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Glycobiology Advance Access originally published online on November 8, 2008
Glycobiology 2009 19(2):182-191; doi:10.1093/glycob/cwn125
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human {alpha}-1,3/4-fucosyltransferase in FVB/N mouse stomach

David Fagerberg3, Jonas Ångström3, Adnan Halim3, Anna Hultberg4,2, Lena Rakhimova5, Lennart Hammarström4, Thomas Borén5 and Susann Teneberg1,3

3 Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, PO Box 440, University of Gothenburg, S-405 30 Göteborg, Sweden
4 Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska Institutet, Karolinska University Hospital, SE 141 86 Huddinge, Stockholm, Sweden
5 Department of Medical Biochemistry and Biophysics, Umeå University, SE 901 87 Umeå, Sweden

1 To whom correspondence should be addressed: Tel: +46-31-786-34-92; Fax: +46-31-413-190; e-mail: Susann.Teneberg{at}medkem.gu.se

Received on July 15, 2008; revised on November 3, 2008; accepted on November 3, 2008


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Material and methods
 Funding
 Conflict of interest statement
 References
 
The "Leb mouse" was established as a model for investigations of the molecular events following Leb-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human {alpha}-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Leb glycoproteins in gastric pit and surface mucous cells was obtained in this "Leb mouse," as demonstrated by binding of monoclonal anti-Leb antibodies. To explore the effects of the human {alpha}-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic {alpha}-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc{alpha}2Galβ3(Fuc{alpha}4)GalNAcβ4Galβ4Glcβ1Cer, i.e., a novel Leb-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAcβ3Gal{alpha}3(Fuc{alpha}2)Galβ4Glcβ1Cer, in which the isoglobo tetrasaccharide has been combined with Fuc{alpha}2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAcβ3(Fuc{alpha}2)Galβ3GalNAcβ4Galβ4Glcβ1Cer.

Key words: transgenic Leb mouse / {alpha}-1,3 / 4-fucosyltransferase / H. pylori-binding glycosphingolipid / glycosphingolipid characterization / novel glycosphingolipid


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Material and methods
 Funding
 Conflict of interest statement
 References
 
Bacterial adherence to target cells is an important virulence trait of pathogenic bacteria. The recognition of the Lewis b blood group antigen (Fuc{alpha}2Galβ3(Fuc{alpha}4)GlcNAc; Leb) by certain strains of Helicobacter pylori was reported over a decade ago (Borén et al. 1993Go), and subsequently the cognate H. pylori Leb-binding adhesin BabA (blood group antigen binding adhesin) was identified (Ilver et al. 1998Go). H. pylori strains expressing BabA together with the vacuolating cytotoxin VacA and the cytotoxin-associated antigen CagA (triple positive strains) are highly associated with severe gastric diseases, as peptic ulcer or gastric adenocarcinoma (Gerhard et al. 1999Go; Rad et al. 2002Go).

Investigations of the molecular events following Leb-mediated adhesion of H. pylori to the gastric epithelium were initially hampered by the lack of animal models since the Leb determinant is only present in humans. However, this obstacle was overcome when Falk et al. (1995Go) created the "Leb mouse" by expressing a human {alpha}-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice. Thereby, a production of Leb glycoproteins in gastric pit and surface mucous cells was obtained, as demonstrated by binding of monoclonal anti-Leb antibodies. Binding of H. pylori to the Leb-positive surface mucous cells was also observed. Colonization trials resulted in equivalent microbial densities in the stomachs of the transgenic Leb mice and their nontransgenic Leb-negative littermates (Guruge et al. 1998Go), while attachment of bacteria to the gastric pit cells was observed in the transgenic Leb mice only. The transgenic animals also had signs of inflammation in terms of chronic gastritis, loss of parietal cells and auto-antibodies to Lewis x carbohydrate epitopes.

The expression of glycoconjugates varies both quantitatively and qualitatively between different species, individuals of the same species, organs, and cells. Also, the glycosylation of the different parts of the gastrointestinal tract varies (reviewed in Hansson (1988Go)). No comprehensive studies of the carbohydrate structures of glycoproteins or glycosphingolipids of the epithelial cells of mouse stomach have been reported. However, in the mouse small intestinal epithelium gangliotetraosylceramide (Galβ3GalNAcβ4Galβ4Glcβ1Cer) is one of the major glycosphingolipids (Suzuki and Yamakawa 1981Go; Umesaki et al. 1981Go; Hansson et al. 1982Go), and gangliotetraosylceramide having a terminal {alpha}2-linked Fuc (fucosyl-gangliotetraosylceramide; Fuc{alpha}2Galβ3GalNAcβ4Galβ4Glcβ1Cer), i.e., a blood group H determinant-carrying compound, has also been described (Umesaki et al. 1981Go), while no glycosphingolipids based on a type 1 core has been characterized in this tissue.

