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.

Helicobacter pylori has been reported to agglutinate erythrocytes and to bind to various other cells in a sialic acid-dependent way. The binding was inhibited by sialyllactose or fetuin and other sialylated glycoproteins. The specificity apparently requires bacterial growth on agar, since we found that it was lost after growth in the nutrient mixture Ham's F12. Instead, the bacteria bound with high affinity and in a sialic acid-dependent way to polyglycosylceramides of human erythrocytes, a still incompletely characterized group of complex glycolipids. Bacteria grown in F12 medium were metabolically labelled with 35S-methionine and analysed for binding to glycolipids on thin-layer chromatograms and to glycoproteins on blots after electrophoresis, with human erythrocyte glycoconjugates in focus. There was no binding to simpler gangliosides including GM3 or sialylparagloboside, or to a mixture of brain gangliosides. In contrast, polyglycosylceramides of human erythrocyte membranes bound at a pmol level. The activity was eliminated by mild acid treatment, mild periodate oxidation or sialidase hydrolysis. Erythrocyte proteins as well as a range of reference glycoproteins did not bind except band 3, which was weakly active. However, this activity was resistant to periodate oxidation. These results indicate a second and novel sialic acid-recognizing specificity which is expressed independently of the previously described specificity.

Helicobacter pylori is a bacterium that colonizes the stomach of a majority of the global human population causing common gastric diseases like ulcers and cancer. It has an unusually complex pattern of binding to various host glycoconjugates including interaction with sialylated, sulfated, and fucosylated sequences. The present study describes an additional binding epitope comprising the neolacto internal sequence of GlcNAc␤3-Gal␤4GlcNAc␤. The binding was detected on TLC plates as an interaction with a seven-sugar ganglioside of rabbit thymus. The glycolipid was purified and characterized as Neu5Gc␣3Gal␤4GlcNAc␤3Gal␤4GlcNAc␤3-Gal␤4Glc␤1Cer with less than 10% of the fraction carrying a repeated lacto (type-1) core chain, Gal␤3Glc-NAc␤3Gal␤3GlcNAc␤. After stepwise chemical and enzymatic degradation and structural analysis of products the strongest binder was found to be the pentaglycosylceramide GlcNAc␤3Gal␤4GlcNAc␤3Gal␤4Glc␤1-Cer, whereas the hexa-and tetraglycosylceramides were less active, and the trihexosylceramide was inactive. Further studies revealed that the terminal GlcNAc␤ of the pentaglycosylceramide may be exchanged for either GalNAc␤3, GalNAc␣3, or Gal␣3 without loss of the activity. Calculated minimum energy conformers of these four isoreceptors show a substantial topographical similarity suggesting that this binding is a result of a molecular mimicry. Although the glycoconjugate composition of human gastric epithelial cells is not known in detail it is proposed that repeating N-acetyllactosamine units of glycoconjugates may serve as bacterial attachment sites in the stomach.

Helicobacter pylori expresses separate binding characteristics depending on growth conditions, as documented by binding to human erythrocyte glycoconjugates. Cells grown in Ham's F12 liquid medium exhibited a selective sialic acid-dependent binding to polyglycosylceramides, PGCs (Miller-Podraza et al.(1996) Glycoconjugate J13:453-60). There was no binding to traditional sialylated glycoconjugates like shorter-chain gangliosides, glycophorin or fetuin. However, cells grown on Brucella agar bound both to PGCs and other sialylated glycoconjugates. Fetuin was an effective inhibitor of haemagglutination caused by agar-grown cells, but had no or a very weak inhibitory effect on haemagglutination by F12-grown bacteria. PGCs were strong inhibitors in both cases, while asialofetuin was completely ineffective. The results indicate that H. pylori is able to express two separate sialic acid-dependent specificities, one represented by binding to fetuin, as described before, and another represented by a selective binding to PGCs.

No abstract