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INTRODUCTION |
Adhesion of microorganisms to target cells is regarded as a first
step in pathogenesis of infections, where the specificity of the
adhesins of the infectious agent on the one hand and the receptor
structures expressed by the epithelial cells of the host target organ
on the other are important determinants of the host range and the
tissue tropism of the pathogen (1).
The human gastric pathogen Helicobacter pylori is an
etiologic agent of chronic active gastritis, peptic ulcer disease, and gastric adenocarcinoma (2, 3). This Gram-negative bacterium has a very
distinct host range and tissue tropism, i.e. it requires human gastric epithelium for colonization (4). In the human stomach
most of the bacteria are found in the mucus layer (5), but selective
association of the bacteria to surface mucous cells has also been shown
(4, 6).
Several different binding specificities of H. pylori have
previously been demonstrated. Thus, the binding of the bacterium to
such diverse compounds as phosphatidylethanolamine and
gangliotetraosylceramide (7), the Leb blood group
determinant (8), heparan sulfate (9), the
GM31 ganglioside and
sulfatide (10, 11), and lactosylceramide (12), has been reported. A
sialic acid-dependent binding of H. pylori to
large complex glycosphingolipids (polyglycosylceramides) has also been
documented (13). However, only one H. pylori adhesin, the
Leb binding BabA adhesin, has been identified to date
(14).
In the present study a number of different H. pylori strains
were labeled with [35S]methionine and examined for
binding to a panel of different naturally occurring glycosphingolipids
separated on thin-layer plates. Two distinct binding specificities were
repeatedly detected by autoradiography. As previously described in
detail, H. pylori bound to lactosylceramide,
gangliotriaosylceramide, and gangliotetraosylceramide (12). The only
binding activity initially detected in human gastrointestinal material
was to a compound in the tetraglycosylceramide region of the non-acid
fraction of human meconium. The isolation and structural
characterization of this H. pylori binding glycosphingolipid and the identification of the same compound in human gastric epithelial cells are described in the present paper.
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MATERIALS AND METHODS |
Bacterial Strains, Culture Conditions, and Labeling--
The
bacteria used and their sources are described in Table
I. In most of the experiments four
strains, the type strains CCUG 17874 and 17875 (obtained from Culture
Collection, University of Göteborg, (CCUG), Sweden, and the
clinical isolates S-002, and S-032, were used in parallel.
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Table I
Helicobacter pylori isolates used in binding assays
Strains I-VII were obtained from the following sources. I. Strains were
obtained from the Culture Collection University of Göteborg
(CCUG), Sweden. II. Strains were obtained from the Department of
Medical Microbiology and Immunology, Örebro Medical Centre
Hospital, Sweden. III. Strains were obtained from the Department of
Medical Microbiology, University of Lund, Sweden. IV. Strains were
obtained from the Department of Microbiology, Medical University of
Wroclaw, Poland. V. These strains were a kind gift of Dr. Thomas
Borén, Umeå University, Sweden. VI. These strains were a kind
gift of Dr. Rainer Haas, Ludwig-Maximilians-Universität, Munich,
Germany. VII. These strains were a kind gift of Dr. Ingrid Bölin,
Göteborg University, Sweden.
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The conditions used for culture and 35S-labeling of the
bacteria have been described previously (12). For binding assays, the
bacteria were suspended to 1 × 108 colony forming
units/ml in phosphate-buffered saline
(PBS),2 pH 7.4. The specific
activities of the suspensions were ~1 cpm/100 H. pylori organisms.
Thin-layer Chromatography--
Thin-layer chromatography was
performed on glass- or aluminum-backed silica gel 60 high performance
thin-layer chromatography plates (Merck) using
chloroform/methanol/water (60:35:8, by volume) as the solvent system.
Chemical detection was accomplished by anisaldehyde (15).
Chromatogram Binding Assay--
The chromatogram binding assays
were done as described (16). Mixtures of glycosphingolipids (20-80
µg/lane) or pure compounds (1-4 µg/lane) were separated on
aluminum-backed silica gel plates. The dried chromatograms were soaked
for 1 min in diethyl ether/n-hexane (1:5, by volume)
containing 0.5% (w/v) polyisobutylmethacrylate (Aldrich). After
drying, the chromatograms were coated to block unspecific binding
sites. Initially different coating conditions were tested,
e.g. 1% polyvinylpyrrolidone (w/v) in PBS (Solution 1), 2%
gelatin (w/v) in PBS (Solution 2), 2% bovine serum albumin (w/v) in
PBS (Solution 3), 2% bovine serum albumin (w/v) and 0.1% (w/v) Tween
20 in PBS (Solution 4), or 2% bovine serum albumin (w/v) and 0.2%
(w/v) deoxycholic acid in PBS (Solution 5). The most consistent results
were obtained with Solution 4, which subsequently was used as the
standard condition. Coating was done for 2 h at room temperature.
Thereafter, a suspension of 35S-labeled bacteria (diluted
in PBS to 1 × 108 colony forming units/ml and
1-5 × 106 cpm/ml) was gently sprinkled over the
chromatograms and incubated for 2 h at room temperature. After
washing six times with PBS and drying, the thin-layer plates were
autoradiographed for 3-120 h using XAR-5 x-ray films (Eastman Kodak
Co.).
Reference Glycosphingolipids--
Acid and non-acid
glycosphingolipid fractions from the sources given in the legend of
Fig. 1 and in Table III were obtained by standard procedures
(17). The individual glycosphingolipids were isolated by acetylation of
the total glycosphingolipid fractions and repeated chromatography on
silicic acid columns. The identity of the purified glycosphingolipids
was confirmed by mass spectrometry (18), proton NMR spectroscopy (19),
and degradation studies (20, 21).
Gal
3GlcNH23Gal4Glc1Cer (No. 3 of Table
III) was generated from Gal3GlcNAc3Gal4Glc1Cer (No. 2) by
treatment with anhydrous hydrazine, as described (12).