Thus, at the glycosphingolipid level the {alpha}-1,3/4-fucosyltransferase expressed in the genetically engineered mice might use the blood group H determinant-carrying fucosyl-gangliotetraosylceramide as an acceptor. This possibility was examined in the present study by isolating neutral glycosphingolipids from stomachs of transgenic {alpha}-1,3/4- fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc{alpha}2Galβ3(Fuc{alpha}4)GalNAcβ4Galβ4Glcβ 1Cer, i.e., a Leb-like glycosphingolipid based on a ganglio core chain. In addition, two novel glycosphingolipids, GalNAcβ3Gal{alpha}3(Fuc{alpha}2)Galβ4 Glcβ1Cer representing a combined isoglobo-blood group B glycotope, and GalNAcβ3(Fuc{alpha}2)Galβ3GalNAcβ4Galβ4 Glcβ1Cer representing an elongated FucGgO4, were isolated and characterized, but were found to be nonbinding with regard to H. pylori.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Material and methods
 Funding
 Conflict of interest statement
 References
 
Glycosphingolipid preparations and binding of H. pylori
From 3.84 g dry weight transgenic {alpha}-1,3/4-fucosyltransferase-expressing mouse stomach tissue, 167 mg (43.5 mg/g) total acid, and 29 mg (7.6 mg/g) total neutral glycosphingolipids were obtained. The major compounds of the acid fraction migrated in the regions of GM3 and GD3 on thin-layer chromatograms. No binding of the H. pylori strain J99, expressing the sialic acid binding SabA adhesin (Mahdavi et al. 2002Go), to the acid glycosphingolipids was obtained, and this fraction was not further characterized.

When the total neutral fraction was analyzed by thin-layer chromatography and chemical detection, the only compound visualized was migrating in the monoglycosylceramide region (data not shown). No binding of H. pylori strain J99 to the total neutral glycosphingolipid fraction was observed. The total neutral fraction was initially separated on a silicic acid column giving 13.5 mg of pure monoglycosylceramides and 10 mg of more slow-migrating compounds. After separation of the fraction with slow-migrating compounds on Iatrobeads columns, 12 glycosphingolipid-containing fractions were obtained (designated fractions n1–n12; Figure 1, lanes 2–13). The amounts of the obtained subfractions are given below the figure.


Figure 1
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  		Fig. 1 Neutral glycosphingolipids isolated from transgenic {alpha}-1,3/4-fucosyltransferase-expressing FVB/N mouse stomach. The glycosphingolipids were separated on glass-supported silica gel 60 HPTLC plates, using chloroform/methanol/water 60:35:8 (by volume) as a solvent system and stained by anisaldehyde. The lanes were: lane 1, reference total neutral glycosphingolipids human blood group O erythrocytes, 40 µg; lanes 2–13, subfractions n1–n12 of transgenic mouse stomach neutral glycosphingolipids 2–4 µg/lane; lane 14, reference A7 type 2 glycosphingolipid (GalNAc{alpha}3(Fuc{alpha}2)Galβ4(Fuc{alpha}3) GlcNAcβ3Galβ4Glcβ1Cer) of human erythrocytes, 4 µg. The amounts obtained of each subfraction are given below the figure (in milligram). The designations to the left indicate the approximate mobility of lactosylceramide (LacCer, Galβ4Glcβ1Cer), globotriaosylceramide (Gb3, Gal{alpha}4Galβ4Glcβ 1Cer), and globotetraosylceramide (Gb4, GalNAcβ3Gal{alpha}4Galβ4Glcβ1Cer), respectively.

 
The glycosphingolipid-containing fractions obtained were tested for binding of BabA-expressing H. pylori strain J99. While the majority of the fractions were nonbinding, an interaction of H. pylori strain J99 with fractions n10 and n11 was obtained (Figure 2B, lanes 2 and 3). The binding-active compound migrated in the hexaglycosylceramide region at the level of the Leb hexaglycosylceramide reference, Fuc{alpha}2Galβ3(Fuc{alpha}4)GlcNAcβ3Galβ4Glcβ1Cer (Figure 2, lane 5).


Figure 2
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  		Fig. 2 Binding of Helicobacter pylori and monoclonal antibodies against the Leb determinant to neutral glycosphingolipids from transgenic {alpha}-1,3/ 4-fucosyltransferase-expressing FVB/N mouse stomach. Thin-layer chromatogram after detection with anisaldehyde (A), and autoradiograms obtained by binding of H. pylori strain J99 (B), monoclonal anti-Leb antibody T218 (C), and monoclonal anti-Leb antibody clone 96 FR2.10 (D). The chromatograms were eluted with chloroform/methanol/water 60:35:8 (by volume), and the binding assays were done as described under "Material and methods." The lanes contained: lanes 1–4, subfractions n9–n12 from transgenic mouse stomach, 4 µg/lane; lane 5, reference Leb hexaglycosy- lceramide (Leb, Fuc{alpha}2Galβ3(Fuc{alpha}4)GlcNAcβ3Galβ4Glcβ1Cer) from human small intestine, 4 µg; lane 6, reference Lea pentaglycosylceramide (Lea, Galβ3(Fuc{alpha}4)GlcNAcβ3Galβ4Glcβ1Cer) from human small intestine, 4 µg; lane 7, reference Ley hexaglycosylceramide (Ley, Fuc{alpha}2Galβ4 (Fuc{alpha}3)GlcNAcβ3Galβ4Glcβ1Cer) from dog small intestine, 4 µg; lane 8, reference fucosyl-gangliotetraosylceramide (fG4, Fuc{alpha}2Galβ3GalNAcβ4Galβ4Glcβ1Cer) from mouse small intestine, 4 µg; lane 9, reference gangliotriaosylceramide (G3, GalNAcβ4Galβ4Glcβ1Cer) from guinea pig erythrocytes, 4 µg. Autoradiography was for 12 h.

