Helicobacter pylori–binding gangliosides of human gastric adenocarcinoma

Niamh Roche1, Thomas Larsson, Jonas Ångström and Susann Teneberg

Institute of Medical Biochemistry, Göteborg University, P.O. Box 440, SE 405 30 Göteborg, Sweden

Received on March 8, 2001; revised on June 5, 2001; accepted on July 24, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Acidic and neutral glycosphingolipids were isolated from a human gastric adenocarcinoma, and binding of Helicobacter pylori to the isolated glycosphingolipids was assessed using the chromatogram binding assay. The isolated glycosphingolipids were characterized using fast atom bombardment mass spectrometry and by binding of antibodies and lectins. The predominating neutral glycosphingolipids were found to migrate in the di- to tetraglycosylceramide regions as revealed by anisaldehyde staining and detection with lectins. No binding of H. pylori to these compounds was obtained. The most abundant acidic glycosphingolipids, migrating as the GM3 ganglioside and sialyl-neolactotetraosylceramide, were not recognized by the bacteria. Instead, H. pylori selectively interacted with slow-migrating, low abundant gangliosides not detected by anisaldehyde staining. Binding-active gangliosides were isolated and characterized by mass spectrometry, proton nuclear magnetic resonance, and lectin binding as sialyl-neolactohexaosylceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer) and sialyl-neolactooctaosylceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer).

Key words: Helicobacter pylori/gangliosides/gastric adenocarcinoma/microbial adhesion


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Helicobacter pylori is a human-specific pathogen found in the gastric mucus or attached to the gastric epithelium (Warren and Marshall, 1983Go). Half of the world’s adult population is infected by this organism, but most of those infected remain asymptomatic, with clinical symptoms occurring in only 10–20% of infected individuals. In the event of clinical symptoms appearing H. pylori is known to be the etiologic agent in the development of chronic active gastritis and peptic ulcer disease and is a risk factor in the development of gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. A recent report even implicates H. pylori infection with Sudden Infant Death Syndrome (Kerr et al., 2000Go).

Attachment of a microbe to a cell surface receptor on the target tissue is considered an essential step in the initiation, establishment, and maintenance of infection. As a result great interest has been shown in the elucidation and identification of potential host receptors, the majority of which appear to be glycoconjugates. Glycoconjugates exhibit a characteristic and specific pattern of expression, which is dependent on the animal species, individual, and cell type, thus explaining the phenomenon of tropism of infection. An understanding of the interaction between the microbe and the carbohydrate moiety of such glyocconjugates offers an alternate mode of treatment of infection in the form of anti-adhesive therapy. Anti-adhesive therapy is particularly attractive in the case of H. pylori infection for a number of reasons. Though H. pylori infection can be combated by antibiotic treatment, resistant strains are emerging (Xia et al., 1996Go; Vakil et al., 1998Go), creating a need for an alternate form of treatment. One alternative to antibiotic therapy is vaccination. However, development of a suitable vaccine has been hindered by the need for a nontoxic adjuvant and the degree of protection offered (reviewed in Banerjee and Michetti, 1999Go). Anti-adhesive therapy is a good second alternative method to combat infection with H. pylori because it neither promotes the emergence of resistant strains nor gives rise to the hurdles presented by vaccine development.

Among potential receptors described for H. pylori are N-acetylneuraminyllactose (Evans et al., 1988Go), lactosylceramide (Ångström et al., 1998Go), sialic acid–containing glycosphingolipids (Miller-Podraza et al., 1997Go) and polyglycosylceramides (Miller-Podraza et al., 1996Go), sulfatide (Saitoh et al., 1991Go; Kamisago et al., 1996Go), Lewis b blood group epitope (Borén et al., 1993Go), and gangliotriaosylceramide and gangliotetraosylceramide (Gold et al., 1993Go). Other potential receptors described for H. pylori include heparan sulfate (Ascencio et al., 1993Go) and phosphatidylethanolamine (Lingwood et al., 1992Go).

A number of different approaches have been employed in the elucidation of target tissue receptors for H. pylori. A combination of in situ analysis of the binding of H. pylori to human gastric surface mucosal cells, blocking of binding by preincubation of bacteria with neoglycoconjugates, and the binding of H. pylori to glycoconjugates immobilized on protein blots were used to reveal the binding of H. pylori to the Leb antigen (Borén et al., 1993Go). In another study, binding of H. pylori to glycolipids obtained from human gastric mucosa separated on thin-layer plates was reported (Saitoh et al., 1991Go). The aim of the present study was to characterize the glycosphingolipids of a human gastric adenocarcinoma, which is derived from the human gastric epithelium, with particular interest in those components with H. pylori–binding activity. We show here that the major acidic glycosphingolipids of human adenocarcinoma, the GM3 ganglioside and sialyl-neolactotetraosylceramide, were not recognized by the bacteria. Instead, H. pylori bound to low abundant, slow-migrating, sialic acid–containing glycosphingolipids, characterized as sialyl-neolactohexaosylceramide and sialyl-neolactooctaosylceramide.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of glycosphingolipids from human gastric adenocarcinoma
From approximately 80 g of starting material, 500 mg of total acidic and 430 mg of total neutral glycosphingolipids were obtained. The sialic acid and sphingosine content of the total acidic glycosphingolipid fraction were 402 and 457 nmol per g dry weight, respectively. The sphingosine content of the total neutral glycosphingolipid fraction was 579 nmol per g dry weight. Anisaldehyde staining of the total neutral glycosphingolipid fraction (Figure 1, lane 4) revealed the presence of three major glycosphingolipids. The dominating component migrated as the lactosylceramide component in the neutral glycosphingolipid fraction of human erythrocytes (lane 1). Two other components of equal intensity were also observed. The faster-migrating of these migrated as globotriaosylceramide (lane 2), and the slower migrated as globotetraosylceramide (lane 3). A number of low abundant, slow-migrating components below the tetraglycosylceramide region were also observed. The dominating glycosphingolipid of the total acidic fraction (lane 5) migrated at the level of the GM3 ganglioside reference (lane 6). Two minor components, of which the faster-migrating one migrated as sialyl-neolactotetraosylceramide (lane 7), were also observed.