Isolation of the H. pylori Binding Tetraglycosylceramide from
Human Meconium--
A total non-acid glycosphingolipid fraction (262 mg) was obtained from 17 pooled meconia by standard methods (17). The
non-acid glycosphingolipids (240 mg) were first separated by HPLC on a 2.2 × 30-cm silica column (YMC SH-044-10, 10 µm particles;
Skandinaviska Genetec, Kungsbacka, Sweden) eluted with a linear
gradient of chloroform/methanol/water 65:25:4 to 40:40:12 (by volume)
for 180 min and with a flow of 2 ml/min. Aliquots of each 2-ml fraction were analyzed by thin-layer chromatography, and the fractions positive
for anisaldehyde staining were further tested for binding of H. pylori using the chromatogram binding assay. The H. pylori binding fractions were collected in tubes 78-88, and after
pooling of these fractions, 14.2 mg were obtained. This material was
acetylated and further separated by HPLC on a YMC SH-044-10 column
eluted with a linear gradient of chloroform/methanol (95:5, by volume) in chloroform for 90 min and with a flow of 2 ml/min. After
deacetylation, aliquots from each 1-ml fraction were analyzed by
anisaldehyde staining on thin-layer chromatograms, and the
glycosphingolipid-containing fractions were examined for H. pylori binding activity. Most of the H. pylori binding
glycosphingolipid was collected in tube 62, and this fraction (2.4 mg)
was used for structural characterization.
Isolation of Non-acid Glycosphingolipids from Human Gastric
Epithelium--
Stomach tissue (10 × 10-cm pieces) were obtained
from the fundus region from patients undergoing elective surgery for
morbid obesity. After washing with 0.9% NaCl (w/v), the mucosal cells were gently scraped off and kept at 
70 °C. The material was
lyophilized, and acid and non-acid glycosphingolipids were isolated as
described (17). In two cases glycosphingolipids were also isolated from the non-mucosal residues. The blood group of the patients and the
amounts of glycosphingolipids obtained from each specimen are given in
Table II.
The non-acid glycosphingolipids from case 4 (2.9 mg) were separated by
HPLC on a 1.0 × 25-cm silica column (Kromasil-Sil, 10-µm
particles, Skandinaviska Genetec) using a gradient of
chloroform/methanol/water 65:25:4 to 40:40:12 (by volume) over 180 min
with a flow rate of 2 ml/min. Aliquots from each fraction were analyzed
by thin-layer chromatography using anisaldehyde as the staining
reagent. The tetraglycosylceramides were collected in tubes 12-17.
Tubes 12-14 also contained a compound with mobility in the
triglycosylceramide region on thin-layer chromatograms and, after
pooling of these three fractions, 0.2 mg was obtained (designated
fraction 4-I). The fractions in tubes 15-17 were pooled separately,
giving 0.5 mg of tetraglycosylceramides (designated fraction 4-II).
Separation of 10.0 mg of the non-acid glycosphingolipid fraction from
case 5 was done using the same system as above, with a gradient of
chloroform/methanol/water 60:35:8 to 40:40:12 (by volume). The fraction
collected in tube 11 (designated fraction 5-I) contained
triglycosylceramides and tetraglycosylceramides (0.1 mg), whereas only
tetraglycosylceramides were obtained in tube 12 and 13. Pooling of the
latter two fractions resulted in 0.3 mg (designated fraction 5-II).
EI Mass Spectrometry--
Before mass spectrometry, the
glycosphingolipids were permethylated, as described (22). The
tetraglycosylceramide isolated from human meconium was analyzed on a VG
ZAB 2F/HF mass spectrometer (VG Analytical, Manchester, UK) using the
in-beam technique (23). Analytical conditions were electron energy 45 eV, trap current 500 µA, and acceleration voltage 8 kV. Starting at
250 °C, the temperature was elevated by 6 °C/min.
The tetraglycosylceramides from the mucosal cells of human stomach were
analyzed by the same technique on a JEOL SX-102A mass spectrometer
(JEOL, Tokyo, Japan). Analytical conditions were electron energy 70 eV,
trap current 300 µA, and acceleration voltage 10 kV. The temperature
was raised by 15 °C/min, starting at 150 °C.
Degradation Studies--
The permethylated glycosphingolipid
from human meconium was hydrolyzed, reduced, and acetylated (20, 21),
and the partially methylated alditol and hexosaminitol acetates
obtained were analyzed by gas chromatography-EI mass spectrometry on a
Trio-2 quadrupole mass spectrometer (VG Masslab, Altrincham, UK). The
Hewlett Packard 5890A gas chromatograph was equipped with an on-column
injector and a 15 m × 0.25-mm fused silica capillary column, DB-5
(J&W Scientific, Ranco Cordova, CA), with 0.25-µm film thickness. The samples were injected on-column at 70 °C (1 min), and the oven temperature was increased from 70 to 170 °C at 50 °C/min and from 170 °C to 260 °C at 8 °C/min. Conditions for mass spectrometry were electron energy 40 eV and trap current 200 µA. The components were identified by comparison of retention times and mass spectra of
partially methylated alditol acetates obtained from reference glycosphingolipids.
Proton NMR Spectroscopy--
Proton NMR spectra were acquired at
7.05 Tesla (300 MHz) on a Varian VXR 300 (Varian, Palo Alto, CA)
and at 11.75 Tesla (500 MHz) on a JEOL Alpha-500 (JEOL, Tokyo, Japan).
Data were processed off-line using NMR1 (NMRi, Syracuse, NY). The
deuterium-exchanged glycosphingolipid fractions were dissolved in
dimethyl sulfoxide-d6/D2O (98:2, by
volume), and spectra were recorded at 30 °C with a 0.4-Hz digital
resolution. Chemical shifts are given relative to tetramethylsilane.
Ceramide Glycanase Treatment of Tetraglycosylceramides from Human
Gastric Epithelium--
The procedure of Hansson et al.
(24) was used for the enzymatic hydrolysis. Briefly, 100 µg of
fraction 4-II from case 4, fraction 5-II from case 5, reference
globoside from human erythrocytes (25), reference
lactotetraosylceramide from human meconium, and reference
lactoneotetraosylceramide (obtained by sialidase treatment of
sialyl-lactoneotetraosylceramide from human erythrocytes; Ref. 26) were
dissolved in 100 µl of 0.05 M sodium acetate buffer, pH
5.0, containing 120 µg sodium cholate and sonicated briefly. Thereafter, 1 milliunit of ceramide glycanase from the leech, Macrobdella decora (Roche Molecular Biochemicals) was added,
and the mixtures were incubated at 37 °C for 24 h. The reaction
was stopped by the addition of chloroform/methanol/water to the final proportions 8:4:3 (by volume). The oligosaccharide-containing upper
phase thus obtained was separated from ceramides and detergent on a
Sep-Pak C18 cartridge (Waters, Milford, MA). The eluant
containing the oligosaccharides was dried under nitrogen and under
vacuum and thereafter permethylated as described (22).
