Redefinition of the carbohydrate binding specificity of Helicobacter pylori BabA adhesin

Abstract

Certain Helicobacter pylori strains adhere to the human gastric epithelium using the blood group antigen-binding adhesin (BabA). All BabA-expressing H. pylori strains bind to the blood group O determinants on type 1 core chains, i.e. to the Lewis b antigen (Fucα2Galβ3(Fucα4)GlcNAc; Le(b)) and the H type 1 determinant (Fucα2Galβ3GlcNAc). Recently, BabA strains have been categorized into those recognizing only Le(b) and H type 1 determinants (designated specialist strains) and those that also bind to A and B type 1 determinants (designated generalist strains). Here, the structural requirements for carbohydrate recognition by generalist and specialist BabA were further explored by binding of these types of strains to a panel of different glycosphingolipids. Three glycosphingolipids recognized by both specialist and generalist BabA were isolated from the small intestine of a blood group O pig and characterized by mass spectrometry and proton NMR as H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer), Globo H hexaglycosylceramide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), and a mixture of three complex glycosphingolipids (Fucα2Galβ4GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, Fucα2Galβ3GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, and Fucα2Galβ4(Fucα3)GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). In addition to the binding of both strains to the Globo H hexaglycosylceramide, i.e. a blood group O determinant on a type 4 core chain, the generalist strain bound to the Globo A heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), i.e. a blood group A determinant on a type 4 core chain. The binding of BabA to the two sets of isoreceptors is due to conformational similarities of the terminal disaccharides of H type 1 and Globo H and of the terminal trisaccharides of A type 1 and Globo A.

FIGURE 1.

Binding of a generalist and a specialist H. pylori strain to non-acid glycosphingolipids of the small intestinal epithelium of a blood group O pig. The glycosphingolipids were separated on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8 by volume) as the solvent system. The chromatogram in A was stained with anisaldehyde. Duplicate chromatograms were incubated with the ³⁵S-labeled H. pylori generalist strain J99 (B) and the H. pylori specialist strain S831 (C) followed by autoradiography for 12 h as described under “Experimental Procedures.” Lane 1, non-acid glycosphingolipids of the intestinal epithelium of a blood group O pig, 40 μg; lane 2, reference H type 2 pentaglycosylceramide (Fucα2Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 4 μg; lane 3, reference Le^a pentaglycosylceramide (Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 4, reference Le^b hexaglycosylceramide (Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 4 μg; lane 5, reference B type 1 heptaglycosylceramide (Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 2 μg.

FIGURE 2.

H. pylori-binding glycosphingolipids isolated from the small intestinal epithelium of a blood group O pig. The glycosphingolipids were separated on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8 by volume) as the solvent system. The chromatogram in A was stained with anisaldehyde. Duplicate chromatograms were incubated with the ³⁵S-labeled H. pylori generalist strain J99 (B), the H. pylori specialist strain S831 (C), the monoclonal anti-H type 1 antibody 17-206 (D), and the monoclonal anti-H type 2 antibody 92FR-A2 (E) followed by autoradiography for 12 h as described under “Experimental Procedures.” Lane 1, fraction P-I isolated from pig intestine, 2 μg; lane 2, fraction P-II from pig intestine, 2 μg; lane 3, fraction P-III from pig intestine, 2 μg; lane 4, reference Le^b hexaglycosylceramide (Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 5, reference H type 2 pentaglycosylceramide (Fucα2Galβ4GlcNAcβ3Galβ4Glcβ1Cer), 2 μg.

FIGURE 3.

Characterization of the H. pylori BabA-binding fraction P-II from the small intestinal epithelium of a blood group O pig. A, base peak chromatogram from LC-ESI/MS of the oligosaccharides obtained by digestion of the H. pylori BabA-binding fraction P-II with Rhodococcus endoglycoceramidase II. B, mass chromatogram of m/z 852. C, mass chromatogram of m/z 1014. D, MS² spectrum of the [M − H⁺]⁻ ion at m/z 852 (retention time (RT), 26.8 min). The interpretation formula shows the deduced oligosaccharide sequence. E, MS² spectrum of the [M − H⁺]⁻ ion at m/z 1014 (retention time, 25.3 min). The interpretation formula shows the deduced oligosaccharide sequence. F, anomeric region of the 600-MHz proton NMR spectrum of fraction P-II (30 °C). The sample was dissolved in dimethyl sulfoxide/D₂O (98:2 by volume) after deuterium exchange.

FIGURE 4.

LC-ESI/MS of the decasaccharides obtained by hydrolysis of H. pylori-binding fraction P-III with Rhodococcus endoglycoceramidase II. A, MS² spectrum of the [M − 2H⁺]²⁻ ion at m/z 864 (retention time (RT), 23.4 min). B, MS² spectrum of the [M − 2H⁺]²⁻ ion at m/z 864 (retention time, 26.3 min). C, MS³ spectrum of the ion at m/z 1201 (retention time, 23.4 min). D, MS³ spectrum of the ion at m/z 1201 (retention time, 26.3 min). E, interpretation formula showing the deduced oligosaccharide sequence.

