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Polyglycosylceramides recognized by Helicobacter pylori: analysis by matrix-assisted laser desorption/ionization mass spectrometry after degradation with endo-beta-galactosidase and by fast atom bombardment mass spectrometry of permethylated undegraded material.
Human erythrocyte polyglycosylceramides (PGCs) are recognized by the gastric pathogen Helicobacter pylori and are based on a successively extended and highly branched N-acetyllactosamine core linked to ceramide and substituted by fucose and sialic acid. As a step in the identification of the binding epitope we earlier characterized intact PGCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS (Karlsson,H., Johansson,L., Miller-Podraza,H., and Karlsson,K-A. [1999] Glycobiology, 9, 765-778). In the present work, PGCs from human blood group O erythrocytes were digested with endo-ss-galactosidase (Bacterioides fragilis), an enzyme which cleaves the bond 3Galss1-4GlcNAc in linear but not branched poly-N-acetyllactosamine chains. The enzymatic digestion resulted in a mixture of neutral and sialic acid-containing glycolipids together with terminal and internal sequences of mainly neutral oligosaccharides. The products were analyzed by MALDI-TOF MS in both positive and negative ion mode which gave spectra where the ions could be assigned to structures of the neutral and acidic components, respectively. Among glycolipids found were [structure in text] where R could be H, Fuc or NeuAc. Also observed were structures as [structure in text] which indicated linear extension along both branches. Observed at higher masses were fully branched structures obtained by stepwise extension with [structure in text] where R could be H, Fuc or NeuAc. Most probably further branching may occur along both the (1-->3)- and the (1-->6)-linked branches to give a partly dendritic structure. Structures with more than one sialic acid substituted could not be observed in the MALDI spectrum. Complementary information of the terminal sequences was obtained by FAB-MS analysis of permethylated undegraded PGCs. High-temperature gas chromatography/mass spectrometry of reduced and permethylated products from enzyme hydrolysis documented that Fuc was present in a blood group O sequence, Fuc-Hex-HexN-. Fucose may be placed on short (monolactosamine) or longer branches, while sialic acid seems to be restricted to monolactosamine branches. The conclusion is that human erythrocyte PGCs display microheterogeneity within terminal and internal parts of the poly-N-acetyllactosamine chains. The first branch from the ceramide end may be located at the second or third Gal and possibly also on the first Gal. Other branches may occur on every N-acetyllactosamine unit in fully branched domains, or there may be linear extensions between branches resulting in incompletely branched structures. The extended linear sequences may be present in both 3- and 6-linked antennae. Terminal structures are based on one, two or maybe higher number of N-acetyllactosamine units.
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