Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures

Halina Miller-Podraza1, Petra Johansson, Jonas Ångström, Thomas Larsson, Marianne Longard and Karl-Anders Karlsson

Institute of Medical Biochemistry, Göteborg University, PO Box 440, SE 405 30 Göteborg, Sweden

Received on June 24, 2003; revised on October 17, 2003; accepted on November 11, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid–dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Galß4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Galß3GlcNAc) and ganglio (Galß3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc{alpha}8NeuAc) and NeuAc{alpha}6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.

Key words: binding epitope / derivatization / Helicobacter pylori / sialic acid


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Helicobacter pylori is a human pathogen implicated in gastric diseases, which include antral gastritis, peptic ulcer, and gastric cancer (Dunn et al., 1997Go; McGee and Mobley, 1999Go; Peek and Blaser, 2001Go). The mechanism by which H. pylori initiates its interactions with target cells seems to be complex, possibly involving more than one receptor-active membrane component (reviewed by Karlsson, K.A., 1998Go, 2000Go). Binding of H. pylori to sialylated carbohydrates was initially described by Evans et al. (1988)Go who proposed Neu5Ac{alpha}3Gal as a binding epitope. Subsequently, sialyllactose and other Neu5Ac-containing structures have been used in inhibition tests and other studies related to binding specificities of H. pylori, and the results were consistent with higher preference of the bacterium for {alpha}3- than {alpha}6-bound sialic acid (Evans et al., 1988Go; Hirmo et al., 1996Go; Johansson and Miller-Podraza, 1998Go; Johansson et al., 1999Go; Miller-Podraza et al., 1997aGo; Roche et al., 2001Go; Simon et al., 1997Go). Several research groups have reported that H. pylori cells contain proteins that bind Neu5Ac (reviewed by Evans and Evans, 2000Go), and a sialic acid–binding adhesin, SabA, has recently been identified using a sialyl-Le x saccharide as part of an affinity cross-linking probe (Mahdavi et al., 2002Go). Our studies have shown that H. pylori–binding gangliosides and sialylated glycoproteins are present in relatively high amounts in human neutrophils (Miller-Podraza et al., 1999Go). Sialylated glycolipids, including complex polyglycosylceramides (PGCs), were detected in plasma membranes of the neutrophils and in different intracellular membrane fractions (Karlsson et al., 2001Go). We have furthermore suggested the existence of two different sialic acid–dependent binding specificities of H. pylori (Miller-Podraza et al., 1997bGo). The first binding was to simple gangliosides like Neu5Ac{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer, sialylparagloboside (S-3PG), and the other was to highly branched PGCs. The binding to PGCs could be expressed separately by some strains of H. pylori grown in liquid cultures.

Helicobacter pylori inflammation in the human stomach is associated with massive phagocyte infiltration of the infected areas (Hatz et al., 1992Go; Warren and Marshall, 1983Go), and it is possible that the bacterium uses the inflammatory products for nutrition (Blaser, 1992Go). In line with this are findings that H. pylori actively recruits neutrophils by synthesizing a neutrophil-activating protein, Hp-NAP (Evans et al., 1995Go), considered a major virulent factor of the bacterium (Satin et al., 2000Go) and shown to bind sialylated sequences (Teneberg et al., 1997Go). Because sialylated oligosaccharides inhibit H. pylori–induced activation of human neutrophils (Teneberg et al., 2000Go), Neu5Ac may be an important factor mediating interaction between H. pylori and inflammatory cells. Also of interest are results indicating that sialic acid contributes to H. pylori resistance to phagocytosis (Chmiela et al., 1994Go)

The sialic acid content in normal human gastric mucosa, the main target tissue for H. pylori, is very low (Filipe, 1979Go; Madrid et al., 1990Go) and the bacterial binding to gastric cells may be through nonsialylated receptors, like Le b anti­genic structures (Borén et al., 1993Go), sulfatide (Kamisago et al., 1996Go; Osawa et al., 2001Go), or lactotetraose (Teneberg et al., 2002Go). However, sialylated structures may be of importance in inflamed human mucosa, where it was shown that the level of sialylation rose with grade of inflammation (Mahdavi et al., 2002Go). Sialyllactose has been reported to inhibit binding of H. pylori to cultured gastrointestinal epithelial cells (Simon et al., 1997Go) and chronic atrophic gastritis in mice has been shown to be associated with increased synthesis of Neu5Ac{alpha}3Gal structures (Syder et al., 1999Go). In addition, inhibition of the sialic acid–specific adhesion of H. pylori to human gastric mucus by some unidentified components of cranberry juice has been reported (Burger et al., 2000Go).