FIGURE 5.

LC-ESI/MS of the undecasaccharide obtained by hydrolysis of H. pylori-binding fraction P-III with Rhodococcus endoglycoceramidase II. A, MS² spectrum of the [M − 2H⁺]²⁻ ion at m/z 937 (retention time (RT), 25.1 min). B, MS³ spectrum of the ion at m/z 1712 (retention time, 25.1 min). C, interpretation formula showing the deduced oligosaccharide sequence.

FIGURE 6.

Anomeric region of the 600-MHz proton NMR spectrum of the H. pylori-binding fraction P-III from porcine small intestinal epithelium (30 °C). The sample was dissolved in dimethyl sulfoxide/D₂O (98:2 by volume) after deuterium exchange.

FIGURE 7.

Comparison of binding of a generalist and a specialist H. pylori strain to reference glycosphingolipids. The glycosphingolipids were separated on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8 by volume) as the solvent system. The chromatogram in A was stained with anisaldehyde. Duplicate chromatograms were incubated with the ³⁵S-labeled H. pylori generalist strain J99 (B) and the H. pylori specialist strain S831 (C) followed by autoradiography for 12 h as described under “Experimental Procedures.” Lane 1, Le^b hexaglycosylceramide (Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 2, B type 1 heptaglycosylceramide (Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 3, A type 1 heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 4, A type 1 hexaglycosylceramide (GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 5, Globo A/A type 4 heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), 2 μg; lane 6, A nonaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 7, A type 2 heptaglycosylceramide (GalNAcα3(Fucα2)Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer), 2 μg.

FIGURE 8.

Comparison of binding of a generalist and a specialist H. pylori strain to dilutions of pure glycosphingolipids on thin-layer chromatograms. The glycosphingolipids were separated on aluminum-backed silica gel plates using chloroform/methanol/water (60:35:8 by volume) as the solvent system. The chromatograms were incubated with the ³⁵S-labeled H. pylori generalist strain J99 (A) and the H. pylori specialist strain S831 (B) followed by autoradiography for 12 h as described under “Experimental Procedures.” Lane 1, Le^b hexaglycosylceramide (Le^b-6; Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 200 ng, and A nonaglycosylceramide (A9-1; GalNAcα3(Fucα2)Galβ3GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer), 200 ng; lane 2, Le^b hexaglycosylceramide, 80 ng, and A nonaglycosylceramide, 80 ng; lane 3, Le^b hexaglycosylceramide, 40 ng, and A nonaglycosylceramide, 40 ng; lane 4, Globo A/A type 4 heptaglycosylceramide (Globo A; GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), 200 ng; lane 5, Globo A/A type 4 heptaglycosylceramide, 80 ng; lane 6, Globo A/A type 4 heptaglycosylceramide, 40 ng; lane 7, A type 1 heptaglycosylceramide (A7-1; GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 200 ng; lane 8, A type 1 heptaglycosylceramide, 80 ng; lane 9, A type 1 heptaglycosylceramide, 40 ng.

FIGURE 9.

Minimum energy models of the H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer) (left), the Globo H hexaglycosylceramide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer) (center), and the H type 2 pentaglycosylceramide (Fucα2Galβ4GlcNAcβ3Galβ4Glcβ1Cer) (right). The terminal disaccharides are colored blue (Fucα2) and purple (Galβ3/4). The terminal part of the BabA-binding H type 1 and Globo H glycosphingolipids can be aligned by assuming different Glcβ1Cer torsion angles, whereas the H type 2 terminal disaccharide is rotated ∼90° in comparison, rendering this glycosphingolipid non-binding with respect to BabA.

FIGURE 10.

Binding of monoclonal antibodies to human gastric glycosphingolipids. Thin-layer chromatogram after detection with anisaldehyde (A) and autoradiograms obtained by binding of the monoclonal anti-Globo H antibody MBr1 (B), the monoclonal anti-Le^b antibody BG-6/T218 (C), and the monoclonal anti-H type 1 antibody 17-206 (D) are shown. The chromatograms were eluted with chloroform/methanol/water (60:35:8 by volume), and the binding assays were done as described under “Experimental Procedures” followed by autoradiography for 12 h. Lane 1, non-acid glycosphingolipids of human stomach (Individual I; blood group O), 40 μg; lane 2, non-acid glycosphingolipids of human stomach (Individual II; blood group A), 40 μg; lane 3, reference Le^b hexaglycosylceramide (Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 4, non-acid glycosphingolipids of the intestinal epithelium of a blood group O pig, 40 μg; lane 5, non-acid glycosphingolipids of the intestinal epithelium of a blood group A pig, 40 μg; lane 6, reference H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer), 2 μg; lane 7, reference Globo H hexaglycosylceramide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), 1 μg; lane 8, reference Globo A heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), 4 μg.