High Temperature Gas Chromatography and Gas Chromatography-EI
Mass Spectrometry of the Permethylated Oligosaccharides--
The
analytical conditions were essentially the same as described in
Karlsson et al. (27). Capillary gas chromatography was performed on a Hewlett Packard 5890A gas chromatograph using a fused
silica column (10 m x 0.25-mm internal diameter) coated with 0.03 µm
of cross-linked PS 264 (Fluka, Buchs, Switzerland) and with hydrogen as
carrier gas. The permethylated oligosaccharides were dissolved in ethyl
acetate, and 1 µl of sample was injected on-column at 70 °C (1 min). A two-step temperature program was used, 70 °C to 200 °C at
50 °C/min followed by 10 °C/min up to 350 °C.
Gas Chromatography-EI mass spectrometry was performed on a Hewlett
Packard 5890-II gas chromatograph coupled to a JEOL SX-102A mass
spectrometer. The chromatographic conditions as well as the capillary
column were the same as for the analyses by gas chromatography, and the
conditions for mass spectrometry were interface temperature 350 °C,
ion source temperature 330 °C, electron energy 70 eV, trap current
300 µA, and acceleration voltage 10 kV.
Inhibition with Soluble Oligosaccharides--
As a test for
possible inhibition of binding by soluble sugars
35S-labeled H. pylori strains S-002 and S-032
were incubated for 1 h at room temperature with various
concentrations (0.05, 0.1, and 0.2 mg/ml) of lactotetraose (Accurate
Chem. and Sci. Corp., Westbury, NY) or lactose (J. T. Baker Inc.)
in PBS. Thereafter the chromatogram binding assay was performed as
described above.
Molecular Modeling--
Minimum energy conformations of the
various glycosphingolipids listed in Table
III were calculated within the Biograf
molecular modeling program (Molecular Simulations Inc., Waltham, MA)
using the Dreiding-II force field (28) on a Silicon Graphics4D/35TG work station. Charges were generated using the charge equilibration method (29), and a distance-dependent dielectric constant
of 3.5 was used for the Coulomb interactions. In addition, a
special hydrogen bonding term was used in which Dhb was set
to 4 kcal/mol (28).
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RESULTS |
Binding to Mixtures of Reference Glycosphingolipids--
A number
of well characterized glycosphingolipid mixtures representing a large
variety of carbohydrate sequences were separated by thin-layer
chromatography. One chromatogram was stained with anisaldehyde, and
duplicate chromatograms were used for binding of
35S-labeled H. pylori. By subsequent
autoradiography only a few bands were visualized, as shown in Fig.
1B. The binding in lane 4 (gangliotriaosylceramide) and lane 7 (gangliotetraosylceramide) was judged to correspond to the "ganglio
binding specificity" of H. pylori described previously in
detail (12).
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Fig. 1.
Detection of a H. pylori
binding glycosphingolipid in the non-acid glycosphingolipid
fraction of human meconium. A, glycosphingolipids
detected with anisaldehyde. B, glycosphingolipids detected
by autoradiography after binding of radiolabeled H. pylori
strain CCUG17875. The glycosphingolipids were separated on
aluminum-backed silica gel plates using chloroform/methanol/water
(60:35:8, by volume) as the solvent system, and the binding assay was
performed as described under "Materials and Methods." The
autoradiogram in B was obtained after coating of the
thin-layer chromatogram with 2% BSA and 0.1% Tween 20 in PBS. The
lanes contained non-acid glycosphingolipids of human blood group A
erythrocytes, 40 µg (lane 1); non-acid glycosphingolipids
of dog small intestine, 40 µg (lane 2); non-acid
glycosphingolipids of guinea pig small intestine, 40 µg (lane
3); non-acid glycosphingolipids of guinea pig erythrocytes, 40 µg (lane 4); non-acid glycosphingolipids of rat small
intestinal epithelium, 40 µg (lane 5); non-acid
glycosphingolipids of human meconium, 40 µg (lane 6);
non-acid glycosphingolipids of mouse feces, 40 µg (lane
7); acid glycosphingolipids of human blood group O erythrocytes,
40 µg (lane 8); bovine brain gangliosides, 40 µg
(lane 9). Autoradiography was for 12 h.
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In addition, selective binding of H. pylori to a compound
migrating in the tetraglycosylceramide region in the non-acid
glycosphingolipid fraction of human meconium was detected (Fig.
1B, lane 6). This binding was only obtained when
detergent (Tween 20 or deoxycholic acid) was present in the coating
buffer. Solution 4 (2% bovine serum albumin and 0.1% Tween 20 in PBS)
was therefore subsequently utilized as standard coating procedure. The
binding-active tetraglycosylceramide from human meconium was isolated
by HPLC and characterized by mass spectrometry, proton NMR, and gas
chromatography-EI mass spectrometry after degradation as follows.
Chemical Structure of the H. pylori Binding Glycosphingolipid from
Human Meconium--
The binding-active tetraglycosylceramide was
isolated from 240 mg of total non-acid glycosphingolipids. By HPLC of
the native glycosphingolipid fraction, 14.2 mg of
tetraglycosylceramides were obtained. The tetraglycosylceramide
fraction was acetylated and further separated by HPLC, giving 2.4 mg of
pure binding-active glycosphingolipid. Each step during the preparative
procedure was monitored by binding of radiolabeled H. pylori
on thin-layer chromatograms.
Structural characterization identified lactotetraosylceramide
(Gal3GlcNAc3Gal4Glc1Cer) as the binding-active component. This conclusion was based on the following observations.
EI mass spectrometry of the permethylated tetraglycosylceramide
(Fig. 2) demonstrated a carbohydrate
chain with Hex-HexNAc-Hex-Hex sequence and d18:1 and t18:0 long chain
bases combined with both hydroxy and non-hydroxy fatty acids of mainly
22 and 24 carbon atoms. A type 1 chain (Hex3HexNAc) was indicated by
the absence of a fragment ion at m/z 182, which
is a dominating ion in the case of 4-substituted HexNAc (30, 31).