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Fig. 1. Crude total acidic and neutral glycosphingolipids of human gastric adenocarcinoma. The crude total acidic and the total neutral glycosphingolipid fractions isolated from a human gastric adenocarcinoma and reference glycosphingolipids were separated on thin-layer plates using chloroform:methanol:water (60:35:8, by volume) as solvent system. Detection was by anisaldehyde staining. Lane 1: total neutral glycosphingolipids of human erythrocytes, 40 µg; lane 2: globotriaosylceramide (Gal{alpha}4Galß4Glcß1Cer with d18:1–16:0 and d18:1–24:0 as ceramides), 2 µg; lane 3: globotetraosylceramide (GalNAcß3Gal{alpha}4Galß4Glcß1Cer with d18:1–16:0 and d18:1–24:0 as ceramides), 2 µg; lane 4: total neutral glycosphingolipids of human gastric adenocarcinoma, 80 µg; lane 5: total acidic glycosphingolipids of human gastric adenocarcinoma, 80 µg; lane 6: GM3 ganglioside (NeuAc{alpha}3Galß4Glcß1Cer with d18:1–18:0 and d20:1–18:0 as ceramides) of human brain, 2 µg; lane 7: sialyl-neolactotetraosylceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer with d18:1–16:0 and d18:1–24:0 as ceramides) of human neutrophils, 2 µg. The bands marked with an X are nonglycosphingolipid contaminants.

 
Binding of H. pylori to glycosphingolipids from human gastric adenocarcinoma
To investigate the presence of glycosphingolipids with H. pylori binding activity, the binding of four H. pylori strains to both the crude total acidic and the total neutral glycosphingolipid fractions from human gastric adenocarcinoma was investigated. The binding of H. pylori to the crude total acidic glycosphingolipid fraction obtained from human gastric adenocarcinoma was initially tested in parallel with the crude total acidic glycosphingolipid fractions of human gall bladder adenocarcinoma and human neutrophils (Figure 2). Thereby, binding of the bacteria to the total acidic glycosphingolipid fraction of human gastric adenocarcinoma, along with binding to the acidic glycosphingolipid fraction of human gallbladder adenocarcinoma, and human neutrophils was observed (Figure 2B, lanes 2–4), whereas no binding to the GM3 ganglioside (lane 1) occurred. In all cases the bacteria bound to slow-migrating gangliosides migrating below the GM3 reference and sialyl-neolactotetraosylceramide of human neutrophils, and which were not detected by the chemical stain (Figure 2A). Of the strains tested, only strains CCUG 17874 and S-032 bound the acidic glycosphingolipids from human gastric adenocarcinoma; no binding of strains CCUG 17875 or 26695 was obtained.



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Fig. 2. Binding of H. pylori to gangliosides of human gastric adenocarcinoma. Gangliosides of the total acidic fraction of human gastric adenocarcinoma as detected by anisaldehyde staining (A) and by autoradiography after binding of 35S-labeled H. pylori strain CCUG 17874 (B). The glycosphingolipids were separated on thin-layer plates using chloroform:methanol:water (60:35:8, by volume) as solvent system, and binding assays were carried out as described under Materials and methods. Autoradiography was for 12 h. Lane 1: GM3 ganglioside (NeuAc{alpha}3Galß4Glc41Cer) of human brain, 4 µg; lane 2: total acidic glycosphingolipid fraction of human gall bladder adenocarcinoma, 40 µg; lane 3: total acidic glycosphingolipid fraction of human gastric adenocarcinoma, 80 µg; lane 4: total acidic glycosphingolipid fraction of human neutrophil granulocytes, 40 µg. The bands marked I and II indicate the migration of GM3 ganglioside and SPG respectively, and bands marked with an X are nonglycosphingolipid contaminants.

 
None of the four strains tested bound to the total neutral glycosphingolipid fraction (not shown); therefore the neutral fraction will not be discussed further.

Purification of acidic glycosphingolipids from human gastric adenocarcinoma
To purify the acidic glycosphingolipids of the human gastric adenocarcinoma, the crude total acidic fraction was acetylated and separated on a silicic acid column followed by deacetylation, Iatrobeads column chromatography, and final purification by high-performance liquid chromatography (HPLC). The fractions obtained were stained by anisaldehyde to select glycosphingolipid-containing fractions and subsequently tested for H. pylori–binding activity using the chromatogram binding assay. Fractions were pooled according to their mobility on thin-layer chromatograms and H. pylori–binding activity, giving rise to four fractions, designated fractions I–IV. Fractions I and II weighed approximately 1 mg each, but only 100 µg of both fractions III and IV was obtained. Fraction I migrated in the GM3 region, fraction II in the sialyl-neolactotetraosylceramide region, and fractions III and IV were more slow-migrating (Figure 3A, lanes 2–5).