In the present article we investigate structural requirements for sialic acid–related binding specificities of H. pylori using a panel of different natural gangliosides, neogangliosides, and chemically modified gangliosides.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Binding studies using various natural glycolipids and neoglycolipids
A panel of different glycosphingolipids and neoglycolipids was tested for interaction with H. pylori on thin-layer chromatography (TLC) plates, and the results are summarized in Table I. Among binding components were natural glycolipids and neoglycolipids of the neolacto series terminated with {alpha}3-linked Neu5Ac. These included nonfucosylated linear gangliosides, sialyl-Le x ganglioside, and highly branched PGCs. S-3PG was the simplest natural ganglioside that was a positive binder in our overlay assays. It is of interest that S-3PG ganglioside as well as longer members of the binding neolacto family are present in appreciable amounts in inflammatory cells (Johansson and Miller-Podraza, 1998Go; Miller-Podraza et al., 1999Go) involved in H. pylori diseases. Binding of H. pylori on TLC plates to the glycolipids was detected at a pmol level, see Table I. The relative strengths of the bindings were evaluated using dilution series of the glycolipids as exemplified in Figure 1. As shown in the table, the binding was stronger to more complex species containing repeated HexHexNAc units. The more effective interaction of H. pylori with more complex glycolipids was confirmed using hemagglutination-inhibition tests, where 50% hemagglutination of human erythrocytes by strain CCUG 17874, was achieved by ~0.3 mM, Neu5Ac{alpha}3Galß4GlcNAcß3Galß4Glc (S-3PG saccharide), 0.3 mM Neu5Ac{alpha}3Galß4(Fuc{alpha}3)GlcNAcß3Galß4Glc (sialyl-Le x saccharide), or ~6 µM Neu5Ac of PGCs.


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Table I. Binding of H. pylori to various glycolipids and neoglycolipids on silica gel thin-layer (TLC) plates

 


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Fig. 1. Detection of glycolipids on TLC silica gel plates: Level of detection of S-3PG (Neu5Ac{alpha}3Galß4GlcNAcß3Galß4Glcß1Cer) using overlay with 35S-labeled H. pylori (strain CCUG 17874). Lanes Ga, bovine brain gangliosides, from top GM1, GD1a, GD1b, GT1b. Lanes 1–9, two-fold dilutions of S-3PG. Total amount of S-3PG in lane 5 is 28 pmoles. The plates were developed in chloroform/methanol/0.25% KCl in water, 50:40:10, and visualized by spraying with anisaldehyde (left) or by overlay with the bacteria (right).

 
Repeated experiments showed no reproducible interaction of the tested H. pylori strains with gangliosides of the ganglio or lacto series. No binding to ganglio series with terminal Neu5Ac{alpha}3Galß3GalNAc also means that very common O-linked sequences of glycoproteins may not be recognized by H. pylori. Negative binding results were also obtained for Neu5Ac{alpha}6-terminated glycolipids and a disialoganglioside, Neu5Ac{alpha}8Neu5Ac{alpha}-3PG. A selective interaction of H. pylori with terminal Neu5Ac{alpha}3Gal of the neolacto carbohydrate chains is exemplified in Figure 2, which shows binding to neogangliosides synthesized from different free oligosaccharides. The structures of the main bands seen in this picture were confirmed by mass spectrometry (MS) after scraping off the material from the plate. The arrows in lane 2 indicate bands corresponding to the hexadecylaniline (HDA) derivative (lower band) and the branched derivative (upper double band). Fast atom bombardment (FAB-) spectra of these fractions are shown in Figure 3. Similar FAB- spectra were obtained for the corresponding bands in lanes 3–5 of Figure 2. The molecular ions and fragment ions were in agreement with calculated masses, confirming the identity of all synthetic substances. The Neu5Ac{alpha}3Galß4GlcNAcß3Galß4Glc saccharide was obtained from a S-3PG preparation that contained minor amounts of larger species of the binding series (see lanes 1 in Figure 2). These minor fractions are apparently strongly binding-active also after derivatization (lane 2 in Figure 2).



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Fig. 2. Binding of 35S-labeled H. pylori (CCUG 17874 strain) to neoglycolipids on TLC plates. The plates were visualized by spraying with anisaldehyde (left) or by overlay with the bacteria (right). For chromatographic conditions see Figure 1. Lane 1, S-3PG. Note that the preparation contains trace amounts of longer members of the binding series. Lane 2, neoglycolipids formed from Neu5Ac{alpha}3Galß4GlcNAcß3Galß4Glc. Lane 3, neoglycolipids formed from Neu5Ac{alpha}3Galß3GlcNAcß3Galß4Glc. Lane 4, neoglycolipids formed from Neu5Ac{alpha}6Galß4GlcNAcß3Galß4Glc. Lane 5, neoglycolipids formed from Galß3(Neu5Ac{alpha}6)GlcNAcß3Galß4Glc. Lane 6, bovine brain gangliosides (from top: GM1, GD1a, GD1b, GT1b). Arrows in lane 2 indicate hexadecylaniline-derivative (lower band) and neoglycolipid with branched lipid chain (upper double band, for formula see Figure 3). The marked fractions in lane 2 and the corresponding fractions in lanes 3–5 were scraped off and tested by MS, see Figure 3. Lc and nLc stand for lacto and neolacto core chains, respectively.

 


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Fig. 3. Negative-ion FAB spectra of neoglycolipids derived from Neu5Ac{alpha}3Galß4GlcNAcß3Galß4Glc. (A) Hexadecylaniline derivative; (B) neoglycolipid with branched lipid part.