 
Next, the binding of monoclonal antibodies directed against the Leb blood group determinant to the H. pylori-binding fractions of transgenic mouse stomach was evaluated. The clone T218 monoclonal anti-Leb antibody bound to reference Leb hexaglycosylceramide, but did not interact with fractions n10 and n11 of transgenic mouse stomach (Figure 2C). In contrast, the clone 96 FR2.10 monoclonal anti-Leb antibody used in the original publication (Falk et al. 1995Go) readily recognized fractions n10 and n11, along with binding to reference Leb hexaglycosylceramide, and also to reference Ley hexaglycosylceramide, Fuc{alpha}2Galβ4(Fuc{alpha}3)GlcNAcβ3Galβ4Glcβ1Cer (Figure 2D).

This differential binding of the monoclonal anti-Leb antibodies suggested that the structure of the H. pylori-binding compound in fractions n10 and n11 was nonidentical to the Leb hexaglycosylceramide. In order to characterize the binding-active compound, fraction n11 was analyzed by mass spectrometry and proton NMR, as described below. In addition, fractions n9, n10, and n12 were characterized by mass spectrometry and proton NMR.

Negative ion FAB mass spectrometry of the H. pylori-binding glycosphingolipid fraction n11 from stomach of transgenic {alpha}-1,3/4-fucosyltransferase-expressing mice
The negative ion FAB mass spectrum of the H. pylori-binding fraction n11 from stomach of transgenic {alpha}-1,3/4-fucosyltransferase-expressing mice (Figure 3) indicated the presence of two different glycosphingolipids. Firstly, the ions at m/z 1517, 1545, 1629, 1647, and 1663 indicated a glycosphingolipid with three hexoses, one N-acetylhexosamine, and two fucoses (Hex3-HexNAc1-Fuc2), and d18:1–16:0, d18:1–18:0, d18:1–24:0, t18:0–24:0, and t18:0–h24:0 ceramides, respectively. Fragment ions derived from the molecular ion at m/z 1663 were seen at m/z 1517 (M–Fuc), m/z 1355 (M–Fuc–Hex), m/z 1006 (M–Fuc–Hex–Fuc–HexNAc), m/z 844 (M–Fuc–Hex–Fuc–HexNAc–Hex), and m/z 682 (M–Fuc–Hex–Fuc–HexNAc–Hex–Hex; not shown).


Figure 3
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  		Fig. 3 Negative ion FAB mass spectrum of the Helicobacter pylori-binding glycosphingolipid fraction n11 from stomach of transgenic {alpha}-1,3/4-fucosyltransferase-expressing mice. Above the spectrum are interpretation formulae for the HexNac–HexNAc–Hex–Hex–Hex glycosphingolipid, and the Fuc–Hex–(Fuc–)HexNAc–Hex–Hex glycosphingolipid, representing the species with t18:0-h16:0 and t18:0-h24:0 ceramides, respectively. The analysis was done as described in "Material and methods".

 
The ion at m/z 1517 may thus represent a molecular ion of Hex3–HexNAc1–Fuc2 with d18:1–16:0 and be a fragment ion due to loss of fucose from the molecular ion at m/z 1663. However, the molecular ions at, e.g., m/z 1545 and 1663 clearly denoted a Hex3–HexNAc1–Fuc2 glycosphingolipid with d18:1–18:0 and t18:0–h24:0 ceramides, respectively.

In addition, the mass spectrum of fraction n11 had two molecular ions at m/z 1462 and 1490, indicating a glycosphingolipid Hex3–HexNAc2 composition and phytosphingosine with hydroxy 16:0 and 18:0 fatty acids. Fragment ions derived from the molecular ion at m/z 1462 were present at m/z 1259 (M–HexNAc), m/z 894 (M–HexNAc–HexNAc–Hex), m/z 732 (M–HexNAc–HexNAc–Hex–Hex; not shown), and m/z 570 (M–HexNAc–HexNAc–Hex–Hex–Hex; not shown).

Thus, negative ion FAB mass spectrometry of fraction n11 showed a mixture of two glycosphingolipids with Fuc–Hex–(Fuc)HexNAc–Hex–Hex and HexNAc–HexNAc– Hex–Hex–Hex carbohydrate sequence, respectively.

