Neu5Ac alpha Gal is part of the Helicobacter pylori binding epitope in polyglycosylceramides of human erythrocytes

The sialic acid dependent binding by the human pathogen Helicobacter pylori to polyglycosylceramides of human erythrocytes was investigated. Polyglyco sylceramides, complex glycosphingolipids with a branched, N-acetyllactosami ne core, were isolated from human erythrocytes, blood group O, and subfract ionated after peracetylation by anion-exchange chromatography. Three subfra ctions were deacetylated, analysed by matrix-assisted laser desorption ioni zation-time of flight MS and 2D H-1 NMR spectroscopy. The observed mass ran ges were m/z = 3093-7622, 3968-7255 and 3459-7987 in the mass spectra of th e first, second and third fractions, respectively. The observed ions agreed with the general formula Hex((x+2))HexNAc(x)Fuc(y)Neu5Ac(z)Cer. Two-dimens ional H-1 total correlation spectra of the mixtures showed that the first f raction contained 3-linked sialic acid and the second and third fractions c ontained both 3-linked and 6-linked sialic acid. Thin-layer chromatogram bi nding assays using the lectins from Maackia amurensis, specific for Neu5Ac alpha 3Gal beta 4GlcNAc; and Sambucus nigra, specific for Neu5Ac alpha 6Gal /GalNAc, were used to confirm this distribution. H. pylori recognized all t hree fractions in the binding assay, indicating that the 3-linked, rather t han 6-linked, sialic acid is essential for binding.