Previous structural investigations performed on
the lipopolysaccharides (LPSs) from the human gastric pathogen
Helicobacter pylori have revealed that these cell surface
glycan molecules express type 2 partially fucosylated, glucosylated, or
galactosylated N-acetyllactosamine O antigen chains
(O-chains) of various lengths, which may or may not be terminated at
the nonreducing end by Lewis X (Lex) and/or Ley
blood group epitopes in mimicry of human cell surface glycoconjugates and glycolipids. Subsequently, serological experiments with
commercially available Lewis-specific monoclonal antibodies also have
recognized the presence of Lex and Ley blood
group antigens in H. pylori but, in addition, have
indicated the presence of type 1 chain Lea,
Leb, and Led (H-type 1) blood group epitopes in
some H. pylori strains. To confirm their presence,
structural studies and additional serological experiments were
undertaken on H. pylori strains suspected of carrying type
1 chain epitopes. These investigations revealed that the O-chain region
of H. pylori strain UA948 carried both Lea
(type 1) and Lex (type 2) blood group determinants. The
O-chain from H. pylori UA955 LPS expressed the terminal
Lewis disaccharide (type 1 chain) and Lex and
Ley antigens (type 2). The O-chain of H. pylori
J223 LPS carried the type 1 chain precursor Lec, the
H-1 epitope (Led, type 1 chain) and an elongated
nonfucosylated type 2 N-acetyllactosamine chain (i
antigen). Thus, O-chains from H. pylori LPSs can also express fucosylated type 1 sequences, and the LPS from a single H. pylori strain may carry O-chains with type 1 and 2 Lewis
blood groups simultaneously. That monoclonal antibodies putatively
specific for the Leb determinant can detect glycan
substructures (Le disaccharide, Lec, and Led)
of Leb indicates their nonspecificity. The expression of
both type 1 and 2 Lewis antigens by H. pylori LPSs mimics
the cell surface glycomolecules present in both the gastric superficial
(which expresses mainly type 1 determinants) and the superficial and glandular epithelium regions (both of which express predominantly type
2 determinants). Therefore, each H. pylori strain may have a different niche within the gastric mucosa, and each individual LPS
blood group antigen may have a dissimilar role in H. pylori adaptation.
 |
INTRODUCTION |
During the past decade, much attention has been directed toward
the Gram-negative bacterium Helicobacter pylori and its
roles in gastritis, peptic ulcer disease, and gastric malignancies in humans (1). One class of molecules produced by Gram-negative enteric
bacteria are the cell surface lipopolysaccharides (LPSs1;
O-chain
core
lipid A), which often play important roles in
bacteria-host interactions (2). In 1994, the first detailed chemical
structure of H. pylori LPS showed that the O-chain region of
H. pylori type strain (NCTC 11637) was composed of an
elongated partially fucosylated type 2 N-acetyllactosamine
(LacNAc) polysaccharide covalently attached at the reducing end by a
core oligosaccharide and terminated at the nonreducing end by mono-,
di-, or trimeric Lewis X (Lex)
(
-D-Gal-(1
4)[
-L-Fuc-(1
3)]
-D-GlcNAc-(1
)
blood-group epitopes in mimicry of normal human cell surface
glycoconjugates and of glycan antigens found in adenocarcinoma tumors
(Refs. 3 and 4; see Fig. 1 for
the molecular structure of O-chain and core regions from
H. pylori type strain LPS). Since then, structural studies
on other H. pylori strains have revealed the presence of the
type 2 Ley
(
-L-Fuc-(1
2)-
-D-Gal-(1
4)[
-L-Fuc-(1
3)]
-D-GlcNAc-(1
)
determinant in the O-chains of strains P466 (5), MO19 (5), O:3, and O:6
(6). This Ley epitope may terminate an elongated partially
fucosylated type 2 LacNAc O-chain polysaccharide as in the cases of
strains P466 (5) and O:3 (6) or may be directly connected to the
remaining LPS molecule as in strains MO19 (5) and O:6 (6). Recently, the LPS of H. pylori strain UA861 was also found to contain
a type 2 LacNAc O-chain polysaccharide, but the lateral fucose was replaced by a glucose unit, and this LPS was terminated by a LacNAc (
-D-Gal-(1
4)-
-D-GlcNAc-(1
) epitope
and did not express terminal Lex or Ley
determinants (7). A preliminary account describing the presence of a
galactosylated N-acetyllactosamine O-chain in H. pylori strain 471 LPS has also been reported (8). This molecular
mimicry displayed by H. pylori LPSs with human cell surface
molecules, which also express Lex and Ley blood
group structures, is now the basis for the hypothesis that there might
be an autoimmune component in H. pylori pathogenesis (9).

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Fig. 1.
The complete structure of O-chain and core
regions of the LPS from H. pylori type strain NCTC 11637 (4). The O-chain region is an elongated (~10 repeating
units) partially fucosylated type 2 N-acetyllactosamine
polysaccharide terminated by the Lex determinant. The
O-chain is covalently linked to the core oligosaccharide through
a side chain DD-Hep. Kdo,
3-deoxy-manno-D-octulosonic acid.