Negative ion FAB mass spectrometry of fractions n9, n10, and n12
The negative ion FAB mass spectra of fractions n9 and n10 (not shown) were very similar and both had a series of molecular ions at m/z 1428–1540, corresponding to a glycosphingolipid with Hex3–HexNAc2 composition combined with sphingosine and nonhydroxy 16:0–24:0 fatty acids. Series of fragment ions were present at m/z 1225–1337 (M–HexNAc), m/z 1022–1134 (M–HexNAc–HexNAc), m/z 860–972 (M–HexNAc–HexNAc–Hex), m/z 698–810 (M–HexNAc–HexNAc–Hex–Hex), and m/z 536–648 (M–HexNAc–HexNAc–Hex–Hex–Hex). Thus, a glycosphingolipid with a HexNAc–HexNAc–Hex–Hex–Hex carbohydrate sequence, and d18:1–16:0–24:0 ceramides, was demonstrated.

The negative ion FAB mass spectrum of fraction n12 is shown in Figure 4. A glycosphingolipid with Hex3–HexNAc2-Fuc composition, and phytosphingosine with hydroxy 16:0–26:0 fatty acids is indicated by the series of molecular ions at m/z 1608–1749. Sequence ions derived from the molecular ions were seen at m/z 1405–1575 (M–HexNAc), m/z 1097–1237 (M–HexNAc–Fuc–Hex), m/z 894–1034 (M–HexNAc–Fuc–Hex–HexNAc), m/z 732–872 (M–HexNAc–Fuc–Hex–HexNAc–Hex), and m/z 732–872 (M–HexNAc–Fuc–Hex–HexNAc–Hex–Hex). Thus, mass spectrometry of fraction n12 demonstrated a glycosphingolipid with the HexNAc–(Fuc)Hex–HexNAc–Hex–Hex carbohydrate sequence combined with phytosphingosine with hydroxy 16:0–26:0 fatty acids.


Figure 4
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  		Fig. 4 Negative ion FAB mass spectrum of fraction n12 from stomach of transgenic {alpha}-1,3/4-fucosyltransferase-expressing mice. Above the spectrum is an interpretation formula representing the species with t18:0-h16:0 to t18:0-h24:0 ceramides. The analysis was done as described in "Material and methods".

 
Proton NMR spectroscopy
Figure 5 shows the anomeric regions of the 600 MHz proton NMR spectra of three different fractions from the FVB/N transgenic mouse stomach. The earliest fraction (n9) is dominated by the Forssman glycolipid (GalNAc{alpha}3GalNAcβ3Gal{alpha}4Galβ4Glcβ1Cer) as shown in the top trace and detailed in Table I. Briefly, the two {alpha}-signals at 4.783 ppm and 4.696 ppm stem from Gal{alpha}4 and GalNAc{alpha}3, respectively, whereas the three β-signals at 4.514 ppm, 4.255 ppm, and 4.17 ppm are attributable to GalNAcβ3, Galβ4, and Glcβ1, respectively (Dabrowski et al. 1980Go). However, there are significant amounts (~2%) also of a minor five-sugar species having two {alpha}-signals at 5.14 ppm and 4.87 ppm corresponding to internal Fuc{alpha}2 (6-CH3 at 1.04 ppm, not shown) and internal Gal{alpha}3, respectively. Furthermore, two β-signals at 4.58 ppm and 4.36 ppm are ascribable to a terminal GalNAcβ3 and a fucose-linked Galβ4 whereas the Glcβ1 at the reducing end tentatively is assigned to the small β-signal seen at 4.21 ppm. These chemical shift values correspond closely to those of isoglobotetraosylceramide (iGb4, Table I) except for the fucose-linked Galβ4, suggesting that the sugar sequence should read GalNAcβ3Gal{alpha}3(Fuc{alpha}2)Galβ4Glcβ1Cer (here termed iGb4-B). This is a novel shorter type 2-based version of the GalNAcβ3-Ganglio-B structure as is clearly reflected in the expected shift differences of the Fuc{alpha}2 and Galβ3/4 residues of type 1- and type 2-based blood group B determinants (Table I).


Figure 5
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  		Fig. 5 Anomeric regions of the 600 MHz proton NMR of three novel glycosphingolipids from transgenic {alpha}-1,3/4-fucosyltransferase-expressing mouse stomach (30°C). The top trace is from fraction n9, the middle trace from fraction n11, and the bottom trace from fraction n12. The Helicobacter pylori-binding glycosphingolipid is found in fraction n11. The sample was dissolved in dimethyl sulphoxide-D2O (98:2, by volume) after deuterium exchange.