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Fig. 2.
EI mass spectrum of the permethylated
H. pylori binding tetraglycosylceramide from human
meconium. The spectrum was recorded at 300 °C. Above the
spectrum is a simplified formula for interpretation, representing the
species with sphingosine and hydroxy 24:0 fatty acid. The carbohydrate
sequence ions at m/z 219 and 187 (219 32), 464, 668, and 872 demonstrated a tetraglycosylceramide with
Hex-HexNAc-Hex-Hex sequence. This was supported by the fragment ion at
m/z 945 (944 + 1), which consisted of the whole
carbohydrate chain and part of the fatty acid. Molecular ions
corresponding to the species with d18:1-24:0, d18:1-h22:0, and
d18:1-h24:0 ceramides were found at m/z 1548, 1550, and 1578, respectively. Loss of the terminal parts of the
carbohydrate chain from the molecular ions were also seen (explained
below the formula for the species with d18:1-h24:0 ceramide).
Immonium ions, containing the complete carbohydrate chain
together with the fatty acid, were found at m/z
1298 and 1326 and also gave evidence of a carbohydrate part composed of
3 Hex and 1 HexNAc combined with h22:0 and h24:0 fatty acids. The ions
at m/z 1342 and 1370 also indicated a compound
with 3 Hex and 1 HexNAc and phytosphingosine with h22:0 (1582 241) and h24:0 (1610 241) fatty acids. Additional information
about the ceramide composition was given by the series of ions at
m/z 548-722, demonstrating a mixture of species
ranging from d18:1-16:0 to t18:0-h24:0.
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The binding positions between the carbohydrate residues were obtained
by degradation of the permethylated tetraglycosylceramide, i.e. the sample was subjected to acid hydrolysis followed by
reduction and acetylation. The resulting partially methylated alditol
acetates were analyzed by gas chromatography-EI mass spectrometry. The reconstructed ion chromatogram thus obtained had four carbohydrate peaks (not shown). The acetate of 2,3,4,6-tetramethylgalactitol identified a terminal galactose, whereas the presence of the acetate of
4,6-dimethyl-2-N-methylacetamidoglucitol (3-substituted
N-acetylglucosamine) indicated a type 1 chain. The two
remaining peaks, acetates of 2,4,6-trimethylgalactitol and
2,3,6-trimethylglucitol, were derived from 3-substituted galactose and
4-substituted glucose, respectively. In combination with the data from
mass spectrometry, a carbohydrate chain with the sequence
Gal1-3GlcNAc1-3Gal1-4Glc1 was deduced.
The anomeric region of the proton NMR spectrum (Fig.
3) contained five large -doublets
(J1,2 
8 Hz). The glucose anomeric proton signal (4.20 ppm, J1,2 = 7.2 Hz) was split into two signals, as is often
the case due to ceramide head group differences. At 4.28 ppm
(J1,2 = 7.2 Hz), the Gal4 anomeric proton appeared, which is indicative of a substitution at the 3-position. The internal GlcNAc anomer was seen at 4.79 ppm (J1,2 = 8.0 Hz) with
its N-acetamido methyl protons resonating at 1.82 ppm.
Finally, the terminal Gal signal was found at 4.15 ppm
(J1,2 = 6.6 Hz), indicating a 1-to-3 linkage. All anomeric
chemical shifts were thus in agreement with published results for
lactotetraosylceramide (32).
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Fig. 3.
The anomeric region of a 300-MHz proton NMR
spectrum of the H. pylori binding
glycosphingolipid from human meconium. 4000 scans were collected
at a probe temperature of 30 °C. The large dispersion-like signal at
5.04 ppm is an instrumental artifact.
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Thus, the H. pylori binding glycosphingolipid from human
meconium was identified as Gal3GlcNAc3Gal4Glc1Cer,
i.e. lactotetraosylceramide, which has previously been
described from the same source (33).
Comparison with Isoreceptors--
Several pure glycosphingolipids
structurally related to lactotetraosylceramide were examined for
H. pylori binding activity using the chromatogram binding
assay (exemplified in Fig. 4). The
results are summarized in Table III. The only binding-active glycosphingolipid was lactotetraosylceramide (No. 2; Fig. 4, lane 2), whereas all the substitutions tested abolished the binding. Thus, the addition of an 
-fucose in the 2-position (No. 4; Fig. 4,
lane 3), an -galactose (No. 6), or an
-N-glycolylneuraminic acid (No. 7) in 3-position, or an
-N-acetylneuraminic acid in 6-position of the terminal
galactose or an -fucose in 4-position of the
N-acetylglucosamine (No. 5; Fig. 4, lane 4) was
not tolerated. No binding to GlcNAc3Gal4Glc1Cer (No. 1; Fig.
4, lane 1) was obtained, demonstrating the importance of the
Gal3GlcNAc part. The acetamido group at 2-position of the
penultimate N-acetylglucosamine contributed substantially to
the interaction, since removal of this moiety (No. 3) completely
abolished the binding.
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Fig. 4.
Binding of H. pylori to pure
glycosphingolipids separated on thin-layer plates. A,
chemical detection by anisaldehyde. B-D, autoradiograms
obtained by binding of 35S-labeled H. pylori
strain CCUG 41936 (B), P1-140 (C), and the
babA2 mutant strain (D). The glycosphingolipids
were separated on aluminum-backed silica gel plates using
chloroform/methanol/water (60:35:8, by volume) as solvent system, and
the binding assay was performed as described under "Materials and
Methods" using 2% BSA and 0.1% Tween 20 in PBS as the coating
buffer. The lanes were GlcNAc3Gal4Glc1Cer
(lactotriaosylceramide), 4 µg (lane 1);
Gal3GlcNAc3Gal4Glc1Cer (lactotetraosylceramide), 4 µg
(lane 2); Fuc2Gal3GlcNAc3Gal4Glc1Cer (H5 type
1 glycosphingolipid), 4 µg (lane 3);
Gal3(Fuc4)GlcNAc3Gal4- Glc1Cer
(Lea-5 glycosphingolipid), 4 µg (lane 4); and
Fuc2Gal4(Fuc3)- GlcNAc3Gal4Glc1Cer (Y-6
glycosphingolipid), 4 µg (lane 5). Autoradiography was for
12 h.