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Following the discovery of Lex and Ley blood
group epitopes in H. pylori LPSs by chemical analysis,
several studies also have detected the presence of these Lewis blood
group antigens in many H. pylori strains by employing
commercially available monoclonal antibodies (mAbs) (10-13). Moreover,
some of these investigations also have described the presence of type 1 Lewis blood group antigens, such as Lea
(
-D-Gal-(1
3)[
-L-Fuc-(1
4)]
-D-GlcNAc-(1
),
Leb
(
-L-Fuc-(1
2)-
-D-Gal-(1
3)[
-L-Fuc-(1
4)]-
-D-GlcNAc-(1
), and H-type 1 (Led)
(
-L-Fuc-(1
2)-
-D-Gal-(1
3)-
-D-GlcNAc-(1
),
in some H. pylori strains (11, 12). Chemically based
structural studies then were initiated on H. pylori strains
suspected of carrying type 1 Lewis antigens (as shown by serology) to
confirm the presence of these epitopes in the LPS. These H. pylori strains are strain UA948, which reacted with both
Lex and Lea mAbs; UA955, which was recognized
by Leb, Ley, and Lex mAbs; and
J223, which reacted with a Leb mAb (12). The results from
the structural and serological investigations on the LPSs from
these H. pylori strains are reported in this paper.
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EXPERIMENTAL PROCEDURES |
Origin and Cell Production of H. pylori Strains--
Clinical
isolates of H. pylori UA948, UA955, and UA1182 were obtained
from dyspeptic patients at the University of Alberta Hospital by
methods described previously (14). These H. pylori strains
were frozen immediately after isolation (
70 °C). Cultures were
subsequently thawed and plated onto brain heart infusion agar (1.2%,
w/v) plates supplemented with 0.5% (w/v) yeast extract and 0.5% (v/v)
fetal bovine sera (Hyclone, Logan, UT). These H. pylori
strains were allowed to grow for 3 days under microaerobic conditions
at 37 °C, subcultured into BHI broth, and allowed to grow for an
additional 3 days under the same conditions with agitation. H. pylori strain J223 was isolated from a duodenal ulcer patient at
the Nashville Veterans Affairs Medical Center, and cells were grown as
described (12). H. pylori strains UA948, UA955, UA1182, and
J223 all were determined to be cagA+.
Isolation of Lipopolysaccharides--
The LPSs were isolated by
the hot phenol-water extraction procedure (15). The water-soluble LPSs
were purified by gel permeation chromatography on a column of Bio-Gel
P-2 (1 m × 1 cm) with water as eluent. In all cases, only one
carbohydrate-positive fraction was obtained which eluted in the high
Mr range (16). These intact H. pylori
LPSs then were used for chemical, spectroscopic, and serological
analyses.
Sugar Composition and Methylation Linkage
Analyses--
Sugar composition analysis was performed by the alditol
acetate method (17). The hydrolysis was done in 4 M
trifluoroacetic acid at 100 °C for 4 h followed by reduction in
H2O with NaBD4 and subsequent acetylation with
acetic anhydride and with residual sodium acetate as the catalyst.
Alditol acetate derivatives were analyzed by gas-liquid chromatography
mass spectrometry using a Hewlett-Packard chromatograph equipped with a
30-m DB-17 capillary column (210 °C (30 min)
240 °C at
2 °C/min), and MS in the electron impact mode was recorded using a
Varian Saturn II mass spectrometer. Enantiomeric configurations of the
individual sugars were determined by the formation of the respective
2-(S)- and 2-(R)-butyl chiral glycosides (18).
Methylation linkage analysis was carried out by the
NaOH/Me2SO/CH3I procedure (19) and with
characterization of permethylated alditol acetate derivatives by
gas-liquid chromatography mass spectrometry in the electron impact mode
(DB-17 column, isothermally at 190 °C for 60 min).
Fast Atom Bombardment-Mass Spectrometry (FAB-MS)--
A
fraction of the methylated sample was used for positive ion FAB-MS,
which was performed on a Jeol JMS-AX505H mass spectrometer with
glycerol (1): thioglycerol (3) as the matrix. A 6-kV xenon beam was
used to produce pseudomolecular ions, which were then accelerated to 3 kV, and their mass was analyzed. Product ion scan (B/E) and precursor
ion scan (B2/E) were preformed on metastable ions created
in the first free field with a source pressure of 5 × 10
5 torr. The interpretations of positive ion mass
spectra of the permethylated LPS derivatives were as described
previously by Dell et al. (20).
NMR Spectroscopy--
1H NMR spectra of the
water-soluble intact LPSs were recorded on a Bruker AMX 500 spectrometer at 300 K using standard Bruker software. Prior to
performing the NMR experiments, the samples were lyophilized
three times with D2O (99.9%). The HOD peak was used as the
internal reference at
H 4.786.