 

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  		Table I Chemical shift data (ppm) for anomeric resonances from proton NMR spectra of novel glycosphingolipids from FVB/N mouse stomach and reference compounds obtained in DMSO-d6/D2O (98:2, by volume) at 30°C

 
Fractions n10 and n11 both contain the same two compounds but in different proportions. In n10, the Forssman glycosphingolipid is very dominant (~95%) whereas in n11 this glycosphingolipid is approximately equal to the second H. pylori-binding component as shown in the middle trace in Figure 5. The anomeric resonances from the Forssman glycosphingolipid are readily identified by comparison with the previous fractions, thus leaving the remaining resonances in the anomeric region originating from the second component which from mass spectrometry is a hexaglycosylceramide with a Fuc–Hex–(Fuc)HexNAc—Hex–Hex carbohydrate sequence. The two {alpha}-signals at 4.896 ppm and 4.783 ppm are assigned to Fuc{alpha}2 and Fuc{alpha}4, respectively, of a Leb-like determinant where the latter fucose differs shiftwise relative to the corresponding residue of a regular Leb determinant by 0.026 ppm, as does its H5 resonance at 4.606 ppm (quartet) by –0.020 ppm (Table I, Clausen et al. 1985Go), indicating a different environment. This supposition is further substantiated when the resonance at 4.560 ppm is inspected. This β-signal has a two-proton intensity suggesting that the Galβ3 resonance has merged with the HexNAcβ signal stemming from the residue to which Galβ3 and Fuc{alpha}4 are attached. By contrast, in the regular Leb hexaglycosylceramide, these two signals are separated by as much as 0.067 ppm (Table I; Leb-6). In order to establish the identity of the overlapping signals at 4.560 ppm, a COSY spectrum was acquired (not shown). Although suffering from an overall poor signal-to-noise ratio, the anomeric β-signals did give rise to sufficiently strong crosspeaks to allow the corresponding H2 resonances to be located. Thus, as expected two crosspeaks were found for the 4.560 ppm resonance yielding H2 resonances at 3.31 ppm and 3.76 ppm. The former resonance originates from Galβ3 but differs significantly from the corresponding H2 in the regular Leb hexaglycosylceramide found at 3.46 ppm (60°C; Dabrowski et al. 1981Go) due to shielding effects mainly from the glycosidic oxygen between the HexNAc and Fuc{alpha}4 residues, suggesting that this oxygen is axial with respect to the HexNAc, which thus would identify the residue as GalNAc. This is further corroborated by the value of the second H2 resonance (3.76 ppm) which is very close to the value found for, e.g., GalNAcβ4 of gangliotetraosylceramide (Galβ3GalNAcβ4Galβ4Glcβ1Cer, Koerner et al. 1983Go). However, final proof that the GalNAc is β4-linked is obtained when examining the shift of the anomeric resonance of the Galβ4 residue to which it is linked. This resonance is found at 4.199 ppm and again comparing with the corresponding sugar in gangliotetraosylceramide (Koerner et al. 1983Go) only a marginal difference is found for this signal as well as for the H2 signal at 4.21 ppm. Finally, the Glcβ1 signal is found at 4.185 ppm strongly overlapping with the H5 resonance (triplet appearance) from the Fuc{alpha}2 at 4.188 ppm. The full sequence of this glycosphingolipid (here termed Ganglio-Leb-6) can thus be established as Fuc{alpha}2Galβ3(Fuc{alpha}4) GalNAcβ4Galβ4Glcβ1Cer.

The final fraction examined by NMR (n12) reveals a single dominating compound also having six carbohydrate residues with the sequence HexNAc–(Fuc)–Hex–HexNAc–Hex–Hex according to mass spectrometry. The interpretation in this case was very straightforward since five of the six anomeric resonances correspond closely to those of fucosyl-gangliotetraosylceramide (Fuc{alpha}2Galβ3GalNAcβ4Galβ4Glcβ1Cer) (FucGgO4, Table I). Fuc{alpha}2 is thus found at 4.937 ppm, Galβ3 at 4.328 ppm, GalNAcβ4 at 4.460 ppm, Galβ4 at 2.213 ppm, and Glcβ1 at 4.198 ppm. The sixth β-anomeric signal at 4.557 ppm must therefore be ascribed to the second HexNAc which by virtue of its chemical shift most likely corresponds to a terminal GalNAcβ3 residue. This is supported by the finding that the shift of the acetamido methyl resonance at 1.810 ppm (not shown) has suffered an upfield shift of 0.013 ppm relative to the corresponding resonance of the terminal GalNAcβ3 residue in the GalNAcβ3-Ganglio-B structure (Teneberg et al. 1994Go) due to crowding by the Fuc{alpha}2.

Comparison of relative binding affinity
The relative binding affinity of H. pylori strain J99 for the traditional Leb hexaglycosylceramide and the Ganglio-Leb hexaglycosylceramide was estimated using dilutions of glycosphingolipids on thin-layer chromatograms, taking into account that the Ganglio-Leb-containing fraction n11 also contains the nonbinding Forssman glycosphingolipid (approximately 50% according to NMR). As shown in Figure 6, H. pylori binding to fraction n11 still occurred when 0.4 µg was applied on the thin-layer plate (lane 5), giving a detection limit for the Ganglio-Leb hexaglycosylceramide at approximately 0.2 µg. In contrast, the bacteria readily bound to the regular Leb hexaglycosylceramide at 0.08 µg (lane 4), and the detection limit for this compound was approximately 0.02 µg (data not shown).