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Inhibition Experiments--
The ability of soluble
oligosaccharides to interfere with the binding of H. pylori
to glycosphingolipids on thin-layer plates was examined by incubating
the bacteria with free lactotetraose or lactose before binding on
chromatograms. The results are shown in Fig.
5. Thus, incubation with lactotetraose
(0.1 mg/ml) inhibited the binding of H. pylori to
lactotetraosylceramide, whereas incubation with lactose had no
inhibitory effect.
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Fig. 5.
Effect of preincubation of H. pylori with oligosaccharides. Radiolabeled H. pylori strain CCUG 17875 was incubated with lactose (0.2 mg/ml) or
lactotetraose (0.1 mg/ml) in PBS for 1 h at room temperature.
Thereafter the suspensions were utilized in the chromatogram binding
assay. Thin-layer chromatogram stained with anisaldehyde
(A), binding of H. pylori incubated with lactose
(B), and binding of H. pylori incubated with
lactotetraose (C). The lanes were non-acid
glycosphingolipids of human blood group AB erythrocytes, 40 µg
(lane 1); Gal3GlcNAc3Gal4Glc1Cer
(lactotetraosylceramide), 4 µg (lane 2); and
GalNAc3Gal4Gal4Glc1Cer (globoside), 4 µg (lane
3). The glycosphingolipids were separated on aluminum-backed
silica gel plates using chloroform/methanol/water (60:35:8, by volume)
as the solvent system, and the binding assay was performed as described
under "Materials and Methods" using 2% BSA and 0.1% Tween 20 in
PBS as the coating buffer. Autoradiography was for 12 h.
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Binding of H. pylori to Non-acid Glycosphingolipids of Whole Human
Stomach--
To examine the expression of binding-active
glycosphingolipids in the target tissue of the bacteria, the binding of
H. pylori to glycosphingolipids isolated from whole human
stomach was first investigated. The tetraglycosylceramide region of
these non-acid fractions was dominated by globoside (Fig.
6A, lane 4), which at least for human small (34) and large intestine (35), is derived from
the non-epithelial part. No binding to these fractions was obtained
(Fig. 6B, lane 4). However, when using the
non-acid glycosphingolipid fraction isolated from the stomach of a
blood group A(Rh+)p individual (36), which lacked the
galactosyltransferase responsible for the conversion of
lactosylceramide to globotriaosylceramide (37) and consequently was
devoid of globoside (Fig. 6A, lane 3), a binding
of H. pylori in the tetraglycosylceramide region was
detected (Fig. 6B, lane 3). The tissue in this
case was obtained after surgery for peptic ulcer disease. Because of
limited amounts available, no chemical characterization of this
binding-active tetraglycosylceramide was possible.
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Fig. 6.
Binding of H. pylori to
non-acid glycosphingolipids of whole human stomach. Thin-layer
chromatogram of separated glycosphingolipids detected with anisaldehyde
(A) and autoradiogram obtained by binding of
35S-labeled H. pylori strain S-002
(B). The lanes were lactotetraosylceramide of
human meconium, 4 µg (lane 1); non-acid glycosphingolipids
of human meconium, 40 µg (lane 2); non-acid
glycosphingolipids of human stomach of a blood group A(Rh+)p
individual, 40 µg (lane 3); and non-acid
glycosphingolipids of human stomach of a blood group A(Rh+)P
individual, 40 µg (lane 4). The glycosphingolipids were
separated on aluminum-backed silica gel plates using
chloroform/methanol/water (60:35:8, by volume) as the solvent system,
and the binding assay was done as described under "Materials and
Methods." The coating buffer contained 2% BSA and 0.1% Tween 20 in
PBS. Autoradiography was for 5 h.
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Binding of H. pylori to Glycosphingolipids of Human Gastric
Epithelium--
Next we examined the binding of H. pylori
to glycosphingolipids isolated from the epithelial cells of human
stomach. Because non-neoplastic tissue rarely is excised during normal
surgical procedures, glycosphingolipids were isolated from specimens
from the fundus region obtained from patients undergoing surgery for obesity. In total, glycosphingolipids were isolated from mucosal scrapings from seven individuals and, in two cases, also from the
non-mucosal residues.
The major compounds of acid glycosphingolipid fractions migrated on
thin-layer chromatograms as sulfatide and the GM3 ganglioside. No
binding of H. pylori to these acid glycosphingolipids was
obtained (not shown). No binding of the bacteria to the non-acid
glycosphingolipids from the non-epithelial stroma was observed.
The non-acid glycosphingolipid fractions isolated from the gastric
epithelial cells from five of the seven individuals are shown in Fig.
7A. In one of the seven
samples a binding of H. pylori in the tetraglycosylceramide
region was obtained (Fig. 7B). The fraction containing the
binding-active tetraglycosylceramide (case 4) and one non-binding
fraction (case 5) were separated by HPLC, and the isolated
tetraglycosylceramides from each case (shown in Fig.
8) were characterized by 1H
NMR, EI mass spectrometry, and gas chromatography-EI mass spectrometry of permethylated tetrasaccharides obtained by ceramide glycanase hydrolysis as follows.
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Fig. 7.
Binding of H. pylori to
non-acid glycosphingolipids from human gastric epithelium.
Lanes 1-5 were non-acid glycosphingolipids (80 µg/lane)
of human gastric epithelium of five individuals (cases 1-5 of Table
II). (A) Chemical detection with anisaldehyde.
(B) Autoradiogram obtained by binding of
35S-labeled H. pylori strain S-032. The
glycosphingolipids were separated on aluminum-backed silica gel plates,
using chloroform/methanol/water (60:35:8, by volume) as solvent system,
and the binding assay was performed as described under "Materials and
Methods," using 2% BSA and 0.1% Tween 20 in PBS as coating buffer.
Autoradiography was for 12 h.
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Fig. 8.
Thin-layer chromatogram showing the
tetraglycosylceramide-containing fractions obtained from gastric
epithelium of cases 4 and 5 of Table II. The lanes were total
non-acid glycosphingolipids of gastric epithelium of case 4, 80 µg
(lane 1); fraction 4-I from case 4, 4 µg (lane
2); fraction 4-II from case 4, 4 µg (lane 3); total
non-acid glycosphingolipids of gastric epithelium of case 5, 80 µg
(lane 4); fraction 5-I from case 5, 4 µg (lane
5); and fraction 5-II from case 5, 4 µg (lane 6). The
glycosphingolipids were separated on glass-backed silica gel plates
using chloroform/methanol/water (60:35:8, by volume) as solvent system
and stained with anisaldehyde.