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Fig. 3. Binding of H. pylori, monoclonal anti-sialyl-Lea antibodies, and S. tuberosum lectin to gangliosides purified from human gastric adenocarcinoma. Thin-layer chromatogram of separated gangliosides after detection with anisaldehyde (A), autoradiograms obtained after binding of 35S-labeled H. pylori strain CCUG 17874 (B), anti-sialyl-Lea monoclonal antibody CA 19–9 (C), and 125I-labeled S. tuberosum lectin (D). The gangliosides were chromatographed using chloroform:methanol:water (60:35:8, by volume) as solvent system, and after drying rechromatographed once using the same solvent system. The binding assays were done as described under Materials and methods. The arrow marks the level on the thin-layer chromatogram plate at which the sample was applied. Lane 1: total acidic glycosphingolipid fraction of human gastric adenocarcinoma, 80 µg; lane 2: fraction I isolated from human gastric adenocarcinoma, 4 µg; lane 3: fraction II isolated from human gastric adenocarcinoma, 2 µg; lane 4: fraction III isolated from human gastric adenocarcinoma, 2 µg; lane 5: fraction IV isolated from human gastric adenocarcinoma, 2 µg; lane 6: reference sialyl-neolactotetraosylceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer) of human erythrocytes, 4 µg; lane 7: reference sialyl-neolactohexaosylceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer) and sialyl-neolactooctaosylceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer) of human erythrocytes 4 µg; lane 8: reference sialyl-Lea hexaglycosylceramide (NeuAc{alpha}3Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer) of human gallbladder adenocarcinoma, 4 µg. Autoradiography was for 12 h. The apparent bands observed in Figure 3C lanes 1 and 4 are artfacts.

 
Binding of H. pylori to purified gangliosides from human gastric adenocarcinoma
To determine which of the four fractions contained H. pylori binding activity, binding assays using H. pylori strain CCUG 17874 were carried out. No binding of H. pylori to fractions I and II was observed (Figure 3B, lanes 2 and 3), while the bacteria bound to two components in both fractions III and IV (Figure 3B, lanes 4 and 5). A comparison of lanes 1, 4, and 5 (Figure 3A) with the corresponding lanes in Figure 3B shows that binding in all three cases is to slow-migrating components, two of which are not detected by chemical staining. In addition, binding of H. pylori to the references sialyl-neolactohexaosylceramide and sialyl-neolactooctoaosylceramide (Figure 3, lane 7), which comigrated with the H. pylori–binding compounds in fractions III and IV, was observed.

Negative ion FAB-MS of the ganglioside fractions isolated from human gastric adenocarcinoma
Fast atom bombardment mass spectrometry (FAB-MS) in the negative mode was used for initial characterization of the four ganglioside fractions isolated from human gastric adenocarcinoma. In the spectrum of fraction I (not shown) characteristic molecular ions at m/z 1152, 1236, and 1252 indicated a glycosphingolipid with one NeuAc and two Hex combined with d18:1–16:0, d18:1–22:0, and d18:1–h22:0 ceramides, respectively. The latter species constituted the dominating ceramide species in this fraction. Sequence ions obtained from the molecular ion at m/z 1252 were seen at m/z 961 (M-NeuAc) and m/z 799 (M-NeuAc-Hex). A peak at m/z 1467, which is the molecular ion of a glycosphingolipid with one NeuAc, one HexNAc, and two Hex, combined with d18:1–24:0 ceramide, was also observed.

The mass spectrum of fraction II (not shown) had molecular ions at m/z 1630 and 1602, which are characteristic of a glycosphingolipid with one NeuAc, one HexNAc, and three Hex, with d18:1–24:0 and d18:1–22:0 ceramides, respectively. Sequence ions derived from the molecular ion at m/z 1630 were observed at m/z 1339 (M-NeuAc), m/z 1177 (M-NeuAc-Hex), m/z 973 (M-NeuAc-Hex-HexNAc), and m/z 811 (M-NeuAc-Hex-HexNAc-Hex), indicating a NeuAc-Hex-HexNAc-Hex-Hex sequence. Molecular ions were also observed at m/z 1354 and m/z 1456, indicating the presence of a glycosphingolipid with one NeuAc, one HexNAc, and two Hex combined with d18:1–16:0 and t18:0–22:0 ceramides, respectively.

Thus, negative ion FAB-MS demonstrated a sequence in agreement with the GM3 ganglioside in fraction I. Minor ions suggesting a HexNAc-NeuAc-Hex-Hex sequence (most likely the GM2 ganglioside) were also found. Fraction II had a compound with NeuAc-Hex-HexNAc-Hex-Hex sequence, which is in agreement with sialyl-neolactotetraosylceramide. Ions suggesting a GM2 ganglioside in this fraction were also observed.

The mass spectrum of fraction III had two molecular ions at m/z 1882 and 1992 (Figure 4A), indicating a glycosphingolipid with one NeuAc, two HexNAc, and four Hex, and with d18:1–16:0 and d18:1–24:1 ceramides, respectively. Two series of fragment ions were observed at m/z 1591/1701 (1882/1992-NeuAc), m/z 1429/1538 (1882/1992-NeuAc-Hex), m/z 1225/1335 (1882/1992-NeuAc-Hex-HexNAc), m/z 1063/1173 (1882/1992-NeuAc-Hex-HexNAc-Hex), and m/z 860/970 (1882/1992-NeuAc-Hex-HexNAc-Hex-HexNAc). In addition, sequence ions at m/z 698/808 and 536/646 (not shown) corresponding to 1882/1992-NeuAc-Hex-HexNAc-Hex-HexNAc-Hex and 1882/1992-NeuAc-Hex-HexNAc-Hex-HexNAc-Hex-Hex, respectively, were also observed. Thus a glycosphingolipid with NeuAc-Hex-HexNAc-Hex-HexNAc-Hex-Hex sequence and d18:1–16:0 and d18:1–24:1 ceramides, respectively, was demonstrated.