 
Binding studies using chemically modified glycolipids
S-3PG was chosen as a main model compound for derivatization studies because this glycolipid is relatively easy to prepare and has a well-defined structure based on one lactosamine unit. The following chemical modifications were performed on S-3PG: (1) mild periodate oxidation of the Neu5Ac glycerol tail followed by reduction or coupling with methylamine or ethanolamine, (2) reduction of the carboxyl group to primary alcohol, (3) conversion of the carboxyl group to various amides, and (4) lactonization. All derivatives were investigated by negative-ion FAB MS to confirm the identity of the structures, see Figures 4GoGo7 and Table II. Molecular ions in all spectra were in agreement with the expected masses, and the changes were limited to the sialic acid residue. The latter was shown by fragment ions indicating sequence of sugars in the core chain at m/z 1338, 1176, 973, and 811 (Y series of ions according to current nomenclature, Domon and Costello, 1988Go; Harvey, 1999Go). The undestroyed ceramide part was indicated by a fragment ion at m/z 649 (Y0 ion corresponding to the ceramide 18:1–24:0). Each of the main ions appeared together with a satellite ion (–28 mass units) due to the presence of some amounts in the preparation of S-3PG with the d18:1–22:0 ceramide (e.g, m/z 1629 and 1601 in Figure 4A or 1582 and 1554 in Figure 4C). The only exception is Figure 4B, where the ions at m/z 1599 and 1569 represent two different derivatives, obtained from the main component 1629 (4A). In Figure 4B the ions with d18:1–22:0 disappear into the background. All derivatives were tested for binding by radiolabeled H. pylori grown on agar using overlay of TLC plates, and the results are summarized in Table II. As shown, most of the modifications of the sialic acid eliminated or drastically reduced the binding. Ethanolamine was not able to mimic glycerol, as was initially thought. There was some binding to the amide and benzylamide derivatives; however, these interactions required higher amounts of the glycolipid material and were not always reproducible. In contrast, a reproducible and strong binding was observed for the octadecylamide derivative of S-3PG, which interacted with H. pylori on TLC plates at similar amounts as underivatized S-3PG (lower pmol level), as shown by experiments with dilution series of the glycolipids.



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Fig. 4. Negative-ion FAB spectra of S-3PG derivatized at the -CHOH-CHOH-CH2OH group. (A) Unmodified S-3PG; (B) S-3PG with oxidized/reduced glycerol chain; (C) S-3PG oxidized and derivatized with methylamine.

 


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Fig. 5. Negative-ion FAB spectra of S-3PG derivatized at the -CHOH-CHOH-CH2OH and -COOH groups. (A) S-3PG after oxidation and derivatization with ethanolamine. (B) S-3PG reduced at -COOH; (C) primary amide.

 


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Fig. 6. Negative-ion FAB spectra of S-3PG derivatized at the -COOH group. (A) Methylamide; (B) ethylamide; (C) propylamide.

 


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Fig. 7. Negative-ion FAB spectra of S-3PG derivatized at the -COOH group. (J) Benzylamide; (K) octadecylamide.

 

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Table II. Binding of H. pylori on silica-gel TLC plates to chemically derivatized glycolipids and MS data

 
Chemical modifications of PGCs included mild periodate oxidation of the glycerol tail of Neu5Ac, followed by reduction, and reduction of the carboxyl group to alcohol (Table II). The modified fractions were tested by electron impact ionization (EI) MS after permethylation and Figure 8 shows ions corresponding to the terminal sialic acid residue before (8A) and after reduction (8B) of COOH. As expected, the ions at m/z 376 and 344 representing Neu5Ac were replaced by ions at m/z 362 and 330, corresponding to the reduced Neu5Ac. Binding tests using overlay of TLC plates with radiolabeled bacteria showed that the modifications abolished the binding. An example of binding of H. pylori to modified PGCs is shown in Figure 9.



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Fig. 8. Example of EI MS of permethylated polyglycosylceramides showing fragment ions corresponding to NeuAc and the reduced NeuAc.

 


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Fig. 9. Binding of H. pylori (strains 17874 and 032) to derivatized PGCs on TLC plates. Left plate was stained for carbohydrates by anisaldehyde, and the right plates were overlaid with 35S-radiolabeled H. pylori. Lanes 1 and 2, underivatized PGCs of human erythrocytes, 1 and 0.5 µg, respectively; lane 3, reduced PGCs (R-COOH->R-CH2OH); lane 4, bovine brain gangliosides (from top: GM1, GD1a, GD1b, GT1b); lane 5, S-3PG. (Note that bacteria 032 from broth do not bind to S-3PG, see also Miller-Podraza et al., 1997bGo.)

 
Molecular modeling
To explain the observed binding pattern of the native and carboxyl-derivatized structures shown in Table II, their preferred conformations were explored by molecular dynamics simulations (1 ns). The principal conformational characteristics of S-3PG determining the presentation of the binding epitope most likely resides in those preferred energy minima of the glycosidic bonds of the Neu5Ac{alpha}3Gal linkage and the Glcß1Cer linkage that are selected by the H. pylori adhesin. In the case of the NeuAc{alpha}3Gal linkage, it is probable that both the anticlinal ({Phi}/{Psi} {approx}-152°/–31°) and synclinal ({Phi}/{Psi}{approx}-75°/9°) conformations are significantly populated as judged from earlier nuclear Overhauser effect–based distance mapping of Neu5Ac{alpha}3Galß4Glcß1Cer (GM3) by nuclear magnetic resonance and molecular dynamics calculations on the constituent disaccharide part of GM3 (Siebert et al., 1992Go). All previous energy mapping of this glycosidic linkage reveal these two minima, but one or two additional minima are observed depending on the force field used (Siebert et al., 1992Go and references therein; Imberty, 1997Go). Use of the CHARMm force field thus gives rise to a third minimum centered around {Phi}/{Psi}{approx}-107°/–53° (after minimization), more closely related to the anticlinal than the synclinal conformation and similar to the minimum seen using the HSEA format (Breg et al., 1989Go).