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Proton NMR of the Tetraglycosylceramide Fractions from Human
Gastric Epithelium--
The proton NMR spectrum of fraction 4-II
isolated from case 4 (data not shown) was dominated by globoside with
its anomeric signals appearing at 4.81 ppm (Gal), 4.52 ppm
(GalNAc), 4.26 ppm (Gal), and 4.20/4.17 ppm (Glc). However, a
small peak on the base of the Gal H1 signal revealed that another
glycosphingolipid was also present in this fraction. This signal was
consistent with GlcNAc H1 of lactotetraosylceramide, the potential
other signals being buried under the globoside resonances. However, the
Gal H1 of globotriaosylceramide would also have a very similar chemical shift. The exact shifts varies with temperature and other factors. To resolve this we compared the reference spectra of lactotetraosyl-, globotetraosyl-, and globotriaosylceramide run under similar conditions at 400 MHz. A reference mixture of
lactotetraosylceramide and globotetraosylceramide was also prepared and
run at 500 MHz. These comparisons clearly showed that the signal at
4.79 ppm belonged to a -anomeric proton from the
N-acetylglucosamine of lactotetraosylceramide. This was
further corroborated when analyzing the more early eluting tetraglycosylceramide-containing fraction (4-I) from case 4. Here two
non-overlapping -anomeric signals from galactose, one corresponding to the internal Gal H1 of globotetraosylceramide (4.81 ppm) and the
other corresponding to terminal Gal H1 of globotriaosylceramide (4.78 ppm) were found.
The presence of lactotetraosylceramide should also give rise to a
different methyl signal from the N-acetamido glucose (38) compared with the N-acetamido galactose of globotria- and
globotetraosylceramide. The GalNAc methyl signal was seen at 1.85 ppm,
and the methyl signal of the GlcNAc in lactotetraosylceramide was seen
at 1.82 ppm, which is identical to our reference spectra and in close agreement with the values reported in Clausen et al. (39).
From the intensities of the methyl signals it was estimated that
fraction 4-II contained ~5% lactotetraosylceramide.
The early eluting tetraglycosylceramide-containing fraction (5-I) from
case 5 contained both globotria- and globotetraosylceramide, as
evidenced by -anomeric signals at 4.81 and 4.78 ppm, respectively. The more late-eluting tetraglycosylceramide-containing fraction (5-II) also contained a -doublet at 4.65 ppm corresponding to GlcNAc of lactoneotetraosylceramide (39). The N-acetamido
glucose of this glycosphingolipid had a methyl signal at 1.82 ppm, in agreement with earlier data on lactoneotetraosylceramide (38).
EI-Mass Spectrometry of the Tetraglycosylceramide Fractions from
Human Gastric Epithelium--
The mass spectra (not shown) obtained by
direct inlet EI mass spectrometry of the permethylated derivatives of
fraction 4-II and 5-II from cases 4 and 5, respectively, were very
similar. In both spectra the ions at m/z 260 and
228 (260 minus 32) were prominent, demonstrating a terminal HexNAc,
whereas no ion indicating a terminal Hex at m/z
219 was found. Terminal HexNAc-Hex was shown by an ion at
m/z 464. A fragment ion at
m/z 945 (944 + 1) containing the whole
carbohydrate chain and part of the ceramide indicated a
HexNAc-Hex-Hex-Hex carbohydrate sequence.
Thus, by EI mass spectrometry only the major compound of the two
samples, most likely globoside was identified, whereas the minor
compounds of the fractions indicated by the proton NMR experiments could not be discerned. However, the increased resolution obtained by
combining chromatographic methods and mass spectrometry permitted the
identification of these minor compounds, as described in the following paragraph.
High Temperature Gas Chromatography-EI Mass Spectrometry of
Permethylated Tetrasaccharides from Human Gastric
Epithelium--
Fraction 4-II from case 4 and fraction 5-II from case
5 were hydrolyzed with ceramide glycanase, and the released
tetrasaccharides were permethylated and analyzed by gas chromatography
and gas chromatography-EI mass spectrometry. The results are summarized in Figs. 9 and
10. Each chromatographic peak was
resolved in - and -conformer.
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Fig. 9.
Reconstructed ion chromatograms of
permethylated oligosaccharides released by ceramide glycanase.
Run A was a reference mixture of globoside,
lactotetraosylceramide, and lactoneotetraosylceramide, whereas
run B was the tetraglycosylceramides from the gastric
epithelium of case 4 of Table II, and run C was the
tetraglycosylceramides from the gastric epithelium of case 5 of Table
II. The analytical conditions are described under "Materials and
Methods." The oligosaccharides of the reference mixture (Run
A) have been marked.
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Fig. 10.
Mass spectra obtained by high temperature
gas chromatography-EI mass spectrometry of permethylated
oligosaccharides released by ceramide glycanase from reference
glycosphingolipids (I and II), tetraglycosylceramide fractions from the
gastric epithelium of case 4 of Table II (III), and of case 5 of Table
II (IV). For analytical conditions, see "Materials and
Methods." The designations Runs A-C refer to the partial
total ion chromatograms shown in Fig. 10. Interpretation formulae are
shown together with the reference spectra.
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The tetrasaccharides of the stomach epithelium of the H. pylori binding case 4 were resolved into two peaks, as shown in
Fig. 9, Run B. The dominating peak eluted at the same
retention time as the saccharide from reference globoside, whereas the
minor peak eluted at the retention time of the saccharide from
reference lactotetraosylceramide.
The tetrasaccharides of the stomach epithelium of the non-binding case
5 (Fig. 9, Run C) were also resolved into two peaks, with
the major peak at the same retention time as the saccharide from
reference globoside. The smaller peak in this case eluted at the
retention time of the saccharide of reference lactoneotetraosylceramide.
To further substantiate the differences in the tetraglycosylceramide
fractions from the H. pylori binding case 4 and the
non-binding case 5, mass spectra of the permethylated oligosaccharides
were obtained (Fig. 10).