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Fig. 4. Negative ion FAB mass spectra of fraction III (A) and fraction IV (B) isolated from human gastric adenocarcinoma. Fractions were dissolved in dimethyl sulfoxide, and FAB-MS was carried out in the negative mode using Xe atoms (6 keV), triethanolamine as matrix, and a resolution of 1000. Two spectra were collected, one using an acceleration voltage of 10 kV (A) and the second using an acceleration voltage of 8 kV (B). For spectrum B a range of 1500–2500 mass units was scanned and data collected for 15 min. Only that part of the spectrum ranging from m/z 1800 to 2350 is shown here.

 
In the spectrum of fraction IV (Figure 4B) six molecular ions were observed. The molecular ions at m/z 1882 and 1992 corresponded to NeuAc-Hex-HexNAc-Hex-HexNAc-Hex-Hex sequence with either d18:1–16:0 or d18:1–24:1 ceramides, as discussed, while the molecular ions at m/z 2010 and 2028 corresponded to the same carbohydrate sequence attached to d18:1–h24:0 and t18:0–h24:0 ceramides, respectively. The molecular ions at m/z 2247 and 2358 represented an analogous compound with NeuAc-Hex-HexNAc-Hex-HexNAc-Hex-HexNAc-Hex-Hex sequence attached to d18:1–16:0 and d18:1–24:1 ceramides. A fragment ion at m/z 1906 corresponding to m/z 2358-NeuAc-Hex was also observed. A series of peaks were also observed between m/z 1906 and 1993. However, these could not be accurately assigned due to poor resolution within this region.

Thus a ganglioside with NeuAc-Hex-HexNAc-Hex-HexNAc-Hex-Hex sequence was indicated by mass spectrometry of H. pylori–binding fraction III. The same ganglioside sequence was present in the spectrum of H. pylori–binding fraction IV, and in addition a ganglioside with NeuAc-Hex-HexNAc-Hex-HexNAc-Hex-HexNAc-Hex-Hex sequence was demonstrated.

Proton NMR of fraction III isolated from human gastric adenocarcinoma
Mass spectrometry data indicate that fraction III contains a glycosphingolipid with the sequence NeuAc-Hex-HexNAc-Hex-HexNAc-Hex-Hex, pointing to the ganglioside sialyl-neolactohexaosylceramide. However, the 600 MHz proton nuclear magnetic resonance (NMR) spectrum of fraction III (Figure 5) clearly reveals the presence of two molecular species with approximate relative intensities 60/40.



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Fig. 5. Proton NMR spectrum of fraction III at 600 MHz. The sample was dissolved in dimethyl sulfoxide/D2O (98:2, by volume) after deuterium exchange, and the spectrum was recorded at 30°C. Readily identifiable resonances have been indicated.

 
A comparison with the reference compound sialyl-Lea (NeuAc{alpha}3Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer) purified from gallbladder adenocarcinoma (unpublished) indicates that the dominating species is identical to this glycosphingolipid (ppm values are given in parentheses). Thus the {alpha}-anomeric signal at 4.78 (4.76) ppm suggests the presence of a Fuc{alpha}4 residue as does the signal at 4.64 (4.66) ppm attributable to the H-5 proton of the same residue. The ß-anomeric signal seen at 4.69 (4.69) ppm is indicative of a GlcNAcß3 residue, whereas the ß-anomeric signal at 4.36 (4.33) ppm most likely is due to an internal Galß3. The anomeric Galß4 signal is seen at 4.27 (4.27) ppm, and the Glcß1 signal is found at 4.16 (4.16) ppm. In addition, the resonance due to the H3eq proton of NeuAc{alpha}3 is found at 2.75 (2.77) ppm. Furthermore, in the N-acetyl region the two methyl signals seen at 1.87 (1.89) ppm and 1.83 (1.81) ppm again reveal the presence of a GlcNAc residue and a NeuAc residue, respectively.

The signals from the minor species most likely corresponds to sialyl-neolactohexaosyl ceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer) as suggested by mass spectrometric data and earlier NMR data (given in parentheses) on the pure compound (Stroud et al., 1996Go). The two partially overlapping GlcNAcß3 anomeric signals from sialyl-neolactohexaosylceramide are expected around 4.64 (4.64) ppm, appearing as a triplet, thus overlapping also with the Fuc{alpha}4 H-5 proton of sialyl-Lea. The two internal Galß4 anomeric signals are expected around 4.25 (4.26) ppm, while the penultimate Galß4 and Glcß1 anomeric signals are seen at 4.22 (4.20) and 4.20 (4.19) ppm, respectively. The resonance due to the H-3eq proton of NeuAc{alpha}3 is found at 2.75 (2.75) ppm, overlapping with the corresponding residue of sialyl-Lea. However, the methyl resonances originating from the N-acetyl moieties of NeuAc{alpha}3 and the two GlcNAcß3 are clearly separated from the corresponding sialyl-Lea signals and are found at 1.89 (1.89) ppm and 1.81 (1.81) ppm, respectively. Due to insufficient material it was not possible to perform NMR analysis on fraction IV.