To minimize energy-costly solvent exposure of the additional hydrophobic tail of the octadecylamide derivative, it is likely that the octadecyl and ceramide tails would have to orient themselves in a coparallel fashion relative to each other when adhering to the polyisobutyl-methacrylate-treated TLC plate. By systematic variation of the glycosidic dihedral angles representing local energy minima of the NeuA5c{alpha}3Gal (Siebert et al., 1992Go) and Glcß1Cer (Nyholm and Pascher, 1993Go) linkages while keeping other glycosidic linkages of S-3PG locked, it is found that with the sialic acid residue in the anticlinal conformation ({Phi}/{Psi}{approx}-155°/–25°) three different Glcß1Cer conformations give rise to a more or less co-parallel arrangement of the hydrophobic tails. The most favorable one in this respect corresponds to the same conformation seen in the crystal structure of cerebroside (Galß1Cer) having dihedral angles of {Phi}//{theta}{approx}50°/175°/-65° (Pascher and Sundell, 1977Go) yielding maximal exposure of the binding epitope as shown in Figure 10. Repeating the same procedure with the sialic acid in the synclinal conformation ({Phi}/{Psi}{approx}-75°/10°) does not yield any configuration compatible with a co-parallel arrangement.



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Fig. 10. The top panel shows a CPK representation of the octadecylamide derivative of S-3PG in its proposed binding-active conformation in which the sialic acid assumes the anticlinal configuration and the hydrophobic tails of the octadecylamide moiety and the ceramide are coparallel, as described further in Results. In the lower panel a close-up view of the binding epitope, constituted by the terminal disaccharide S-3PG in its binding-active configuration (left). The glycosidic torsion angles are in the anticlinal conformation with the O1 oxygen of the carboxyl group forming a hydrogen bond with the 8-OH of the glycerol tail. For comparative purposes the nonbinding propylamide derivative is shown also in the anticlinal conformation but with propylamide moiety directed toward the glycerol tail which is the most favored orientation according to dynamics simulations (right).

 
Reliable dynamics simulations of the octadecylamide derivative are not possible due to the long octadecyl tail, but simulations of the conformational preferences of the smaller derivatives should be compatible with the picture if correct. Although the performed simulations are too short to yield reliable statistics, they do indicate which of the glycosidic conformations may coexist with different orientations of the modified carboxyl function. Regarding the terminal disaccharide of native S-3PG, which constitutes the binding epitope (see Discussion), bascially the same transitional behavior was observed as found before (Siebert et al., 1992Go) with a slight preference for the anticlinal conformation (A) over the synclinal one (B) irrespective of the starting conformation (Figure 11, top). The third conformation (C) was, as expected, also represented in three brief instances. The octadecylamide derivative is, as already mentioned, found to be binding-positive, most likely due to its long hydrophobic tail, influencing the conformational selectivity without affecting binding site accessibility. It is therefore reasonable to assume that the preferred conformations of the much shorter but structurally related methyl-, ethyl-, and propylamide derivatives, all three of which are nonbinding with regard to H. pylori (Table II) and behave similarly in the simulations (represented by the propylamide derivative in Figure 10, bottom), may consequently be excluded as the ones presenting the binding epitope properly.



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Fig. 11. Trajectories from molecular dynamics simulations (300 K, {varepsilon} = 8) of the terminal disaccharide NeuAc{alpha}3Galß of native S-3PG (top), the amide derivative (middle), and propylamide derivative (bottom) of the same disaccharide. Solid and open squares indicate the glycosidic torsion angles defined as {Phi} = C1-C2-O2-C'3 and {Psi} = C2-O2-C'3-H'3, respectively, whereas the orientation of the modified carboxyl moiety is defined as {omega} = O5-C2-C1-O1 and indicated by solid triangles. The approximate duration of three different glycosidic conformations in the top panel have been indicated by horizontal lines and denoted A, B, and C, respectively. Starting conformations was the anticlinal one for the native structure whereas the other two simulations used the synclinal one.