The spectra of the dominant peaks of both cases were in agreement with
that of standard globoside (not shown). However, the spectra of the
minor tetrasaccharides of the H. pylori binding case 4 (Fig.
10, III) and the non-binding case 5 (Fig. 10, IV)
showed some dissimilarities. Fragment ions demonstrating a terminal
Hex-HexNAc-Hex carbohydrate sequence were seen at
m/z 187 (219 32), 219, 432 (464 32), 464, and 668 in both spectra. However, in the spectrum of the
late-eluting peak of case 5, the fragment ion at
m/z 182 was prominent, as it was in the reference
spectrum Fig. 10, II. In contrast, this ion was absent in
the spectrum of the late-eluting peak of case 4 as well as in the
reference spectrum Fig. 10, I. The fragment ion at
m/z 182 is characteristic for type 2 carbohydrate chains, Gal4GlcNAc (30, 31). The fragment ion at
m/z 432 (464 minus 32) was also prominent in the
spectrum of the saccharide from case 5 as in the spectrum of reference
lactoneotetraosylceramide (Fig. 10, II), indicating that
methanol is more readily eliminated from Gal4GlcNAc chains than
from Gal3GlcNAc chains, most probably from C2-C3. The saccharide
from case 4 gave a strong fragment ion at m/z
228. This ion was also predominant in the spectrum of reference
lactotetraosylceramide (Fig. 10, I) and probably originated from the internal GlcNAc, since no ion at m/z 260 was seen.
In conclusion, by gas chromatography and gas chromatography-EI mass
spectrometry of permethylated oligosaccharides from the tetraglycosylceramides of cases 4 and 5, the results from proton NMR
spectroscopy of these fractions were confirmed. The predominant compound of both fractions was identified as globotetraose, whereas the
minor components differed. In the case of the H. pylori
binding case 4, the minor compound was identified as lactotetraose,
whereas the non-binding case 5 had neolactotetraose.
Frequency of Lactotetraosylceramide Binding among H. pylori
Strains--
The frequency of expression of the lactotetraosylceramide
binding property was estimated by analyzing the binding of the 74 H. pylori isolates listed in Table I to glycosphingolipids
on thin-layer chromatograms. For the binding assays the bacteria were
grown from stock cultures and examined for binding of
lactotetraosylceramide of human meconium by the chromatogram binding
assay. A positive binding indicated a pattern identical to that seen in
lane 6 of Fig 1B. The strains that failed to bind
were re-cultured twice from storage and re-assayed by the chromatogram
binding assay, i.e. no binding to lactotetraosylceramide was
detected in three consecutive assays of the strains assigned as
non-binding. By these criteria, 9 of the 74 isolates analyzed (strains
15, 65, 176, 198, 239, 269, 271, and 272 and BH000334 of Table I) were
non-binding, whereas 65 isolates (88%) expressed the
lactotetraosylceramide binding capacity. A further notation was that
lactotetraosylceramide was recognized also by the mutant strains
lacking the Leb binding adhesin (babA2 mutant
strain; Fig. 4D) or lacking the Alp protein (strain P1-140;
Fig. 4C) or lacking the HpaA protein (strain
SS1(
hpaA); not shown).
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DISCUSSION |
The glycosphingolipid composition of the human gastric epithelium
has not been well characterized. In a study of glycosphingolipids of
the mucosal cells and submucosal tissue of the human gastrointestinal tract (40), an enrichment of sulfatides in the fundic and antral mucosa
of the stomach was reported. The major non-acid
glycosphingolipids migrated as galactosylceramide, lactosylceramide,
globotriaosylceramide, and globoside on thin-layer plates, whereas the
main gangliosides migrated as GM3, GM1, and GD3. H. pylori
binding lactosylceramide with phytosphingosine and hydroxy fatty acids
has also been characterized in the human gastric epithelium (12). In
addition, the blood group Cad-active ganglioside
(GalNAc4(NeuAc3)Gal4GlcNAc3Gal4Glc1Cer) has been identified in the fundus region of human stomach (41), whereas
it was not found in the pyloric region (42), indicating a differential
expression of glycosphingolipids in different regions of the human stomach.
Because of limited access to human gastric tissue, we initially
concentrated on the H. pylori binding glycosphingolipid
detected in human meconium, which is the first sterile feces of the
newborn and consists mainly of extruded mucosal cells from the
developing gastrointestinal tract. After isolation, this H. pylori binding glycosphingolipid was characterized by mass
spectrometry, proton NMR, and methylation analysis as
Gal3GlcNAc3Gal4Glc1Cer (lactotetraosylceramide). The tissue
distribution of this glycosphingolipid is very limited. Until recently
lactotetraosylceramide had only been identified in human meconium (33)
in the small intestine of an individual previously resected
according to Billroth II (34) in normal human gastric mucosa and
in human gastric cancer tissue (43). However, the "normal" mucosa
in 4 of the 5 cases described in the latter report was obtained by
antrectomy due to duodenal or gastric ulcer. Immunohistochemical
studies, using the monoclonal antibody K-21 demonstrated a selective
expression of the Gal3GlcNAc sequence in superficial human gastric
mucosa of non-secretor individuals (44) coinciding with the
localization of H. pylori binding to tissue sections (4, 6).
An immunohistochemical study utilizing polyclonal antibodies binding to
the Gal3GlcNAc sequence showed the presence of
lactotetraosylceramide in the brush border cells of human jejunum and
ileum of blood group OLe(ab)non-secretor individuals and also of
one individual with the blood group OLe(a+b+)non-secretor (45).
The relevance of the lactotetraosylceramide binding specificity was
substantiated by the binding of H. pylori to the
tetraglycosylceramide region of the non-acid glycosphingolipids
isolated from the target epithelial cells of human stomach. By proton
NMR and gas chromatography-mass spectrometry of permethylated
tetrasaccharides obtained by ceramide glycanase hydrolysis, it was
demonstrated that the binding-active fraction contained
lactotetraosylceramide. This binding-active lactotetraosylceramide was
only found in one of seven individuals analyzed, which is suggestive in
view of the fact that although infection with H. pylori and
the associated chronic gastritis are very common, only a small fraction
of those infected develops any further consequences such as peptic
ulcer or gastric adenocarcinoma (46). The presence of
lactotetraosylceramide on the gastric epithelial cells may be one of
the co-factors necessary for the development of the severe
consequences of the infection. An interesting notation in this context
is that the stomach of the blood group A(Rh+)p individual, where
H. pylori binding in the tetraglycosylceramide region was
observed, was obtained after surgery for peptic ulcer disease.