Binding of Solanum tuberosum lectin to gangliosides of human gastric adenocarcinoma
To further characterize the H. pylori–binding compounds in fractions III and IV, binding assays using S. tuberosum lectin PL-I, which binds to sialic acid–terminated glycosphingolipids carrying an internal dimeric Galß4GlcNAcß sequence (Ciopraga et al., 2000Go), were subsequently carried out. The lectin bound to the total acidic fraction of human gastric adenocarcinoma (Figure 3D, lane 1), to fractions III and IV (lanes 4 and 5) and to the references sialyl-neolactohexaosylceramide and sialyl-neolactooctaosylceramide (lane 7). Indeed, the binding pattern of the S. tuberosum lectin was identical to the binding patterns obtained with H. pylori strains CCUG 17874 (Figure 3B) and S-032 (not shown).

Binding of Erythrina corallodendron lectin to desialylated gangliosides of human gastric adenocarcinoma
The lectin from E. corallodendron binds to glycosphingolipids with terminal Galß4GlcNAcß and Fuc{alpha}2Galß4GlcNAcß sequences (Teneberg et al., 1994Go). Binding of 125I-labeled E. corallodendron lectin to the de-sialylated fractions III and IV of human gastric adenocarcinoma is shown in Figure 6. Thus desialylation of fraction III resulted in a compound with E. corallodendron–binding activity, migrating in the hexaglycosylceramide region (lane 2), while the desialylated fraction IV (lane 4) had two compounds with E. corallodendron–binding activity, migrating as hexa- and octaglycosylceramides, respectively.



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Fig. 6. Binding of E. corallodendron lectin to desialylated fractions III and IV from human gastric adenocarcinoma. Thin-layer chromatogram of separated glycosphingolipids after detection with anisaldehyde (A), and autoradiogram obtained after binding of 125I-labeled E. corallodendron lectin (B). The glycosphingolipids were chromatographed using chloroform:methanol:water (60:35:8, by volume) as solvent system. The binding assays were done as described under Materials and methods. Lane 1: fraction III isolated from human gastric adenocarcinoma, 4 µg; lane 2: fraction III after mild acid hydrolysis, 4 µg; lane 3: fraction IV isolated from human gastric adenocarcinoma, 4 µg; lane 4: fraction IV after mild acid hydrolysis, 4 µg; lane 5: reference neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer) of human neutrophil granulocytes, 4 µg; lane 6: reference total neutral glycosphingolipids of human neutrophil granulocytes, 40 µg. Autoradiography was for 12 h.

 
Binding of monoclonal antibody CA 19-9 to gangliosides of human gastric adenocarcinoma
Because the proton NMR analysis suggested the presence of sialyl-Lea in fraction III binding by the monoclonal antibody CA 19-9, which recognizes the sialyl-Lea epitope (Magnani et al., 1982Go) was tested. However, the monoclonal antibody bound only to the reference sialyl-Lea hexaglycosylceramide (Figure 3C, lane 8; Figure 7C, lanes 2 and 3), while no binding to the total acidic fraction of human gastric adenocarcinoma or to fractions I–IV isolated there from (Figure 3C, lanes 1–5; Figure 7C, lane 1) was obtained. The absence of binding of H. pylori to the sialyl-Lea hexaglycosylceramide (Figure 3B, lane 8; Figure 7B, lanes 2 and 3) should also be noted.



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Fig. 7. Comparison of binding of H. pylori and anti-sialyl-Lea monoclonal antibody CA 19-9. Thin-layer chromatogram of separated glycosphingolipids after detection with anisaldehyde (A), and autoradiograms obtained after binding of 35S-labeled H. pylori strain CCUG 17874 (B) and CA 19-9 monoclonal antibodies (C). Glycosphingolipids were separated on thin-layer plates using chloroform:methanol:water (60:35:8, by volume) as solvent system. The binding assays were done as described under Materials and methods. Lane 1: acidic glycosphingolipids of human gastric adenocarcinoma, 40 µg; lane 2: acidic glycosphingolipids of human gall bladder adenocarcinoma, 40 µg; lane 3: reference sialyl-Lea hexaglycosylceramide (NeuAc{alpha}3Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer) of human gall bladder adenocarcinoma, 2 µg; lane 4: reference Lea pentaglycosylceramide (Galß3(Fuc{alpha}4)GlcNAcß3Galß4Glcß1Cer) of human meconium, 4 µg. Autoradiography was for 12 h. Nonglycosphingolipid contaminants are marked with an X.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
H. pylori has a very narrow host and tissue range and has only been found in connection to gastric cells from humans and monkeys (Dubois et al., 1991Go). In the present study, a human gastric adenocarcinoma was used as a model for human gastric epithelium, to obtain sufficient material to allow isolation and structural characterization of potential H. pylori–binding glycosphingolipids. Acidic and neutral glycosphingolipid fractions obtained from the adenocarcinoma were tested for H. pylori–binding activity, and two slow-migrating H. pylori–binding gangliosides were thereby detected. After isolation the binding-active gangliosides were characterized by mass spectrometry, proton NMR, and lectin binding as sialyl-neolactohexaosylceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer) and sialyl-neolactooctaosylceramide (NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcNAcß3Galß4GlcNAcß3Galß4Glcß1Cer). Sialyl-neolactohexaosylceramide has been tentatively identified in normal human gastric epithelium (Teneberg et al., unpublished data). The structures of the gangliosides isolated from human gastric adenocarcinoma and their H. pylori–binding activity are summarized in Table I. The nonbinding of the GM3 ganglioside and NeuAc{alpha}6neolactotetraosylceramide, sialyl-Lea hexaglycosylceramide, along with binding to NeuAc{alpha}3neolactohexaosylceramide and NeuAc{alpha}3neolactooctaocylceramide is in agreement with the NeuAc{alpha}3Galß4GlcNAcß-epitope previously suggested by Johansson and Miller-Podraza (1998)Go.