 
The only conformational combination not seen in the dynamics run is the anticlinal one in which the amide carbonyl forms a hydrogen bond with the 8-OH of the Neu5Ac glycerol tail and/or the 4-OH of the galactose (Figure 10). This is identical to the proposed conformation of the octadecylamide derivative, and if allowed would most likely be binding positive. In support of this supposition, the weakly binding amide derivative does display this conformation for approximately 100 ps when starting the simulation from the synclinal conformation (Figure 8, middle) and similarly when starting from the two other glycosidic conformations. Furthermore, the requirement of an intact glycerol tail (Table II) indicates that steric inter­ference from the aliphatic portion of the methyl-, ethyl-, or propylamide moieties having the opposite conformation would indeed render these derivatives nonbinding. Moreover, the observation that the hydroxyl group of the alcohol derivative overwhelmingly interacts with the 8-OH of the glycerol tail or the 4-OH of the galactose (not shown) but nevertheless is nonbinding suggests that the hydrogen bond acceptor function of the corresponding carboxyl oxygen in underivatized S-3PG is essential for binding to occur. Last, in the benzylamide derivative, the orientation of the benzyl group is always toward the glycerol tail when the disaccharide is in its dominating anticlinal configuration (not shown) suggesting that this derivative should be binding negative. However, this rather bulky moiety may well reorient itself to interact with the hydrophobic plastic coating of the TLC plate in the binding assay, thereby rendering this derivative partially active. In summary, the conformational preferences of the smaller derivatives are consistent with the binding conformation deduced for the octadecylamide derivative.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Structural requirements for recognition of sialic acid by H. pylori has been discussed by several research groups (Evans et al., 1988Go; Hirmo et al., 1996Go; Johansson and Miller-Podraza, 1998Go; Johansson et al., 1999Go; Miller-Podraza et al., 1996Go, 1997bGo; Simon et al., 1997Go), and the results of this article are in line with these reports, confirming the preference of H. pylori for {alpha}3-linked Neu5Ac and ß4-linked Gal. Derivatization of glycolipids followed by binding studies showed dependence of the binding on the glycerol tail and carboxyl group. The fact that free carboxyl may be replaced by some amide forms indicates that only one oxygen of COO- (or carbonyl oxygen in the amide case) is crucial for the interaction. It is likely that Neu5Ac{alpha}3Galß4GlcNAc, which is part of many human and animal glycoconjugates, carries the natural binding epitope for H. pylori. However, unmodified GlcNAc does not seem to be a requirement for binding because {alpha}Fuc linked to O3 of GlcNAc and other modifications of GlcNAc like N-deacetylation (Johansson and Miller-Podraza, unpublished data) do not abolish the binding. According to these results the strength of the binding to sialyl-Le x is similar to that observed for S-3PG, and sialyl-Le x was recently used in an affinity approach for identification of the sialic acid–binding adhesin SabA (Mahdavi et al., 2002Go). Besides, sialyllactose (Neu5Ac{alpha}3-Galß4Glc) has been shown by several groups to inhibit the interaction of H. pylori with sialylated structures and target tissues (Evans et al., 1988Go; Hirmo et al., 1996Go; Simon et al., 1997Go). From these data it is still impossible to conclude how much of Neu5Ac{alpha}3Galß4GlcNAc(Glc) may be accommodated by the putative binding site of SabA. However, no binding to Neu5Ac{alpha}3Galß3GlcNAc or NeuA5c{alpha}3Gal-ß3GalNAc indicates that in natural receptors the terminal trisaccharide element, rather than disaccharide, is decisive for the binding. One should also note that only two bacterial strains were routinely used in the present study. Testing more strains may well reveal somewhat different results. A variation in binding specificity between clinical isolates was clearly documented when binding to albumin-based sialyl-Le x (Neu5Ac{alpha}3Galß4(Fuc{alpha}3)GlcNAc-R) and sialyl-Le a (Neu5Ac{alpha}3Galß3(Fuc{alpha}4)GlcNAc-R) was tested (Mahdavi et al., 2002Go). Out of 89 clinical isolates tested, 35 bound sialyl-Le x, and of these 15 (43%) isolates also bound sialyl-Le a. It is likely that isolates which recognize sialyl-Le a also bind to NeuAc{alpha}3Galß3GlcNAc-R, which was negative for our strains (Table I). Therefore more isolates should be analyzed for potential differences in fine specificities.

As shown in Table I, there is a relatively stronger binding to more complex gangliosides, as compared with S-3PG, which suggests that structural factors connected with core carbohydrate parts enhance affinity of the sialylated epitope. The more effective interaction may also depend on the combined action of different bacterial adhesins recognizing both terminal and internal parts of the extended core chains. Helicobacter pylori is known to display binding specificities associated with both sialylated and neutral saccharide chains (Karlsson, 1998Go). Recently, Roche et al. (2001)Go reported binding of H. pylori to gangliosides with repeated lactosamine units prepared from human gastric carcinoma. It is thus likely that Neu5Ac{alpha}3Galß4GlcNAc and its extended and fucosylated counterparts represent functioning epitopes during contact of H. pylori with inflammatory cells that are enriched in these structures.

On the other hand, the simpler 3-sugar-containing ganglioside GM3 was negative in our TLC assays, although Neu5Ac{alpha}3Galß4Glc (sialyl lactose), both free (Evans et al., 1988Go) and coupled to albumin (Simon et al., 1997Go), is active as inhibitor of the binding. No binding to GM3 in the present studies might be due to an inaccessible binding epitope at the TLC assay surface. Our experience indicates that use of very short glycolipids on TLC plates may lead to false negative results and that spacers in neoglycolipids may influence the binding. It should also be mentioned that GM3 is not expected as a strong binder because Neu5Ac{alpha}3Galß4Glc saccharide is less effective as inhibitor of hemagglutination by H. pylori than Neu5Ac{alpha}3Gal-ß4GlcNAcß3Galß4Glc (Johansson and Miller-Podraza, unpublished data).