Serological Procedures--
For ELISA experiments on H. pylori strains UA948, UA955, and UA1182, cells were harvested from
cultures by centrifugation (5000 × g for 5 min) and
washed once with coating buffer (0.01 M sodium carbonate,
pH 9.5). The whole cell protein concentration was adjusted to 10 µg/ml and 100 µl of solution and added to each well of a microtiter
plate. The microtiter plate was incubated overnight at 4 °C, and
then nonadherant cells were removed by washing three times with
phosphate-buffered saline (pH 7.4) containing 0.05% (w/v) bovine serum
albumin, 0.05% (v/v) Tween 20, and 0.004% (w/v) Thimersol (wash
buffer). The reaction wells were blocked with blocking buffer
(phosphate-buffered saline, pH 7.4, containing 2.5% (w/v) bovine serum
albumin, 5% (v/v) fetal bovine serum, 0.05% (v/v) Tween 20, and
0.004% (w/v) Thimersol overnight at 4 °C. The microtiter plates
were washed three times with wash buffer and then incubated with the
primary antibodies against Lewis antigens. The antibodies used were
anti-Lea (mAb BG-5, clone T174), anti-Leb (mAb
BG-6, clone T218), anti-Lex (mAb BG-7, clone P12),
anti-Ley (mAb BG-8, clone F3). All mAbs were obtained from
Signet Laboratories Inc. (Dedham, MA). The primary antibodies were
diluted 1:100 in phosphate-buffered saline containing 0.5% (w/v)
bovine serum albumin, 1% (v/v) fetal bovine serum, 0.05% (v/v) Tween
20, and 0.004% (w/v) Thimersol for 2 h at 37 °C and washed as
described above. The secondary antibody (1:2000 dilution of anti-mouse
IgG plus IgM conjugated to horseradish peroxidase (Biocan, catalog
number 115 035 068, Mississauga, Ontario, Canada) was added to the
wells and incubated for 1.5 h at 37 °C. The microtiter plate
was developed at room temperature with 1 mM
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma, catalog
number A-1888), H2O2 (0.03%) in 0.01 M citrate buffer (pH 4.2) for 20-30 min with agitation
(150 rpm). The reaction was stopped with 4 mM sodium azide,
and the absorbance was recorded at 405 nm using a Titretek (Helsinki,
Finland) Multiscan MC microtiter plate reader.
For the acrylamide gel electrophoresis and immunoblot studies,
proteinase K-treated whole cells extracts of H. pylori
strains UA948, UA955, and UA1182 were prepared as described previously by Hitchcock and Brown (21). For the LPS analysis, a 15% (w/v) acrylamide separating gel containing urea (Life Technologies, Inc.) and
a 5% (w/v) polyacrylamide stacking gel were used. Electrophoresis was
conducted with a constant current of 35 mA for 1 h. These gels
were either silver-stained according to the method of Tsai and Frasch
(22) or electroblotted onto a nitrocellulose membrane (Micron
Separations Inc. Westboro, MA; pore size, 0.22 µm) according to the
methods described by Towbin et al. (23). Nitrocellulose membranes with transferred LPSs were probed with the antibodies described previously. Anti-Lewis (Signet Laboratories Inc.) antibodies diluted 1:100 were used as the primary antibody, and goat anti-mouse IgG plus IgM conjugated to horseradish peroxidase diluted 1:2000 was
the secondary antibody as described above. Reactions were visualized
using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech)
according to the manufacturer's specifications, and blots were
developed on BioMax film (Eastman Kodak Co.).
The LPS from strain J223 was serotyped in ELISA using procedures that
have been previously described (9, 11). For serotyping of H. pylori J223, the following mAbs were employed (11): CB-10 and
54.1F6A (both anti-Lex), 1E52 (anti-Ley), 7-Le
(anti-Lea), 225-Le (anti-Leb), 4D2 (anti-H type
1), 3-3A (anti-blood group A), and NAM61-1A2 (anti-i antigen) (24).
H. pylori J223 LPS was subjected to SDS-polyacrylamide gel
electrophoresis, silver-stained, and immunoblotted as described previously (11).
Polyclonal antisera against the core of LPS of four H. pylori strains were used whose specificity had been determined in
a previous study (25). Antisera were raised in rabbits immunized with
formalin-treated heat-killed H. pylori strains that
expressed rough form LPS lacking O-chain. Following incubation with the relevant primary antibody, the nitrocellulose membranes were washed as
described above and incubated with goat anti-mouse IgG-horseradish peroxidase conjugate (Sigma) and goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad), as appropriate, for 1 h at room temperature. After washing, reactions were visualized with the Bio-Rad
premixed enzyme substrate kit (2.5 ml of 4-chloro-1-naphtol in
diethylene glycol, 25 ml of Tris-buffered saline, and 15 µl of
H2O2) according to the manufacturer's
instructions.
 |
RESULTS |
All chemical, spectroscopic, and serological experiments were
performed on intact water-soluble LPS to prevent any aberrant results
that might arise due to the inadvertent loss of acid-labile glycose
units (such as fucose and sialic acid) that could take place during the
standardized mild acetic acid treatment of LPS that is normally used to
liberate the polysaccharide from the insoluble lipid A moiety of the
LPS molecule. Contrary to LPSs from typical Gram-negative bacteria,
H. pylori high Mr smooth-form LPSs
are soluble in water. The structural studies were performed on the LPSs
isolated from the same H. pylori cells with which the
serological experiments were carried out.
Characterization of the O-chain Region of H. pylori Strain
UA948--
The criteria of Wirth et al. (12), that an
absorbance value of <0.1 absorbance units was considered a negative
result in the ELISA, whereas higher values were positive, was used in
these studies. The serological experiments shown in Figs.
2 and 3
indicated that the LPS from H. pylori strain UA948 expressed
both Lea and Lex antigenic determinants.
H. pylori strain UA1182, whose LPS expresses a terminal
Ley epitope that terminates an O-chain of approximately
eight repeating internal Lex units, Ley-[
Lex-]8
core
lipid A2, used
as a control, was recognized by mAbs to Lex and
Ley, as expected. The reactions of the Lex and
Lea mAbs were directed at the O-chain region of UA948 LPS
as shown by the SDS-polyacrylamide gel electrophoresis and immunoblot
profiles (Fig. 2).

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Fig. 2.
SDS-polyacrylamide gel electrophoresis and
immunoblots of LPSs from H. pylori strains UA948, UA955,
andUA1182 with Lewis blood group mAbs. Lane A, strain UA948;
lane B, strain UA955; lane C, strain UA1182.