Figure 6
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  		Fig. 6 Binding of Helicobacter pylori to dilutions of glycosphingolipids on thin-layer chromatograms. Autoradiogram obtained by binding of H. pylori strain J99. The chromatogram was eluted with chloroform/methanol/water 60:35:8 (by volume), and the binding assay was done as described under "Material and methods." Lanes 1–4 were dilutions (0.8–0.08 µg) of the Leb hexaglycosylceramide (Fuc{alpha}2Galβ3(Fuc{alpha}4)GlcNAcβ3Galβ4Glcβ1Cer) and lanes 5–8 were dilutions (0.8–0.08 µg) of the Ganglio-Leb hexaglycosy- lceramide (Fuc{alpha}2Galβ3(Fuc{alpha}4)GalNAcβ4Galβ4Glcβ1Cer, fraction n11). Autoradiography was for 12 h.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Material and methods
 Funding
 Conflict of interest statement
 References
 
In the present study, an H. pylori-binding glycosphingolipid was isolated from stomach of transgenic {alpha}-1,3/4- fucosyltransferase-expressing mice and characterized by mass spectrometry and proton NMR as Fuc{alpha}2Galβ3 (Fuc{alpha}4)GalNAcβ4Galβ4Glcβ1Cer, i.e. a Leb-like glycosphingolipid on a ganglio core structure. The binding affinity of the H. pylori strain J99 for this ganglio-based Leb epitope was lower than for the "traditional" Leb epitope, indicating that a GalNAc instead of a GlcNAc is not optimal for the BabA adhesin. In this context, it should also be noted that the BabA adhesin of the J99 strain is of the generalist type and, in addition to the Leb and H type 1 epitopes, binds to the blood group A and B type 1 epitopes (Aspholm-Hurtig et al. 2004Go). When using the specialist strain P466, with BabA recognizing the Leb and H type 1 epitopes only, no binding to the Ganglio-Leb hexaglycosylceramide was obtained (data not shown).

The occurrence of "ganglio-B," Gal{alpha}3(Fuc{alpha}2)Galβ 3GalNAcβ4Galβ4Glcβ1Cer, in rat bone marrow, spleen, thymus, gastric mucosa, and rat hepatoma ascites has been reported (Taki et al. 1985Go; Hansson et al. 1987Go). However, the ganglio-based Leb-glycosphingolipid is to our knowledge a novel structure.

In addition, two novel glycosphingolipids were characterized. The first was a minor compound identified as GalNAcβ3Gal{alpha}3(Fuc{alpha}2)Galβ4Glcβ1Cer, i.e., a Fuc{alpha}2-substi- tuted isoglobotetraosylceramide having an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAcβ3(Fuc{alpha}2)Galβ3GalNAcβ- 4Galβ4Glcβ1Cer. None of these novel glycosphingolipds were recognized by H. pylori, and although isolated from transgenic {alpha}-1,3/4-fucosyltransferase-expressing mouse stomach, it is not likely that the human {alpha}-1,3/4-fucosyltransferase is involved in the synthesis of these two glycosphingolipids since no {alpha}3- or {alpha}4-linked fucose is present in these compounds.

The transgenic Leb mouse was established by the expression of a human {alpha}-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice. During the studies preceding the creation of the transgenic Leb mouse, the presence of an acceptor blood group H determinant in gastric pit cells and surface mucous cells of the FVB/N mice was established by binding of a monoclonal antibody directed against the blood group H type 2 determinant (Fuc{alpha}2Galβ4GlcNAc). Monoclonal antibody clone 96 FR2.10 binding to histo-sections and isolated glycoproteins was also used to establish the creation of the Leb epitope upon the expression of the human {alpha}-1,3/4-fucosyltransferase in the pit cell lineage (Falk et al. 1995Go).

In the chromatogram binding assay, the T218 anti-Leb monoclonal antibody bound only to reference Leb hexaglycosylceramide and did not recognize the H. pylori-binding glycosphingolipid of transgenic Leb mice stomach. In contrast, the H. pylori-binding glycosphingolipid was recognized by the clone 96 FR2.10 monoclonal antibody, along with binding to the reference Leb and Ley hexaglycosylceramides. Thus, the binding specificity of the clone 96 FR2.10 anti-Leb monoclonal antibody is broader, and it tolerates both a type 2 core chain (Galβ4GlcNAc) and a ganglio core chain (Galβ3GalNAc), in addition to the type 1 core chain (Galβ3GlcNAc) of Leb. Cross-reactivity of the clone 96 FR2.10 monoclonal antibody with blood group H type 1 determinant (Fuc{alpha}2Galβ3GlcNAc) has been reported previously (Good et al. 1992Go; Larson et al. 1999Go).