Serologic typing using erythrocytes and saliva demonstrated that the
blood group status of case 4 was ALe(a+b)non-secretor (data not
shown), and this is in agreement with the presence of H. pylori binding-unsubstituted lactotetraosylceramide in the gastric
epithelium of this individual. The non-secretor status of this
individual is interesting in view of the increased prevalence of
duodenal ulcer among non-secretors (47-49). One study (50) has
demonstrated that non-secretion is not associated with increased susceptibility to infection with H. pylori. However, one may
speculate that the secretor status determines the outcome of the
colonization, i.e. that the increased liability
of non-secretors to develop peptic ulcer disease may be due to the
presence of the H. pylori binding lactotetraosylceramide on
the gastric epithelium of these individuals.
Lactotetraosylceramide is also known as the Lec antigen,
present in red cell Lewis-negative ABH non-secretors (for review, see
Ref. 51). However, to our knowledge no studies of the frequency of
H. pylori infection among Le(ab)non-secretor individuals have been reported.
Among the 74 H. pylori isolates analyzed in this study, 65 strains (88%) were found to express the lactotetraosylceramide binding
specificity, whereas 9 strains were non-binding. The high prevalence of
the lactotetraosylceramide binding property among the H. pylori isolates demonstrates that it is a conserved property of
this gastric pathogen and may, thus, represent an important virulence factor.
Under the experimental conditions of the present study, H. pylori did not bind to the glycosphingolipids tentatively
identified as sulfatide and the GM3 ganglioside in the acid fractions
from human gastric epithelium. The binding of H. pylori to
lactotetraosylceramide was not affected by changing the growth
conditions, since this binding was obtained both when the bacteria were
grown on agar and in broth. Also, binding to lactotetraosylceramide was
obtained both with bacteria grown for 12 and 120 h. The binding to
lactotetraosylceramide was inhibited by incubating the bacteria with
free univalent lactotetraose but not with lactose.
Huesca et al. (52) report that upon treatment of H. pylori with acidic pH or heat the binding of this bacterium to
sulfatide is induced. In our hands, when the chromatogram binding assay was conducted at pH 5, the bacteria failed to bind to any
glycosphingolipid, including gangliotetraosylceramide, sulfatide,
lactotetraosylceramide, and the Leb
hexaglycosylceramide (data not reproduced). Alternatively, a binding to a multitude of glycosphingolipids with diverse sequences was
observed. However, the pH gradient of the human gastric mucus layer
ranges from pH 2 on the luminal side to almost pH 7 on the epithelial
cell surface (53), suggesting that binding assays conducted at pH 7.3 may be of relevance for attachment of H. pylori to
epithelial receptors.
The Leb determinant (Fuc2Gal3(Fuc4)GlcNAc) is
based on the type 1 disaccharide unit, which is the terminal part of
lactotetraosylceramide. Binding to lactotetraosylceramide was, however,
also obtained with strains devoid of Leb binding activity,
as e.g. the CCUG 41936 strain (identical to the 26695 strain) and the MO19 strain (14). Furthermore, inactivation of the
babA gene coding for the Leb binding adhesin
(14) did not abolish the binding of lactotetraosylceramide. Thus, the
binding of H. pylori to the Leb determinant and
to lactotetraosylceramide represents two separate binding
specificities and not a cross-binding.
This was further substantiated by inspection of the minimum
energy molecular model of lactotetraosylceramide in comparison with the
Leb-6 glycosphingolipid, as shown in Fig.
11. In trying to discern the important
parts making up the binding epitope of lactotetraosylceramide, two
observations, the non-binding of lactotriaosylceramide
(GlcNAc3Gal4Glc1Cer) and of lactotetraosylceramide in which
the acetamido moiety had been converted to an amine
(Gal3GlcNH23Gal4Glc1Cer), indicate that the
terminal disaccharide Gal3GlcNAc3 constitutes the epitope. The
non-binding of the latter structure further indicates either that an
intact acetamido group is essential for binding to occur or that an
altered conformation results since an amine no longer may
participate in hydrogen bond interactions with the 2-OH group of the
internal Gal4. A combination of these two effects is also possible.
Moreover, extension of the terminal Gal of lactotetraosylceramide by
Gal3 or Fuc2 or substitution of the penultimate GlcNAc by Fuc4
yields structures that are inactive, suggesting that the major part of
the terminal disaccharide Gal3GlcNAc3 is directly involved in
interactions with the adhesin responsible for binding. In the
Leb structure, the GlcNAc3 residue is inaccessible, and
the penultimate Gal3 partly so since they are covered by the two
fucoses, as seen in the top view of Fig. 11. Furthermore,
since the binding of H. pylori to Leb is
inhibited by the free oligosaccharide of the Ley
isostructure (8), the GlcNAc3 residue of Leb is not
essential for binding to this compound. Alignment of the minimum energy
structures of the terminal tetrasaccharide part of Leb-6
and Ley-6 shows that the only difference is an ~180°
turn of the GlcNAc3 residue, thus proving the non-requirement of the
acetamido moiety of the GlcNAc3 residue (or even more likely the
whole residue) in the Leb structure, whereas in
lactotetraosylceramide the opposite is true. It may be further noted
that the angle between the ring plane of the terminal Gal3 in
lactotetraosylceramide and the corresponding plane in the
Leb structure is close to 40°, due to the crowding caused
by the two additional fucose units, affording an additional reason as to why these structures should be regarded as separate receptors for
H. pylori.
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Fig. 11.
Minimum energy conformers of the H. pylori binding lactotetraosylceramide
(Gal3GlcNAc3Gal4Glc1Cer,
left) and the Leb-6
glycosphingolipid
(Fuc2Gal3(Fuc4)GlcNAc3- Gal4Glc1Cer,
right) (B). The top charts
(A) show the same structures viewed from above. The
Glc1Cer linkage is shown in an extended conformation. A detailed
comparison between these two structures is made in the text.
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§,
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TOP
ABSTRACT
INTRODUCTION