Positive interactions can be seen between UA948 and Lea and
Lex mAbs, between UA955 and Lex and
Ley mAbs, and between UA1182 (control) and Ley
mAb.
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Fig. 3.
H. pylori whole cell ELISA of strains
UA948, UA955, and UA1182. These data suggest that H. pylori
UA948 expresses both Lea and Lex antigens;
H. pylori UA955 expresses the Leb,
Lex, and Ley antigens; and H. pylori
UA1182 expresses Lex and Leyantigens. Only
absorbance values over 0.1 atomic unit were considered positive
(12).
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Sugar compositional analysis on the water-soluble intact UA948 LPS by
the alditol acetate method showed the presence of L-Fuc, D-Glc, D-Gal, D-GlcNAc,
D-glycero-D-manno-heptose
(DD-Hep), and L-glycero-D-manno-heptose
(LD-Hep) in the approximate molar ratio 1:1.4:3.6:3.4:1.5:1.3,
respectively. The methylation linkage analysis performed on the intact
UA948 LPS (Table I) revealed the presence of the following Lewis blood group-related units, terminal Fuc and Gal,
3-linked Gal, and 4- and 3,4-linked GlcNAc. No 2-linked Gal was
detected. Other permethylated alditol acetate derivatives of Glc, Gal,
DD-Hep, and LD-Hep were also detected (Table I) and, as shown later,
are placed within the core oligosaccharide framework of H. pylori LPS architecture (Fig. 1). To deduce the sequence of the
glycose units within the chain, FAB-MS was performed on the methylated
intact LPS. The FAB-MS spectrum of the methylated UA948 LPS derivative
(Fig. 4a) showed primary
fragment (A-type) glycosyl oxonium ions, from preferential cleavage at
the GlcNAc units (20, 26) and of defined compositions (Table
II), at m/z 638 (deoxy-Hex,
Hex, HexNAc), m/z 1087 (deoxy-Hex, Hex2,
HexNAc2), and at m/z 1261 (deoxy-Hex2, Hex2, HexNAc2). The
FAB-MS spectrum also showed two possible secondary fragment ions from
m/z 638, arising from
-elimination of the substituent at
O-3 of the branched 3,4-linked GlcNAc unit, those being, m/z
402 through loss of terminal Gal-OH (236 atomic mass units), which is a
characteristic pattern of the Lea structure (26), and
m/z 432 from the elimination of Fuc-OH (206 atomic mass
units), which is associated with the presence of the Lex
structure (26) (Table II). To demonstrate that ions m/z 638, 432, and 402 were in fact related, MS/MS experiments were performed on
these ions. Product (parent) ion MS/MS on m/z 638 (Fig.
4b) showed that both m/z 402 and 432 stem from
m/z 638, indicating that m/z 638 represented both
terminal Lex and Lea epitopes. Also, when
m/z 402 was subjected to a precursor (daughter) ion MS/MS it
showed m/z 638 has its origin, thus confirming the presence
of the Gal-1
3-[Fuc-1
4]GlcNAc (Lea) trisaccharide.
The higher mass primary ions at m/z 1087 and 1261 and the
secondary ion at m/z 1055 (m/z 1087-32 and
m/z 1261-206) represent a further type 2 chain extension of
the terminal Lewis epitope (638 atomic mass units) by a LacNAc unit
(449 atomic mass units),
3-Gal-1
4-GlcNAc-1
, and by an
additional internal Lex trisaccharide (623 atomic mass
units), respectively (Table II). These data do not differentiate
whether the type 2 LacNAc and Lex chain extensions are
attached to the type 1 terminal Lea or to the type 2 terminal Lex epitope. No evidence was obtained indicating
that a type 1,
3-Gal-1
3-GlcNAc-1
, chain extension existed, nor
could prominent m/z ions, which would have pointed toward a
connection of the Lex/a
LacNAc and Lex/a
Lex regions to units of the core be detected.
However, one dominant A-type primary fragment ion of defined
composition and that must include one heptose residue in addition to a
terminal Lewis trisaccharide was seen at m/z 886 (deoxy-Hex,
Hex, Hep, HexNAc) (Fig. 4a). Since there were no terminal
heptose and no 2-substituted galactose derivatives detected, this
primary ion must originate from cleavage at the heptosyl glycosidic
bond to give Gal
[Fuc
]GlcNAc
Hep+. Either the
terminal Lea or Lex antigen may be attached to
this heptose core-related unit. The primary fragment ion m/z
886 has also been observed in previous FAB-MS experiments with other
H. pylori strains, that showed the attachment of the first
Lex O-chain repeating unit to the core oligosaccharide (4,
5). The 1H NMR spectrum of the intact UA948 LPS confirmed
the presence of several Fuc residues (6CH3,
1.15-1.4) and GlcNAc (COCH3,
2.02 s) units. These
structural and serological studies were consistent in showing that the
O-chain of the LPS from H. pylori UA948 carried type 1 Lea and type 2 Lex terminal epitopes, which may
be extended by a further LacNAc or internal Lex units
(Table II).
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Table I
Sugar linkage analyses of the intact LPSs of H. pylori UA948, UA955,
and J223 showing approximate molar ratios
These ratios are approximate values, and any variances with ratios
from composition analysis represent either minor structural
differences between the samples used for each analyses due to molecular
heterogeneity or are simply a shortfall of the permethylated alditol
acetate derivatives in the linkage analysis procedures (an inherited
analytical feature of this type of analysis). In addition, composition
analysis by the alditol acetate method will detect GlcNAc from the
lipid A GlcN units, but these units will not be observed in the
methylation linkage analysis procedure, thus leading to some
discrepancy between these two ratios.