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Table I. Binding of H. pylori to gangliosides isolated from a human gastric adenocarcinoma
 
Interestingly, gangliosides with terminal NeuAc{alpha}3Galß4GlcNAcß3Galß4GlcNAcß sequence are also recognized by the neutrophil-activating protein of H. pylori, HPNAP (Teneberg et al., 1997Go). However, although it was recently reported that HPNAP is a major immunogen of H. pylori (Satin et al., 2000Go), the presence of this protein on the bacterial cell surface has not yet been demonstrated. Further investigations are thus necessary to determine if the HPNAP is the adhesin involved in binding of H. pylori to the sialylated sequences described here.

The normal human gastric epithelium has a low content of sialylated glycoconjugates (Filipe, 1979Go; Madrid et al., 1990Go). However, ablation of parietal cells in transgenic mice leads to an increased proliferation of NeuAc{alpha}3Galß-bearing cells to which H. pylori bind (Syder et al., 1999Go). Following binding of H. pylori to NeuAc{alpha}3Galß structures, stimulation of the inflammatory response was observed. This earlier study suggests that binding of H. pylori to sialylated glycoconjugates seems to be intricately connected to the inflammatory response. It can therefore be envisaged that binding by H. pylori to the linear sialylated neolacto series gangliosides described here may act in a similar manner. Infection by H. pylori is persistent, and the ability of the organism to adapt to changes in its environment may be deemed necessary for its survival. In an environment where receptors are appearing and disappearing, the ability of H. pylori to bind to a range of receptors in its environment may be a prerequisite for not just its survival but also the maintenance of persistent infection and the development of gastritis (Syder et al., 1999Go).

With respect to binding of H. pylori to the GM3 ganglioside, there are conflicting reports in the literature. Binding by H. pylori to GM3 ganglioside and sulfatide was reported in one study (Saitoh et al., 1991Go), but the binding by H. pylori to sulfatide but not to GM3 was later reported by the same group (Kamisago et al., 1996Go). Other studies report that binding to GM3 ganglioside by strain NCTC 11637 is occasional (Miller-Podraza et al., 1997Go) or is lost on growth in liquid culture (Kamisago et al., 1996Go; Miller-Podraza et al., 1996Go). During the course of this study, no binding to the GM3 ganglioside was observed in spite of the fact that the bacteria were cultured on solid media. These conflicting findings may in part be explained by poor exposure of this ganglioside on the thin-layer plate surface.

Binding of H. pylori to sialyl-neolactotetraosylceramide has also been reported (Miller-Podraza et al., 1997Go; Johansson and Karlsson, 1998Go). However, no binding to the ganglioside with the NeuAc-Hex-HexNAc-Hex-Hex sequence (fraction II), and most likely sialyl-neolactotetraosylceramide, was observed in the present study. H. pylori binds to sialyl-neolactotetraosylceramide when cultured on agar but not in liquid media (Miller-Podraza et al., 1996Go). As all strains used in this study were cultured on agar plates, this cannot explain the lack of binding to NeuAc-Hex-HexNAc-Hex-Hex sequence observed. A preferential binding of many strains to terminal NeuAc{alpha}3 over NeuAc{alpha}6 have been described (Hirmo et al., 1996Go; Johansson and Miller-Podraza, 1998Go; Johansson et al., 1999Go). An increase in the expression of NeuAc{alpha}6-terminated gangliosides in human colonic and liver adenocarcinoma as opposed to the corresponding normal tissue has been reported (Hakamori et al., 1983aGo,b; Nilsson et al., 1985Go). Thus a possible explanation for the nonbinding of H. pylori to fraction II may be that the sequence isolated during the course of this work contains NeuAc{alpha}6 instead of NeuAc{alpha}3. Due to insufficient material, it was not possible to clarify this point.

The sialyl-Lea-epitope has been detected in lipid extracts from four out of five human gastric adenocarcinomas (Magnani et al., 1982Go). In the present study the proton NMR data demonstrated the presence of sialyl-Lea in fraction III. However, no ions indicating a NeuAc-Hex-(Fuc)-HexNAc-Hex-Hex were found by negative ion FAB-MS. In addition, no binding by the monoclonal antibody CA 19-9 was observed to this fraction. The nondetection of the sialyl-Leaglycosphingolipid by negative ion FAB-MS might be due to the failure of this glycospshingolipid to enter the gas phase during the negative ion FAB-MS experiment. Although no binding by the monoclonal antibody CA 19-9, specific for the sialyl-Lea-epitope, to the total acidic or the isolated fractions from human gastric adenocarcinoma was obtained, binding of the CA 19-9 antibody was instead achieved to the acidic glycosphingolipids of human gallbladder adenocarcinoma and the reference Lea (Figure 7C). Although binding of both H. pylori and the monoclonal antibody CA 19-9 to the acidic glycosphingolipids of human gallbladder adenocarcinoma was observed, binding was to different regions (Figure 7B, lanes 2 and 3). This, together with the fact that H. pylori did not bind to the sialyl-Lea reference, suggests that even if the sialyl-Lea epitope is present in fraction III it is not involved in the binding of H. pylori.