As mentioned, H. pylori is highly variable regarding binding activities, and it has been shown that the expression of different specificities depends both on bacterial strains and growth conditions. We have previously suggested two sialic acid–dependent binding specificities of this bacterium (represented by interactions with S-3PG and PGCs, respectively), based on comparison of bacterial cells grown on agar and in liquid cultures (Miller-Podraza et al., 1997bGo). We have also noticed that binding to S-3PG by H. pylori is stronger after longer growth times on agar plates. Both S-3PG and PGCs contain Neu5Ac{alpha}3Galß4GlcNAc (Karlsson H. et al., 2000Go), and the question arises whether different presentations of this structural element may result in formation of two different binding epitopes. To better understand this issue, molecular modeling of S-3PG and its chemical derivatives was employed, the results of which suggest that the sialic acid has to adopt the anticlinal conformation for the binding epitope to be presented correctly. However, molecular dynamics investigations of various hypothetical PGC fragments (Ångström et al., unpublished data) indicate, on the other hand, that the synclinal conformation is preferred. The results further indicated that in the S-3PG case a direct involvement of the glycerol tail at the binding interface is likely, whereas in the PGC case the same group is stabilizing an intramolecular conformation in which two neighboring branches become spatially fixed relative each other.

Another important question is whether the binding to different sialylated glycoconjugates is through one or more bacterial adhesins. To investigate this issue we performed binding experiments using H. pylori knockout strains lacking the sialic acid–binding protein SabA (Mahdavi et al., 2002Go). We found that genetically modified (SabA–) strains lose the ability to bind to both PGCs and S-3PG. This result favors the presence in H. pylori of a single adhesin responsible for interactions with both S-3PG and PGCs. Consequently, the two modes of binding observed in our studies (Miller-Podraza et al., 1997bGo) could depend on two different binding sites on the SabA adhesin and/or different densities of SabA on H. pylori cells grown under different conditions. The apparently more rigid presentation of Neu5Ac in branched PGCs, as compared to S-3PG (Ångström, unpublished data) may result in an apparent higher affinity of the epitope requiring lower concentrations of the protein. An answer to this may be given when the binding site of the adhesin has been identified.

Helicobacter pylori is a gastric pathogen that causes ulceration and confers a greater risk of stomach cancer. Current treatment is based on the use of antibiotics and this is connected to the development of resistant bacterial strains (Adamsson et al., 2000Go; Kate and Anathakrishnan, 2001Go). Thus new ways of therapeutical strategies are needed, and carbohydrates and their chemical analogs are relevant candidates for antiadhesion therapy. As discussed in the Introduction, various sialylated structures have been reported to inhibit H. pylori binding to gastric cells and artificial surfaces (Burger et al., 2000Go; Simon et al., 1997Go; Wang et al., 2001Go), supporting the relevance of such an approach. An important experiment using rhesus monkeys showed that oral administration of sialyllactose (Neu5Ac{alpha}-3Galß4Glc) can eradicate H. pylori or decrease bacterial colonization in animal stomachs (Mysore et al., 1999Go). The epitope dissection of this work was limited to two groups of Neu5Ac, the glycerol tail and the carboxyl group. The third functional group of interest is N-acetamido group, which also is essential for binding as will be discussed separately (Johansson and Miller-Podraza, unpublished data).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Source of natural glycolipids
PGCs (human erythrocytes) were isolated in our laboratory according to the peracetylation method (Miller-Podraza et al., 1993Go). S-3PG (human erythocytes and human leukocytes), disialylparagloboside (human erythrocytes), S-6PG (human leukocytes), 7-sugar neolacto ganglioside (human erythrocytes and leukocytes), and globoside (human erythrocytes) were also prepared in our laboratory (Karlsson, 1987Go). GQ1b of human brain was from the Department of Neurochemistry of Göteborg University (Miller-Podraza et al., 1992Go). Gangliosides GM1, GD1a, GD1b, and GT1b of bovine brain were purchased from Calbiochem (San Diego, CA).

Source of oligosaccharides
Neu5Ac{alpha}3Galß4GlcNAcß3Galß4Glc was prepared in our laboratory from S-3PG (human erythrocytes) using ceramide glycanase (from leech, Boehringer Mannheim GmBH, Germany) digestion (Ito and Yamagata, 1989Go) and phase partition in chloroform/methanol/water, 2:1:0.6. The pentasaccharide was recovered from the upper phase. Neu5Ac{alpha}3Galß3GlcNAcß3Galß4Glc, Neu5Ac{alpha}6Galß-4GlcNAcß3Galß4Glc and Galß3(Neu5Ac{alpha}6)GlcNAcß3-Galß4Glc were purchased from IsoSep (Tullinge, Sweden).

Other reagents
4-HDA, methylamine, ethylamine, propylamine, butylamine, and benzylamine were from Aldrich Chemical (Milwaukee, WI). Glycolic acid, ethanolamine, and octadecylamine were purchased from Sigma-Aldrich (Germany). Sephadex LH 20 was from Pharmacia (Uppsala, Sweden) and ethylene glycol from Fluka (Sweden).

Chemical modifications of the sialic acid glycerol tail
Mild periodate oxidation (Veh et al., 1977Go) followed by reduction (R-CHOH-CHOH-CH2OH->R-CHOH-CH2OH/R-CH2OH): The material (0.5–1 µmol) was incubated in 500 µl 0.05 mM acetate buffer, pH 5.5, containing 1–2 mM NaIO4, for 40 min on ice. The reaction was terminated with an excess of ethylene glycol. The sample was then concentrated by freeze-drying (about fivefold) and reduced with an excess of NaBH4 at room temperature overnight. Finally the sample was dialyzed against distilled water for 2 days and freeze-dried.