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Fig. 4.
FAB-MS spectra of H. pylori UA948
methylated intact LPS. a, the complete FAB-MS spectrum of
UA948. b, the product ion MS/MS of m/z 638.
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Table II
Interpretation of the ions from the FAB-MS spectrum of methylated
intact H. pylori UA948 LPS
The secondary ions shown originate from -elimination of the residue
at the O-3 position of the GlcNAc at the reducing end. The shill
indicates that either structure is possible.
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Characterization of the O-chain Region of H. pylori Strain
UA955--
The whole cell ELISA (Fig. 3) study performed on H. pylori UA955 suggested the presence of Lex,
Ley, and Leb epitopes in the LPS. However, in
immunoblots only a positive reaction with Lex and
Ley mAbs with the O-chain regions could be observed, but
not with the Leb mAb (Fig. 2).
Sugar compositional analysis of the intact UA955 LPS showed the
presence of L-Fuc, D-Glc, D-Gal,
D-GlcNAc, DD-Hep, and LD-Hep in the approximate ratio of
4:2:7:6:2:1, respectively. Lewis blood group-related derivatives were
the major components observed in the linkage analysis of UA955 LPS
(Fig. 5 and Table I), those being
terminal Fuc, 3-linked Gal, and 3,4-linked GlcNAc. Other detected
derivatives of the Lewis blood group lineage were terminal and 2-linked
Gal and 4-linked GlcNAc. The FAB-MS spectrum of the methylated UA955
LPS (Fig. 6a) showed the
following primary glycosyl oxonium ions (see Table
III for interpretation of ions),
m/z 434 (deoxy-Hex, HexNAc), 638 (deoxy-Hex, Hex, HexNAc),
812 (deoxy-Hex2, Hex, HexNAc), 1057 (deoxy-Hex2, Hex, HexNAc2), 1087 (deoxy-Hex2, Hex2, HexNAc2), 1261 (deoxy-Hex, Hex2, HexNAc2), 1435 (deoxy-Hex3, Hex2, HexNAc2), 1884 (deoxy-Hex3, Hex3, HexNAc3), and
2058 (deoxy-Hex4, Hex3, HexNAc3).
Notwithstanding the small quantities of material available, selective
MS/MS experiments were performed on three critical m/z ions,
those being the primary fragment ions m/z 638 (characteristic of Lea and Lex epitopes),
m/z 812 (typical of Leb and Ley
units), and the secondary fragment ion m/z 402, which may
arise from m/z 638 (Lea (638-236)), 812 (Leb (812-410)), or from m/z 434 (Lewis
disaccharide, Fuc-1
4-GlcNAc (434-32)). First, the product ion MS/MS
spectrum of m/z 638 (Fig. 6b) showed only one
secondary ion at m/z 432 due to
-elimination of Fuc-OH
(206) from one Lex epitope (26), but no m/z 402, which would have implicated Lea, was observed. Next, the
product ion MS/MS spectrum of ion m/z 812 (Fig.
6c) revealed that it lost 206 atomic mass units (Fuc-OH) to
yield m/z 606, which indicated that m/z 812 originated from a Ley determinant and not from
Leb (26). Finally, the precursor ion MS/MS spectrum of
m/z 402 (Fig. 6d) showed that this secondary ion
emanated from m/z 434 (Fuc-1
4-GlcNAc+) (Lewis
disaccharide) through
-elimination of methanol (434-32) and, as
shown in the product ion MS/MS experiments discussed above, did not
originate from m/z 638 nor 812, ruling out the presence of
terminal Lea and Leb epitopes in UA955 LPS.
Also seen in the higher m/z region of the FAB-MS spectrum of
UA955 LPS (Fig. 6a) are the primary and their corresponding
secondary ions that represent extensions of the O-chain region, which
can vary in glycosylation patterns (Table III). Of particular interest
is the presence of m/z 1057 and 851 (1057-206), whose
composition must include the Fuc-1
4-GlcNAc Lewis disaccharide
terminal epitope covalently attached to one internal Lex
repeat. The presence of these two different fucosylated GlcNAc derivatives, Fuc-1
4-GlcNAc and Fuc-1
3-GlcNAc, implies that the same O-chain molecule can express type 1 and type 2 structural regions
simultaneously. The longest chain observed in the FAB-MS spectrum of
UA955 LPS was a tetrafucosylated Ley
Lex
Lex sequence similar to that found in H. pylori strain P466 (5). The 1H NMR spectrum of the
intact UA955 LPS confirmed the presence of several Fuc residues
(6CH3,
1.2-1.4
J5,6,6' ~ 6 Hz) with one of major intensity at
H 1.2 belonging to the more abundant
-L-Fuc-1
3-
-D-GlcNAc and GlcNAc
(COCH3,
2.02 s) units. These investigations on
H. pylori UA955 LPS showed that the O-chain region consisted
of type 1 and 2 chain sequences. The type 1 chain was exemplified by
the
-L-Fuc-1
4-
-D-GlcNAc-1
(Lewis
disaccharide) terminal segment, and Lex and Ley
determinants represented the type 2 class in this LPS (Table III). No
Leb epitope was detected in the LPS from H. pylori UA955.

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Fig. 5.
Gas-liquid chromatogram of the permethylated
alditol acetates from H. pylori UA955 intact LPS. The
major components are terminal Fuc, 3-linked Gal, and 3,4-linked
GlcNAc.
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Fig. 6.