H. pylori strains may be classified as type I or type II, depending on their degree of virulence (Xiang et al., 1995Go; Censini et al., 1996Go). Type I strains express both cytotoxin-associated protein (CagA) and vacuolating cytotoxin (VacA) and are more virulent than type II strains, which, by definition, express neither CagA nor VacA cytotoxin. Other strains, including some type I strains, have been shown to express the blood group antigen-binding adhesin (BabA) (Gerhard et al., 1999Go). The Lewis b blood group epitope (Fuc{alpha}2Galß3(Fuc{alpha}4)GlcNAcß) is expressed on human gastric epithelial cells (Borén et al., 1993Go) and acts as a receptor for H. pylori strains expressing the BabA adhesin but not for strains lacking it (Ilver et al., 1998Go). In a recent study, it was demonstrated that H. pylori strains expressing the BabA adhesin in addition to CagA and VacA had high correlation to duodenal ulcer and adenocarcinoma but low correlation to MALT lymphoma and gastritis (Gerhard et al., 1999Go). On the other hand, type I strains that were both CagA and VacA positive but BabA negative were associated with only duodenal ulcer but not adenocarcinoma. This illustrates the role that not just strain but also the expression/nonexpression of a particular adhesin play in determining the outcome of infection with H. pylori.

The observed binding in this study by H. pylori strains CCUG 17874 and S-032 to both the crude acidic fraction and to slow migrating gangliosides isolated from this fraction (see Tables I and II), and the lack of binding as observed for strain CCUG 17875 are in agreement with previously published results that report the binding of strains CCUG 17874 and S-032 to sialic acid–containing glycoconjugates (Hirmo et al., 1996Go), while strain CCUG 17875 does not bind sialic acid (Hirmo et al., 1996Go; Miller-Podraza et al., 1997Go). It should also be noted that strain 26695 does not bind the total acidic fraction (see Table II). This difference in binding may be explained by the expression of different adhesins by the different strains. Because H. pylori strains show a high degree of variability with respect to binding, caution should be exercised when attempting to extrapolate the binding pattern of one strain to the general H. pylori population.


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Table II. Binding of different strains of H. pylori to the total acid glycosphingolipid fraction of human gastric adenocarcinoma
 
NeuAc{alpha}3Galß4Glcß-terminated glycoconjugates inhibit the binding of H. pylori to human gastrointestinal adenocarcinoma–derived cell lines (Simon et al., 1997Go), and it was recently reported that anti-adhesive therapy with NeuAc{alpha}3Galß4Glcß in H. pylori–infected Rhesus monkeys gave suppression of infection (Mysore et al., 1999Go). As discussed above, a number of H. pylori–binding gangliosides have to date been reported. To address their relevance for the adhesion of H. pylori to its target tissue, we are currently investigating the distribution of H. pylori–binding gangliosides in normal human stomach.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of glycosphingolipids from human gastric adenocarcinoma
A human gastric adenocarcinoma (dry weight 80 g) was obtained at autopsy, and after lyophilization the tissue was kept at –70°C for several years. Extraction of glycosphingolipids was carried out as previously described (Karlsson, 1987Go) using chloroform:methanol (2:1 and 9:1, by volume) in a Soxhlet apparatus. The fractions were pooled, subjected to mild alkaline hydrolysis, and dialyzed before separation on a silicic acid column. Acidic and neutral glycosphingolipids were then separated on a DEAE-cellulose column. The neutral glycosphingolipids were acetylated and separated from alkali-stable phospholipids on a second silicic acid column. This was followed by deacetylation and dialysis of the neutral glycosphingolipids. Final purification of the total neutral fraction was achieved using DEAE and silicic acid columns. Approximately 500 mg of crude acidic glycosphingolipids and 430 mg of neutral glycosphingolipids was obtained.

The crude total acidic fraction (approximately 480 mg) was acetylated (Handa, 1963Go), and initially purified on a silicic acid column. After deacetylation, further separation using an Iatrobeads column (Iatron Laboratories, Tokyo) was carried out, before final purification by HPLC on a Kromosil 5 silica column of length 250 mm, ID 10 mm, and particle size of 5 µm (Phenomenex, Torrance CA) using a linear gradient of chloroform:methanol:water (60:35:8 to 40:40:12, by volume) over 180 min, with a flow rate of 2 ml/min. The 2-ml fractions collected were analyzed by thin-layer chromatography and anisaldehyde staining, and the H. pylori–binding activity was assessed using the chromatogram binding assay. The fractions were pooled according to the mobility on thin-layer chromatograms and their H. pylori–binding activity.

Chemical determinations
The sialic acid content was determined essentially using the resorcinol/HCl method (Svennerholm, 1957Go) with minor modifications. The absorbance of the upper phase at 620 nm was read. Determination of the sphingosine content of both the neutral and acidic fractions was carried out according to the method of Lauter and Trams (1960)Go with minor modifications.

Reference glycosphingolipids
The reference glycosphingolipids were isolated and characterized at the Institute of Medical Biochemistry, Göteborg University, Sweden. Structural characterization was performed using proton NMR spectroscopy (Falk et al., 1979aGo,b,c; Koerner et al., 1983Go), mass spectrometry (Samuelsson et al., 1990Go), and degradation studies (Yang and Hakomori, 1971Go; Stellner et al., 1973Go).

Mild acid hydrolysis
Desialylation was done by incubating the gangliosides in 1% (by volume) acetic acid for 1 h at 100°C.

