1,4-Fuc-T associated with the same samples.
The Triton X-100-solubilized membrane fraction gave rise to slightly
higher
1,3-Fuc-T activity than the untreated extract (Table I). No
1,3-Fuc-T activity was obtained from either the membrane or the
cytoplasmic fractions prepared from cells producing the truncated
HpFuc-T protein encoded by pBKHp763fucT38. This result indicated that
the C-terminal 115 amino acids of HpFuc-T is crucial for this
activity.
|
The reaction products synthesized by the H. pylori
1,3-fucosyltransferase were characterized by capillary
electrophoresis with laser-induced fluorescence detection using
tetramethylrhodamine (TMR)-labeled acceptors (18). The reaction mixture
containing the membrane fraction of cells harboring pBKHp763fucT39,
GDP-fucose, and LacNAc-TMR produced a new peak (Fig. 5a,
Lewis-X peak) which co-migrated with a
synthetic Lex-TMR in the
electropherogram (Fig. 5c, peak 3), indicating
that this new peak represents the Lex product synthesized
by this bacterial
1,3-fucosyltransferase. The synthesis of
Lex with this enzyme was further demonstrated by digestion
with fucosidase which cleaved the Lex product and released
LacNAc-TMR. As a result, the concentration of LacNAc in the reaction
mixture increased (Fig. 5b, LacNAc peak), whereas
the concentration of the Lex product decreased (Fig.
5b, Lewis-X peak).
In this study we have cloned and sequenced the H. pylori 1,3-fucosyltransferase gene HpfucT. There are
two unique features found in the DNA sequence of HpfucT as
well as its flanking regions. First, in addition to a prokaryotic
ribosomal binding site, the Kozak's consensus context (ribosomal
binding site) also exists around the translation codon AUG. It will be
of interest to determine whether or not this Kozak's context is
functional in eukaryotic cells. Second, 10 direct 21-nucleotide repeats
are present in the region close to the 3
end of the HpfucT
gene, which encode 10 corresponding 7 amino acid repeats in the HpFuc-T
protein. Sequence analysis and expression of HpfucT in
E. coli indicated that this gene coded for a protein of
~56 kDa comprising 478 amino acids. Fucosyltransferase activity of
HpFuc-T produced in E. coli cells was assayed by in
vitro biochemical characterization, which demonstrated that this
enzyme strictly utilizes LacNAc as an acceptor. No fucose transfer was
detected when either Gal
1-3GlcNAc-R or Phenyl-Gal was used as an
acceptor. Although the activity and the substrate specificity of
HpFuc-T determined in vitro may or may not represent the
in vivo situation of this enzyme, the lines of evidences
presented here confirmed that the HpFuc-T represents the novel
bacterial
1,3-fucosyltransferase.
Amino acid sequence comparison revealed that HpFuc-T exhibits a
significant sequence similarity to the highly conserved eukaryotic Fuc-T C-terminal catalytic domain. Beyond this region, the sequences between this bacterial Fuc-T and the eukaryotic Fuc-Ts extensively diverge. This is not surprising since the N-terminal amino acid sequences among these eukaryotic Fuc-Ts are relatively variable (33).
However, several unique structural features indicate that the HpFuc-T
is distinct from eukaryotic Fuc-Ts. First, in comparison with the
primary sequences of mammalian and chicken Fuc-Ts, the HpFuc-T protein
contains an additional C-terminal ~100amino acid region including
the 10 direct repeats. Our preliminary characterization indicated that
this region is crucial for Fuc-T activity since the deletion of this
portion from the HpFuc-T protein completely abolishes the Fuc-T
activity (Table I). This result is consistent with the previous finding
that removal of one or more amino acids from the C terminus of Fuc-TV
dramatically reduced or completely abolished enzyme activity (35).
Second, HpFuc-T does not contain a N-terminal transmembrane segment, a
typical feature for mammalian Fuc-Ts, primarily consisting of
hydrophilic residues (Fig. 2B). It should be noted that the
majority of the HpFuc-T activity is present in the membrane fraction,
whereas Triton X-100 solubilization of the membrane fraction did not
significantly increase such activity. This suggests that HpFuc-T could
be associated with the cytoplasmic membrane through electrostatic
forces as was found for the E. coli phosphatidylserine
synthase whose overall amino acid sequence is also hydrophilic (53).
Third, five potential asparagine-linked glycosylation sites lie within
the HpFuc-T sequence, three of which are located in the C-terminal
region downstream of the Fuc-T catalytic domain. These three C-terminal
glycosylation sites are not found in mammalian and chicken
1,3-Fuc-Ts. An unexpected finding was that sites III and IV are
comparable to those in
1,2-Fuc-Ts from humans and rabbit (43).
It is interesting that the sequence of the region containing the 10 direct repeats is extensively similar to the leucine-zipper domain of
the plant HD-Zip proteins (ATHB-1 and -5 to -7) found in A. thaliana (46-48). These highly conserved leucine residues also
correspond to those in the leucine motif of the eukaryotic bZip
proteins (Fig. 3B). The leucine-zipper motif of plant HD-Zip proteins is closely linked to the homeodomain which consists of 61 amino acids with DNA-binding properties (54, 55), whereas this motif in
the bZip proteins is located immediately downstream of a basic region
responsible for the sequence-specific DNA binding (49, 50). In
contrast, the putative leucine zipper domain in HpFuc-T follows the two
potential glycosylation sites. Leucine zippers are responsible for
dimerization in a separate class of transcription factors found in some
eukaryotes (reviewed in Ref. 56). More recently, a bZip motif was
identified in a bacterial histidine kinase TodS regulating toluene
degradation in Pseudomonas putida F1 (52). The bZip
motif-mediated dimerization of TodS for DNA binding was suggested by
in vitro DNA binding assays using a short dimerized peptide
derived from the N-terminal region of TodS to mimic the full-length
protein; this short peptide dimer could bind to the 196-base pair DNA
fragment PCR-amplified from the intergenic region between
todH and todS (52). In addition, the leucine
zipper repeats of HpFuc-T exhibit significant sequence similarity (31%
identity) to the multiple leucine zipper motifs of EAP-300, a
developmentally regulated embryonal protein found in chicken (51). This
protein has been implicated in a role of neural development, and it was
postulated that existence of the multiple leucine-zipper motifs might
enable EAP-300 to form dimers or multimers (51). We hypothesize that
this leucine-zipper domain may dimerize the HpFuc-T protein. Such a
dimer could act as either a transcription regulator controlling
Lex production or a functional enzyme complex or both.
Construction of mutations in this region would test this hypothesis.
Additional studies on relationships between these structures and the
1,3-fucosyltransferase functions, and transcriptional and
translational regulation of this gene, should shed light on the
pathogenic role of this gene product during H. pylori
infection.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF008596.
We thank Margaret Deschiffart for technical assistance.