The clone 96 FR2.10 monoclonal antibody was utilized to demonstrate the presence of the Leb epitope in a glycoprotein from the transgenic Leb mouse stomach by binding to stomach protein extracts (Falk et al. 1995Go). We also detected a glycoprotein recognized by this antibody in protein preparations from transgenic Leb mouse stomach. However, no binding of the clone T218 monoclonal antibody or H. pylori strain J99 to this glycoprotein was obtained (data not shown), indicating that clone 96 FR2.10 monoclonal antibody-binding carbohydrate of this glycoprotein differs from the "traditional" Leb epitope and also from the ganglio-based Leb epitope. Further studies are, thus, needed to identify the Leb-like glycoprotein epitope from the transgenic Leb mouse stomach and also to clarify if the binding of H. pylori is mediated by this Leb-like glycoprotein epitope or by the ganglio-Leb glycosphingolipid.

Transgenic animals with re-modeled carbohydrates have also been developed in the context of xenotransplantation. Here, three different approaches have been utilized, all targeting the xenoantigen Gal{alpha}3Galβ4GlcNAc, produced by the {alpha}-1,3-galactosyltransferase. Firstly, the level of the Gal{alpha}3Galβ4GlcNAc epitope has been reduced by the introduction of an {alpha}-1,2-fucosyltransferase, which competes with the {alpha}-1,3-galactosyltransferase for the common N-acetyllactosamine (Galβ4GlcNAc) substrate (Costa et al. 1999Go). Secondly, the {alpha}-1,3-galactosyltransferase gene has been inactivated, leading to elimination of the xenoantigen (Tearle et al. 1996Go; Phelps et al. 2003Go; Kolber-Simonds et al. 2004Go). A recent biochemical characterization of small intestinal and pancreatic glycosphingolipids from galactosyltransferase knock-out pigs confirmed the absence of Gal{alpha}3Galβ4GlcNAc-terminated compounds (Diswall et al. 2007Go). The third approach is overexpression of lysosomal {alpha}-galactosidase, which also decreased the level of the Gal{alpha}3Galβ4GlcNAc epitope (Osman et al. 1997Go).

In conclusion, manipulation of the glycome by the introduction of glycosyltransferases in transgenic animals may result in novel carbohydrate sequences. The resulting carbohydrate expression of transgenic animals has mainly been monitored by binding of antibodies and lectins (Shinkel et al. 1997Go; Phelps et al. 2003Go; Kolber-Simonds 2004Go). However, as recently described by Manimala et al. (2007Go), there are severe specificity problems with many carbohydrate-binding monoclonal antibodies currently in use. Thus, biochemical methods are still necessary for the characterization of carbohydrate sequences, and antibody binding data only are not sufficient.


   Material and methods
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 Abstract
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 Results
 Discussion
 Material and methods
Funding
 Conflict of interest statement
 References
 
Preparation of glycosphingolipids
FVB/N transgenic mice, expressing the human {alpha}-1,3/4-fucosyltransferase gene in the stomachs, were used (Falk et al. 1995Go). The mice were bred and kept at Astrid Fagreaus Laboratory in Stockholm, Sweden. They were kept on a 12 h light-dark cycle and water and food (standard pellet diet) was provided ad libitum. To ensure transgenicity, all breeding couples were tested for the presence of the transgene as described by Falk et al. (1995Go). All animal experiments were approved by the local animal ethical committee at Karolinska Institutet.

The mice were sacrificed with cervical dislocation under inhalation anesthesia with isofluran and stomachs were recovered and kept at –70°C. In total, stomach tissue was collected from 209 transgenic Leb-mice. Acid and neutral glycosphingolipids were isolated as described (Karlsson 1987Go). Briefly, the pooled stomach tissue was lyophilized (dry weight 3.84 g), and then extracted in two steps in a Soxhlet apparatus with chloroform and methanol (2:1 and 1:9, by volume, respectively). The material obtained was subjected to mild alkaline hydrolysis and dialysis, followed by separation on a silicic acid column. Acid and neutral glycosphingolipid fractions were obtained by chromatography on a DEAE-cellulose column. In order to separate the neutral glycolipids from alkali-stable phospholipids, this fraction was acetylated and separated on a second silicic acid column, followed by deacetylation and dialysis. After further chromatographies on DEAE-cellulose and silicic acid columns, 167 mg of acid and 29 mg of neutral glycosphingolipids were obtained.

The neutral fraction was first separated on a silicic acid column eluted with increasing volumes of methanol in chloroform. Thereby, 13.5 mg of pure monoglycosylceramides was obtained. The more slow-migrating compounds (10 mg) were further separated on an Iatrobeads’ (Iatrobeads 6RS-8060; Iatron Laboratories, Tokyo) column (10 g), first eluted with chloroform/ methanol/water 65:25:4 (by volume), 5 x 5 mL, followed by chloroform/methanol/water 60:35:8 (by volume), 7 x 5 mL, and chloroform/methanol/water 40:40:12 (by volume), 2 x 10 mL. Aliquots of the fractions that were colored by anisaldehyde on thin-layer plates were tested for binding of H. pylori and monoclonal anti-Leb antibodies using the chromatogram binding assay (see below).