Thin-layer chromatography
Total acidic or neutral glycosphingolipid fractions (40–80 µg) and pure glycosphingolipids (2–4 µg) were separated on aluminium-backed silica gel 60 high-performance thin-layer chomatography plates (Merck, Darmstadt, Germany) using chloroform:methanol:water (60:35:8, by volume) as solvent system. To obtain better separation, the acidic glycosphingolipid fractions were occasionally first chromatographed using chloroform:methanol:water (60:35:8, by volume) as solvent system, and after drying rechromatographed once using the same solvent system. Chemical detection of glycosphingolipids on thin-layer chromatograms was carried out using anisaldehyde (Waldi, 1962Go). Gangliosides were detected using the resorcinol reagent (Svennerholm, 1963Go).

Bacterial strains and growth conditions
H. pylori strains 17874 and 17875 were obtained from the Culture Collection, University of Göteborg (CCUG). Strain S-032, a clinical isolate, was a kind gift from Dr. Dan Danielsson, Örebro Medical Centre, Örebro, Sweden. Strain 26695 (Tomb et al., 1997Go) was obtained from Dr. Thomas Borén, University of Umeå, Umeå, Sweden. H. pylori were grown at microaerophilic conditions on Brucella medium (Difco Laboratories, MI) containing 10% fetal calf serum (Harlan Sera-Lab, UK) inactivated in a 56°C waterbath for 1 h and BBL IsoVitaleX Enrichment (Becton Dickson Microbiology Systems, USA). The bacteria were radiolabeled by addition of 7.15 mC/ml of 35S-methionine (Amersham Pharmacia Biotech, UK) to the culture plates. The bacteria were harvested by scraping and were washed four times by centrifugation at 3500 rpm for 10 min in phosphate-buffered saline (PBS), pH 7.3. Finally the bacteria were resuspended in PBS containing 2% (w/v) bovine serum albumin (PBS/BSA) to approximately 1 x 108 CFU/ml. The specific activities of the suspensions were approximately 1 cpm per 100 bacteria.

Lectins and antibodies
S. tuberosum lectin (designated PL-I) was a gift from Dr. Jeana Ciopraga, Romanian Academy, Bucharest, Romania. E. corallodendron lectin was purchased from Sigma (St. Louis, MO). Anti-sialyl-Lea monoclonal antibodies (CA 19-9) were purchased from Signet Laboratories (Dedham, MA). Rabbit anti-mouse immunoglobulins were purchased from Dakopatts AB (Sweden). The lectins and the anti-mouse antibodies were labeled with 125I, using the Iodogen method (Aggarwal et al., 1985Go). Approximately 5 x 103 cpm/µg protein was obtained.

Chromatogram binding assay
The binding specificities of the carbohydrate binding ligands used are summarized in Table III. The chromatogram binding assay was essentially carried out as described previously (Karlsson and Strömberg, 1987Go). Dried thin-layer chromatograms with separated glycosphingolipids were treated in 0.5% polyisobutylmethacrylate (Aldrich Chemical Company, USA) (w/v) in diethylether/n-hexane (1:5, v/v) for 1 min and then air-dried. To reduce nonspecific binding chromatograms were incubated in PBS/BSA containing 0.1% (w/v) Tween 20 for 1.5–2 h at room temperature. The plates were then incubated for 2 h at room temperature with 35S-labeled H. pylori diluted in PBS/BSA (2–5 x 106 cpm/ml) or 125I-labeled lectin (approximately 2 x 103 c.p.m./µl). The plates were then washed three times with PBS/BSA and allowed to dry. Binding assays using monoclonal antibodies were carried out in a similar manner (Magnani et al., 1983Go). Following incubation with the relevant antibody and washing with PBS/BSA, the chromatograms were incubated with 125I-labeled anti-mouse antibodies (approximately 2 x 103 cpm/µl). Blocking and all incubations were carried out for 2 h at room temperature. Following the final wash and drying, autoradiography was carried out overnight using either the Biomax MR or X-OMAT film (Eastman Kodak Company, NY).


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Table III. Carbohydrate binding specificities of bacteria, lectins, and monoclonal antibodies used in thin-layer chromatogram binding assays
 
Mass spectrometry
Mass spectra were obtained on a JEOL SX 102A mass spectrometer (JEOL, Tokyo). Negative ion FAB mass spectra were obtained of native gangliosides dissolved in dimethyl sulfoxide using Xe atom bombardment (6 keV), an acceleration voltage of 8 kV or 10 kV, triethanolamine as matrix, and a resolution of 1000.

Proton NMR spectroscopy
1H-NMR spectra were acquired on a Varian 600 MHz spectrometer at 30°C. The samples were dissolved in dimethyl sulfoxide:D2O (98:2, by volume) after deuterium exchange.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This study was supported by the Swedish Medical Research Council (Grant Nos. 12628, 3967, and 10435), the Swedish Cancer Foundation and by a grant from the program "Glycoconjugates in Biological Systems" sponsored by the Swedish Foundation for Strategic Research. The glycosphingolipid nomenclature follows the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for lipids: Eur. J. Biochem. [1977], 79, 11–21, J. Biol. Chem. [1982], 257, 3347–3351, and J. Biol. Chem. [1987], 262, 13–18). It is assumed that Gal, Glc, GlcNAc, GalNAc, and NeuAc are of the D-configuration, Fuc of the L-configuration, and all sugars present in the pyranose form.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BabA, blood group antigen-binding adhesin; BSA, bovine serum albumin; CagA, cytotoxin-associated protein; CFU, colony forming units; FAB-MS, fast atom bombardment mass spectrometry; HPLC, high-performance liquid chromatography; HPNAP, neutrophil-activating protein of H. pylori; MALT, mucosa-associated lymphoid tissue; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; VacA, vacuolating cytotoxin


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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