FAB-MS spectra of H. pylori UA955
methylated intact LPS. a, the complete FAB-MS spectrum of
UA955. b, the product ion MS/MS of m/z 638. c, the product ion MS/MS spectrum of m/z 812. d, the precursor ion MS/MS spectrum of m/z
402.
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Table III
Interpretation of the ions from the FAB-MS spectrum of methylated
intact H. pylori UA955 LPS
The secondary ions shown originate from -elimination of the residue
at the O-3 position of the GlcNAc at the reducing end.
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Characterization of the O-chain Region of H. pylori Strain
J223--
In a previous study (12) H. pylori J223 was
recognized by a Leb-specific mAb (mAb BG-6 from Signet
Laboratories, clone T218). To confirm the presence of the type 1 Leb determinant in the LPS of this strain, the following
structural experiments were performed.
D-Gal and D-GlcNAc were the major glycose units
present in the J223 LPS (Fig. 7),
L-Fuc, D-Glc, DD-Hep, and LD-Hep were also present in J223 LPS, the approximate molar ratio being
7.5:8:1:1.5:1.2:1.2, respectively. The Lewis blood group related
permethylated alditol acetate derivatives detected by the sugar linkage
analysis of J223 LPS (Table I) were terminal Fuc and Gal, 2- and
3-linked Gal, and 3- and 4-linked GlcNAc. The FAB-MS spectrum (Fig.
8a) of the methylated J223 LPS
derivative displayed prominent primary glycosyl oxonium ions at
m/z 464 (Hex, HexNAc), 638 (deoxy-Hex, Hex, HexNAc), 913 (Hex2, HexNAc2), and 1087 (deoxy-Hex,
Hex2, HexNAc2); the interpretation of these
ions is shown in Table IV. The product
ion MS/MS spectrum of m/z 464 (Hex-1
3- or
4-HexNAc+) showed m/z 432 as its major
precursor ion (Fig. 8b) arising from
-elimination of
methanol from O-3 of HexNAc, suggesting the presence of
Gal-1
4-GlcNAc (type 2 LacNAc) as a terminal sequence. Another
secondary ion arising from m/z 464 was m/z 228 (464-236) from a Gal-1
3-GlcNAc segment (type 1 precursor,
Lec). m/z 638 produced m/z 228 (638-410), from
-elimination of Fuc
Gal-OH, as the sole secondary
ion (Fig. 8c) that established the blood group H-type 1 epitope (Led) (Fuc-1
2-Gal-1
3-GlcNAc) (26) as a
terminal antigen in the J223 LPS. There was no evidence pointing toward
the presence of an H-type 2 epitope (i.e. m/z 638
606).
The primary ions at m/z 913 and 1087 represent extended
N-acetyllactosamine chains (Table IV). m/z 913 represents two sequential fucose-free LacNAc repeats, which, due to the
fact that only slightly more than one unit of 3-linked GlcNAc
derivative was detected (Table I), is consistent with the presence of a
type i antigen (Gal-1-[
4-GlcNAc-1
3-Gal-1-]n
) (Table
IV), which correlates to the significant number of 4-linked GlcNAc
units observed (Table I). However, since only a trace of m/z
881 (913-32) was observed in the J223 LPS FAB-MS, the possibility of a
type 1 chain extension with a
3-Gal-1
3-GlcNAc-1
disaccharide cannot be excluded. The absence of a branched 3,4-substituted GlcNAc
unit and, consequently, of any terminal Lex or
Lea blood group epitopes implies that m/z 1087 must be composed of a terminal H-1 unit (Led) and one
Gal
GlcNAc repeat as shown in Table IV. The 1H NMR
spectrum of the intact J223 LPS showed the characteristic deoxy
resonances from the sole Fuc residue (6CH3,
1.25 J5,6,6' ~ 6 Hz) and the acetamido signals
from the GlcNAc (COCH3,
2.02 s) units.

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Fig. 7.
Gas-liquid chromatogram of the alditol
acetate derivatives from H. pylori J223 intact LPS.
The major components are Fuc, Gal, and GlcNAc.
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Fig. 8.
FAB-MS spectra of H. pylori J223
methylated intact LPS. a, the complete FAB-MS spectrum J223.
b, the product ion MS/MS of m/z 464. c, the product ion MS/MS spectrum of m/z
638.
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Table IV
Interpretation of the ions from the FAB-MS spectrum of methylated
intact H. pylori J223 LPS
The secondary ions shown originate from -elimination of the residue
at the O-3 position of the GlcNAc at the reducing end. The shill
indicates that either structure is possible.
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Purified H. pylori J223 LPS was then used in a series of
additional serological experiments with mAbs specific for Lewis blood group antigens. In ELISA, J223 LPS reacted strongly
(A492 > 2.6) with mAbs 4D2 (anti H-type 1) and
NAM61-1A2 (anti-i antigen). No reactions (A <0.3) were
observed between J223 LPS and mAbs specific for Lex,
Ley, blood group A, or Lea, nor with
Leb mAb 225-Le. The immunoblot (Fig.
9) shows the recognition of H-type 1 (Led) and of the i antigen in J223 LPS. Both mAbs in Fig. 9
recognize the "runs" of the J223 LPS ladder which represent the
O-chain region. Collectively, structural and serological investigations performed on the J223 LPS revealed that the O-chain region was composed
of an H-type 1 terminal antigen (Led) and of a
nonfucosylated type 2 LacNAc chain
-D-Gal-1
4-
-D-GlcNAc-1-[
3-
-D-Gal-1
4-
-D-GlcNAc-1-]n
(i antigen); no Leb antigen was detected.