Reference glycosphingolipids
Total acid and neutral glycosphingolipid fractions were obtained by standard procedures (Karlsson 1987Go). The individual glycosphingolipids were isolated by repeated chromatography on silicic acid columns of the native glycosphingolipid fractions, or acetylated derivatives thereof. The identity of the purified glycosphingolipids was confirmed by mass spectrometry (Samuelsson et al. 1990Go), proton NMR spectroscopy (Koerner et al. 1983Go), and degradation studies (Yang and Hakomori 1971Go; Stellner et al. 1973Go).

Thin-layer chromatography
Precoated silica gel 60 HPTLC plates with either a glass or aluminum support (Merck, Darmstadt, Germany) were used for thin-layer chromatography. The solvent system used for separation of neutral glycosphingoipids was chloroform/ methanol/water 60:35:8 (by volume). Glycosphingolipids were detected by the anisaldehyde reagent (Waldi 1962Go).

Chromatogram binding assay
Mouse monoclonal anti-Leb IgM antibodies from clone BG-6 (T218) were purchased from Signet/Covance (Princeton, NJ). Mouse monoclonal anti-Leb IgM antibodies from clone 96FR2.10 were from Immucor (Rödermark, Germany). The BabA-expressing H. pylori strain J99 was described by Mahdavi et al. (2002Go). Culture conditions and 35S-labeling of the H. pylori strains was described in Roche et al. (2004Go).

Binding of monoclonal antibodies to glycosphingolipids separated on thin-layer chromatograms was performed according to Hansson et al. (1983Go). In short, 20–40 µg aliquots of mixtures of glycolipids, or 0.08–4 µg of pure compounds, were separated on aluminum-backed thin-layer plates. Dried chromatograms were dipped for 1 min in diethylether/n-hexane (1:5, by volume) containing 0.5% (w/v) polyisobutylmethacrylate (Aldrich Chem. Comp., Milwaukee, WI). After drying, the chromatograms were soaked in phosphate-buffered saline, pH 7.3 (PBS), containing 2% bovine serum albumin and 0.1% NaN3 (Solution A), for 2 h at room temperature. Suspensions of monoclonal antibodies from clone T218 (Signet/Covance) diluted 1:100 in Sol. A, or from clone 96 FR2.10 (Immucor Gamma, Norcross, GA) diluted 1:1000 in Sol. A, were gently sprinkled over the chromatograms, followed by incubation for 2 h at room temperature. After washing with PBS followed a second 2 h incubation with 125I-labeled (labeled by the IODO-GEN method; Aggarwal et al. 1985Go) rabbit anti-mouse antibodies (DakoCytomation Norden A/S, Glostrup, Denmark) diluted to 2 x 106 cpm/mL in Sol. A. Finally, the plates were washed six times with PBS.

Binding of radiolabeled H. pylori to glycosphingolipids on thin-layer chromatograms was done as described (Roche et al. 2004Go).

Dried chromatograms were autoradiographed for 12–24 h using XAR-5 X-ray films (Eastman Kodak, Rochester, NY).

Negative ion FAB mass spectrometry
Negative ion FAB mass spectra were recorded on a JEOL SX-102A mass spectrometer (JEOL, Tokyo, Japan). The ions were produced by 6 keV xenon atom bombardment, using triethanolamine (Fluka, Buchs, Switzerland) as a matrix, and an accelerating voltage of –10 kV.

Proton NMR spectroscopy
1H NMR spectra were acquired on a Varian 600 MHz spectrometer at 30°C. Samples were dissolved in dimethyl sulfoxide/D2O (98:2, by volume) after deuterium exchange. Two-dimensional double quantum-filtered correlated spectroscopy (DQF-COSY) spectra were recorded by the standard pulse sequence (Marion and Wüthrich 1983Go).


   Funding
Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Funding
Conflict of interest statement
 References
 
The Swedish Medical Research Council (to S.T. and T.B.); the Swedish Cancer Foundation (to S.T. and T.B.); Umeå University Biotechnology Fund (to T.B.); the JC Kempe and Seth M Kempe Memorial Foundation (to T.B.); Magnus Bergvalls Foundation (to S.T.); and the program "Glycoconjugates in Biological Systems/Swedish Foundation for Strategic Research (to S.T. and T.B.).


   Conflict of interest statement
Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Funding
 Conflict of interest statement
References
 
None declared.


   Acknowledgements
 
The use of the Varian 600 MHz machine at the Swedish NMR Centre, Hasselblad Laboratory, Göteborg University, is gratefully acknowledged.


   Footnotes
 
2 Present address: Cellular Architecture and Dynamics (CA&D), Utrecht University, The Netherlands. Back


   Abbreviations
 
BabA, blood group binding adhesin; FAB, fast atom bombardment


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Funding
 Conflict of interest statement
 References
 
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A. Magalhaes, J. Gomes, M. N. Ismail, S. M Haslam, N. Mendes, H. Osorio, L. David, J. Le Pendu, R. Haas, A. Dell, et al.
Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa
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