The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.

The cell surface is covered by a dense layer of protein-and lipid-linked glycans. Although it has been known that distinct glycan structures are associated with cancer, the whole spectrum of cancer-associated glycans has remained undiscovered. In the present study, we analyzed the protein-linked cancer glycome by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric glycan profiling of cancer patient tissue samples. In lung cancer, we detected accumulation of a novel group of tumor-associated glycans. These protein-linked glycans carried abnormal nonreducing terminal B-N-acetyl-D-glucosamine (GlcNAc) residues. A similar phenomenon was also detected in structural analyses of tumor-derived glycosphingolipids. This showed that glycan biosynthesis may dramatically change in cancer and that direct glycome analysis can detect the resulting marker glycans. Based on the structural knowledge, we further devised a covalent labeling technique for the detection of GlcNAcexpressing tumors with a specific transferase enzyme. In normal tissues, terminal GlcNAc antigens are capped by galactosylation. Similarly to common cancer-associated glycan antigens T, Tn, and sialyl-Tn, the newly discovered GlcNAc antigens result from incomplete glycosylation. In conclusion, the identified terminal GlcNAc glycans should be recognized as a novel class of tumor markers. [Cancer Res 2009;69(14):5811-9]

The binding of Helicobacter pylori to glycosphingolipids was examined by binding of 35 S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. In addition to previously reported binding specificities, a selective binding to a non-acid tetraglycosylceramide of human meconium was found. This H. pylori binding glycosphingolipid was isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, were identified as Gal␤3GlcNAc␤3-Gal␤4Glc␤1Cer (lactotetraosylceramide). When using non-acid glycosphingolipid preparations from human gastric epithelial cells, an identical binding of H. pylori to the tetraglycosylceramide interval was obtained in one of seven samples. Evidence for the presence of lactotetraosylceramide in the binding-active interval was obtained by proton NMR spectroscopy of intact glycosphingolipids and by gas chromatography-electron ionization mass spectrometry of permethylated tetrasaccharides obtained by ceramide glycanase hydrolysis. The lactotetraosylceramide binding property was detected in 65 of 74 H. pylori isolates (88%). Binding of H. pylori to lactotetraosylceramide on thin-layer chromatograms was inhibited by preincubation with lactotetraose but not with lactose. Removal of the terminal galactose of lactotetraosylceramide by galactosidase hydrolysis abolished the binding as did hydrazinolysis of the acetamido group of the Nacetylglucosamine. Therefore, Gal␤3GlcNAc is an essential part of the binding epitope.Adhesion of microorganisms to target cells is regarded as a first step in pathogenesis of infections, where the specificity of the adhesins of the infectious agent on the one hand and the receptor structures expressed by the epithelial cells of the host target organ on the other are important determinants of the host range and the tissue tropism of the pathogen (1).The human gastric pathogen Helicobacter pylori is an etiologic agent of chronic active gastritis, peptic ulcer disease, and gastric adenocarcinoma (2, 3). This Gram-negative bacterium has a very distinct host range and tissue tropism, i.e. it requires human gastric epithelium for colonization (4). In the human stomach most of the bacteria are found in the mucus layer (5), but selective association of the bacteria to surface mucous cells has also been shown (4, 6).Several different binding specificities of H. pylori have previously been demonstrated. Thus, the binding of the bacterium to such diverse compounds as phosphatidylethanolamine and gangliotetraosylceramide (7), the Le b blood group determinant (8), heparan sulfate (9), the GM3 1 ganglioside and sulfatide (10, 11), and lactosylceramide (12), has been reported. A sialic aciddependent binding of H. pylori to large complex glycosphingolipids (polyglycosylceramides) has also been documented (13). However, only one H. pylori adhesin, the Le b binding BabA adhesin, has been identified to date (14).In the present study a number of different H. pylori strains were labeled with [35 S]methionine and examined for binding to a panel of...

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