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Fig. 9.
SDS-polyacrylamide gel electrophoresis and
immunoblot of J223 LPS. Left lane, silver stain of J223 LPS;
middle lane, immunoblot with anti-H-type 1 (Led)
mAb; right lane, immunoblot with anti-i antigen mAb.
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Core Regions of H. pylori Strains UA948, UA955, and J223--
In
addition to the O-chain Lewis blood group glycose units, for all three
strains, all chemical analysis revealed the presence of LD-Hep, DD-Hep,
D-Glc, and D-Gal derivatives, which were
surmised to originate from the core region (Table I; Figs. 5 and 7).
The LD-Hep, DD-Hep, D-Glc, and D-Gal
permethylated alditol acetate derivatives (Table I) emanating from
the core regions of the LPS molecules studied here were of the same
type as previously found in the core area of other H. pylori
strains (4-7). However, in strains UA948, UA955, and J223, the same
type of core may carry type 1 and 2 blood group epitopes as members of
the O-chain. To add assurance to this apparent similarity between the
core regions of H. pylori strains, H. pylori J223
LPS was subjected to immunodot analysis with antisera against the core
of four strains, the type strain NCTC 11637 (4), C-5437, S-24, and K1
(25). H. pylori J223 LPS reacted with the four antisera,
confirming the presence of similar epitopes as in the core of these
H. pylori LPSs.
The chemical analyses performed in these studies were not aimed at
obtaining information about the residues belonging to the lipid A
moiety. However, in the 1H NMR spectrums of the intact LPSs
of the strains investigated here, different patterns of resonances
emanating from the lipid A fatty acids were observed, implying that
there exists some variety in fatty acid substitution between H. pylori strains. Two recent investigations have dealt with the
structural features of H. pylori lipid A molecules (27,
28).
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DISCUSSION |
This investigation on the chemical composition of LPSs from
H. pylori has added new insight into the structure of these
molecules. The most significant new finding is that H. pylori LPSs can express type 1 Lewis blood group determinants,
namely, Lea, H-1 (Led), and the type 1 chain
precursor (Lec). Type 2 epitopes, Lex and
Ley, have been described to be present in H. pylori LPSs in earlier studies (Refs. 3-6; Table
V). Chemical analyses in combination with
serological experiments (mAbs) showed that the O-chain from H. pylori strain UA948 LPS possessed both Lea (type 1 chain) and Lex (type 2 chain) antigens (Figs. 2-4; Table
II). The O-chain of H. pylori strain UA955 LPS (Figs. 2, 3,
5, and 6; Table III) expressed both type 2 chain Lex and
Ley determinants and a sequence composed of type 1 and 2 chain regions in Fuc-1
4-GlcNAc-1
Lex. The LPS of
H. pylori UA955 also was composed of elongated sundrily fucosylated LacNAc chains (Table III). H. pylori strain J223
LPS (Figs. 7-9; Table IV) also showed the ability to express type 1 chains by carrying the type 1 chain precursor Lec, the H-1
epitope (Led), and type 2 chain molecules (LacNAc chain (i
antigen)). The core oligosaccharide regions of H. pylori
UA948, UA955, and J223 appear to have the same structure as those
from H. pylori strains previously investigated (Fig. 1 and
Table I) (4-7), despite their ability to carry a variety of type 1 and
2 Lewis blood group antigens representing the O-chain segment.
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Table V
Molecular makeup of H. pylori LPSs investigated by structural analysis
to date and their classification into glycotypes
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The expression of type 1 Lewis blood group antigens suggests that for
the biosynthesis of some H. pylori LPS molecules there must
be glycosyltransferases of the type 1 family, such as a fucosyl transferase(s) that places the
-L-Fuc unit at O-4 of
-D-GlcNAc and a galactosyl transferase that adds
-D-Gal to O-3 of GlcNAc. The LPS from the type strain of
H. mustelae, the Helicobacter gastric pathogen
from ferrets, also carries a type 1 histo-blood group epitope, the
monofucosyl blood group A type 1 (
-D-GalNAc-1
3[
-L-Fuc-1
2]-
-D-Gal-1
3-
-D-GlcNAc) (29). The concurrent expression of type 1 and 2 chains and of various
chains with different glycosylation patterns within the LPS of a single
H. pylori strain represents a complex biosynthesis of these
molecules, which differs from the well known "block-by-block" O-chain repeating unit addition mechanism of Escherichia and
Salmonella LPS biosynthesis (30). Various factors, such as
differential enzyme kinetics, regulation, and mutation or clonal
variation may control the assembly of H. pylori LPS
molecules.
The genome of H. pylori strain 26695 and other strains
contain two copies of the
-1-3-fucosyltransferase and multiple
copies may play a role in the expression of the Lewis antigens
(31-33). Recently, the presence of a possible
-1-2-fucosyltransferase gene in H. pylori has also been
described (34). The
-1-4-fucosyltransferase genes to produce the
Lea and Leb epitopes have not yet been
identified. Many strains express Lex and Ley,
but few seem to express type 1 epitopes (11, 12). This phenomenon might
be due to regulation whereby all H. pylori strains contain the genes for the assembly of these alternative structures, but in
certain instances some transferases are not expressed; alternatively, only certain strains may contain the appropriate genes required to
produce the type 1 antigens. Separately grown bacterial culture batches
of the same H. pylori strain may express different degrees of fucosylation (Fuc-1
3-GlcNAc and Fuc-1
2-Gal), suggesting that the transferases responsible for these glycosylations are variably expressed.2 Diversity in
fucosylation and thus in Lex and Ley expression
among single colonies derived from the same gastric biopsy suggests
that this phenomenon might occur in vivo as well (35).
