The lactosylceramide binding specificity of Helicobacter pylori

Abstract

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.

Lactotetraosylceramide, a Novel Glycosphingolipid Receptor for Helicobacter pylori, Present in Human Gastric Epithelium

“…Two distinct binding specificities were repeatedly detected by autoradiography. As previously described in detail, H. pylori bound to lactosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide (12). The only binding activity initially detected in human gastrointestinal material was to a compound in the tetraglycosylceramide region of the non-acid fraction of human meconium.…”

“…1B. The binding in lane 4 (gangliotriaosylceramide) and lane 7 (gangliotetraosylceramide) was judged to correspond to the "ganglio binding specificity" of H. pylori described previously in detail (12).…”

“…The conditions used for culture and 35 S-labeling of the bacteria have been described previously (12). For binding assays, the bacteria were suspended to 1 ϫ 10 8 colony forming units/ml in phosphate-buffered saline (PBS), 2 pH 7.4.…”

“…3 of Table III) was generated from Gal␤3GlcNAc␤3Gal␤4Glc␤1Cer (No. 2) by treatment with anhydrous hydrazine, as described (12).…”

“…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).…”