Mild periodate oxidation of S-3PG followed by coupling with methylamine or ethanolamine (R-CHOH-CHOH-CH2OH->R-CH2-NH-CH3 or R-CH2-NH-CH2CH2-OH). After oxidation of S-3PG with mild periodate (see previous methods) and addition of ethylene glycol, the material was dialyzed for 2 days against distilled water and freeze-dried. The oxidized S-3PG was coupled with methylamine or ethanolamine under the following conditions: the glycolipid (0.5 mg) was dissolved in 200 µL methanol/chloroform 3:1 and mixed with 50 µL amine, 200 µL NaBH3CN in methanol (62 mg/mL) and 200 µL glycolic acid in water (136 mg/mL). In the case of methylamine, 50 µL tetrahydrofuran was added to improve solubility. The sample was incubated at 30°C for 4 h and evaporated under nitrogen. The residue was suspended in chloroform/methanol/water, 60:30:4.5 and desalted using Sephadex LH-20 column packed in methanol. After application of the sample (about 0.6 mL per 0.5 x 15 cm column), the column was eluted with methanol, and the glycolipid was recovered by collecting sugar-positive fractions (monitored by TLC and anisaldehyde). For final purification of molecular species (see formulas), the material was separated by preparative TLC using chloroform/methanol/water, 60:35:8, as developing system. The main band (detected with anisaldehyde after cutting off a strip from the plate) was scraped out and extracted with the same solvent.

Modifications of the carboxyl group (Lanne et al., 1995Go)
S-3PG (0.5–5 mg) was first converted to the methylester (R-COOH->R-COOCH3) by incubation with methyl iodide (100 µL) in dimethylsulfoxide (0.5 mL) for 1 h at room temperature. The product was purified using Sephadex LH-20, as already described. To prepare the alcohol derivative (R-COOCH3->R-CH2OH) of S-3PG, the methylester (0.5 mg) was dissolved in 0.5 mL methanol, followed by addition of 5 mg NaBH4. After 1 h at room temperature, the reduced S-3PG was desalted using Sephadex LH-20 (see previous methods). For synthesis of the amide and the methyl-, ethyl-, propyl-, benzyl-, and stearylamide of S-3PG, the methyl ester (0.5 mg in 0.5 mL methanol) was mixed with 0.2 mL 30% NH3 in water, 0.5 mL 40% methylamine in water, 1 mL 70% ethylamine in water, 100 µL propylamine, 100 µL benzylamine, or 200 µL stearylamine in tetrahydrofuran (saturated solution), respectively. After incubation overnight at room temperature, the products were evaporated under nitrogen. Benzyl and stearyl amides were further purified by extraction with hexane/acetone 1:1. The samples were washed with excess of the solvent mixture and centrifuged, and the supernatants were discarded.

PGC derivatives were prepared in the same way with the following exceptions. First, the reduced PGC preparation was separated on a DEAE-Sephadex column and only the neutral fraction was further investigated. Second, the oxidation/reduction procedure was performed twice.

Coupling of HDA to free saccharides
The synthesis was performed as described, with some modifications (Evangelista et al., 1996Go). Saccharide (0.5 mg) was dissolved in 100 µL methanol and mixed with 100 µL NaBH3CN in methanol (62 mg/mL), 100 µL HDA in tetrahydrofuran (40 mg/mL), and 100 µL of 1.8 M glycolic acid in water (136 mg/mL). Additional 100–200 µL tetrahydrofuran was added to improve solubility of the precipitating hexadecylaniline. The sample was incubated at 30°C overnight and the product purified by Sephadex LH 20 chromatography (see previous method description). The yield of this reaction was more than 90%.

Synthesis of branched lipid parts
To obtain neoglycolipids with branched lipid parts, the HDA-derivatized saccharides were further modified by N-acylation (Magnusson et al., 1994Go; Read et al., 1977Go). P-nitrophenylpalmitate (500 µL in dry dimethysulfoxide, saturated solution) was added to 200–300 µg of the dried HDA-saccharide. Four drops of triethylamine were added to the sample, which was incubated in nitrogen atmosphere at 37°C for 3 days. Glycolipids were purified using Sephadex LH-20 column chromatography (see described methods) and preparative TLC. The yield was 10–20%.

Preparation of lactones
S-3PG was transformed into its lactone form as described elsewhere (Laferriere and Roy, 1994Go). S-3PG (1 mg) was dissolved in concentrated acetic acid, and the reaction was allowed to proceed at room temperature for 2 days. The acetic acid was then evaporated and the remaining material dissolved in chloroform/methanol/water, 60:35:8 (0.5 mL). The yield of the reaction was controlled by TLC on aluminum-backed high-performance TLC plates coated with 0.1 mm silica gel 60 (Merck, Darmstadt, Germany) and was ~50%.