The serological studies that showed recognition of the Leb
epitope in H. pylori LPSs with the Leb-specific
mAb BG-6 (clone T218) in strains UA955 and J223 were recognizing only
substructures of the Leb antigen. In H. pylori
UA955, mAb BG-6 presumably detected the
-L-Fuc-1
4-
-D-GlcNAc Lewis
disaccharide, which is a region of the Leb determinant
(
-L-Fuc-1
2-
-D-Gal-1
3[
-L-Fuc-1
4]-
-D-GlcNAc) (Fig. 10), and in H. pylori
J223, mAb BG-6 either recognized the H-1 antigen (Led)
(
-L-Fuc-1
2-
-D-Gal-1
3-
-D-GlcNAc)
or Lec
(
-D-Gal-(1
3)-
-D-GlcNAc), which are
biosynthetic precursors of Leb
(
-L-Fuc-1
2-
-D-Gal-1
3[
-L-Fuc-1
4]-
-D-GlcNAc)
(Fig. 10) (see Ref. 36 for a review concerning Lewis biosynthetic
pathways and Leb mAb cross-reactivity). The
Fuc-1
4-GlcNAc terminal unit in H. pylori UA955 may either
be a biosynthetic precursor of Lea or Leb in
H. pylori LPS biosynthesis, or it represents a dead end
product due to premature fucosylation of GlcNAc at O-4, inhibiting
further galactosylation of GlcNAc at O-3. The cross-reactivity observed between Leb mAb BG-6 and H. pylori J223 LPS
seems to be a mAb-specific phenomenon, since Leb mAb 225-Le
did not react with J223 LPS. Imberty et al. (37) has
reported that blood group determinants have certain conformational dependent "microepitopes" toward which different mAbs have
dissimilar reactivities. It is also worth noting that the same Le mAb
might have different sensitivity toward the same LPS epitopes when
tested under ELISA or immunoblot conditions, as indicated by the
reaction of Leb mAb with UA955 in ELISA (Fig. 3), but not
in immunoblot after removal of the proteins by proteinase K (Fig. 2).
Isolated H. pylori UA955 proteins did not react with the
Leb mAb, suggesting that the observed activity in Fig. 3 is
not to the protein. Also, when whole cells of UA955 were tested in
immunoblot, the Leb mAb BG-6 showed no reaction, suggesting
that recognition of UA955 LPS by mAb BG-6 is lost under immunoblot
conditions. The same type of phenomenon can also be observed with
strain UA1182, whose internal Lewis X epitopes are well recognized by
the Lewis X mAb in ELISA (Fig. 3), but show only a weak interaction in
immunoblot (Fig. 2). We suspect that these two differences in
reactivities are caused by conformational changes in the LPS molecules.
These studies illustrate the necessity for performing structural rather than serological studies to define H. pylori LPS
antigens.

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Fig. 10.
The Leb structure. The
Lewis disaccharide
-L-Fuc-1 4- -D-GlcNAc-1 is present in
H. pylori UA955, and the H-type 1 (Led)
-L-Fuc-1 2- -D-Gal-1 3- -D-GlcNAc-1
and Lec
-D-Gal-1 3- -D-GlcNAc-1 epitopes are
part of the LPS from H. pylori J223.
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Using the structural information for H. pylori LPSs
available to date (Table V) permits their classification into six
different groups (glycotypes A-F; see Table V). (i) Glycotype A
contains LPSs with long fucosylated type 2 LacNAc O-chains attached at the reducing end to the core oligosaccharide, as in strains NCTC11637 (4), P466 (5), and UA11822; (ii) glycotype B contains LPSs
with long fucosylated type 2 LacNAc O-chains attached to a heptan
domain, as in strain O:3 (6); (iii) glycotype C contains LPSs with
short O-chains (one repeat), which are attached to a heptan domain, as
in strains MO19 (5) and O:6 (6); (iv) glycotype D contains LPSs with glucosylated type 2 LacNAc O-chains as in strain UA861 (7); (v)
glycotype E contains LPSs with galactosylated type 2 LacNAc O-chains as
in strain 471 (8); and (as found in this study) (vi) glycotype F
contains LPSs, which, in addition to type 2 Lewis determinants, also
possess chains composed of type 1 Lewis epitopes, as in strains UA948,
UA955, and J223.
The most striking feature of the O-chains of H. pylori LPSs
is their ability to mimic human cell surface glycoconjugates and glycolipids that have Lewis structures. Past structural investigations revealed that H. pylori LPSs mimicked type 2 Lex
and Ley human cell surface glycoforms (Refs. 3-6; Table V)
and that this molecular imitation may be an antecedent for autoimmunity (9). Alternatively, host-related microbial Lewis expression could help
avoid host immune responses (38). The molecular mimicry between
H. pylori LPSs and host molecules has now been extended to
include the type 1 determinants Lea, Led, and
Lec. This ability of H. pylori to produce
various Lewis isoforms permits mimicking of all regions of the gastric
epithelium, those being the gastric superficial and glandular
epithelium, which display mainly type 2 molecules, and the superficial
epithelium, which expresses predominantly type 1 chains (39-41).
Consequently, each H. pylori strain, depending on the
antigens expressed by its LPS, may have a different ecological niche
within the gastric mucosa, and ultimately the role of LPS in
pathogenesis and adaptation may differ between H. pylori
strains.