Synthesis and preparation of ganglioside GM1b
Gangliotetraosylceramide, prepared by desialylation of the ganglioside GM1, was sialylated at the terminal galactose using an {alpha}-2,3-(0)-sialyltransferase, EC number 2.4.99.4 (recombinant, rat liver-Spodoptera frugiperda) from Calbiochem (Darmstadt, Germany). The conditions were as described in Lee et al. (1994)Go with slight modifications, as follows. Gangliotetraosylceramide (100 µg) was dissolved in 10 µL 500 mM 4-morpholineethane–sulfonic acid buffer, pH 6.0, containing Triton CF-54 (2%) and diluted with 74 µL water. Then, 10 µL CMP-NeuAc (15 mM in water) and 6 µL {alpha}-2,3-sialyltransferase (338 mU/mL) were added to the reaction mixture, and the sample was left at room temperature overnight. The progress of the reaction was analyzed by TLC using chloroform/methanol/0.25% KClaq (50:40:10) as eluent. The reaction mixture was then evaporated under a stream of nitrogen, dissolved in chloroform/methanol/H2O (60:30:4.5) (2 mL) and applied to a small column (~2.5 x 0.6 cm) packed with Sephadex G-25 (prewashed with 5 mL of the same solvent system). The glycolipid material was eluted with 2.5 mL of the solvent mixture and 2.5 mL chloroform/methanol (2:1, by volume). Finally, the material was evaporated and dissolved in a small volume of chloroform/methanol/H2O (60:30:4.5).

H. pylori strains
Helicobacter pylori strain CCUG 17874 was from Culture Collection (Göteborg University, Sweden) and H. pylori strain 032 was a gift from Prof. T. Wadström (Department of Medical Microbiology, Lund University, Sweden). Helicobacter pylori strains J99 and J99(SabA–) were kindly donated by Dr. Thomas Borén from Department of Odontology/Oral Microbiology (Umeå University, Sweden) (Mahdavi et al., 2002Go).

Overlay of TLC plates with H. pylori
Overlay of gangliosides on silica-gel TLC plates with 35S-labeled H. pylori was performed as described elsewhere (Miller-Podraza et al., 1996Go).

MS
FAB MS of glycolipids was performed on a JEOL SX-102 mass spectrometer in the negative ion mode. The spectra were produced by Xe atoms using triethanolamine as a matrix. EI MS of permethylated PGCs was performed using the same mass spectrometer. Samples were evaporated in the ion source between 150 and 410°C, and the spectra were recorded at different points of broad peaks in the end of the run. The electron energy was 70 eV and ion current 300 µA.

Molecular modeling
Minimum energy conformations of the glycosphingolipids and chemical derivatives thereof used in this study were calculated within the Quanta2000/CHARMm25 software (Accelrys) on an Indigo2Extreme workstation (Silicon Graphics) using a dielectric constant of {varepsilon} = 8. Glycosidic dihedral angles are defined as follows: {Phi} = H1-C1-O1-C'X and {Psi} = C1-O1-C'X-H'X for 2-, 3-, or 4-linked residues other than NeuAc; {Phi} = C1-C2-O2-C'3 and {Psi} = C2-O2-C'3-H'3 for {alpha}3-linked NeuAc; {Phi} = H1-C1-O1-C'1, {Psi} = C1-O1-C'1-C'2, and {theta} = O1-C'1-C'2-C'3 for the Glcß1Cer linkage, whereas the orientation of the modified carboxyl moiety is defined as {omega} = O5-C2-C1-O1. The Glcß1Cer glycosidic dihedral angles of S-3PG were varied according to Nyholm and Pascher (1993)Go, whereas the angles of the Galß4GlcNAcß3Galß4Glc segment were kept constant at their respective constituent disaccharide global minima (Imberty et al., 1991Go; Poppe et al., 1990Go). The terminal disaccharides of native S-3PG and its chemical derivatives were subjected to molecular dynamics simulations (1 ns) after initial heating (300 K) and equilibration periods using a 1 fs time step to determine the conformational preferences of the modified sialic acid. All bonds to hydrogens were constrained by the SHAKE algorithm. Frames were written to the trajectories once every ps. Simulations were carried out starting from both the anticlinal and synclinal conformations.

The carbohydrate and glycosphingolipid nomenclature are according to recommendations of the Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature of carbohydrates: Carbohydr. Res. (1997) 297, 1. Nomenclature of glycolipids: Carbohydr. Res. (1998) 312, 167 and Eur. J. Biochem. (1998) 257, 293. All solvent mixtures were prepared volume by volume, unless otherwise stated.


    Acknowledgements
 
This work was supported by the Swedish Medical Research Council (grant number 06X-12628), the Swedish Cancer Foundation, the Wilhelm and Martina Lundgrens Research Foundation, the Adlerbertska Research Foundation, the Ingabritt and Arne Lundberg Foundation, the Medical Faculty of Sahlgrenska Academy, and Biotie Therapies Corporation.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: halina.miller-podraza{at}medkem.gu.se Back


    Abbreviations
 
EI, electron impact ionization; FAB, fast atom bombardment; GM3, Neu5Ac{alpha}3Galß4Glcß1Cer; HDA, hexadecyla­niline; MS, mass spectrometry; PGC, polyglycosylceramide; S-3PG, 3'-sialylparagloboside (Neu5Ac{alpha}3Galß4GlcNAcß­ 3Galß4 Glcß1Cer); TLC, thin-layer chromatography; Cer, ceramide; Ncu5Ac(NeuAc), N-acetylneuraminic acid; Glc, glucose; Gal, galactose; GLcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Fuc, Fucose


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
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