Selective binding by Helicobacter pylori of leucocyte gangliosides with 3-linked sialic acid, as identified by a new approach of linkage analysis.
The human gastric pathogen Helicobacter pylori has been shown to bind to glycoconjugates of human leucocytes in a sialic acid-dependent way. In order to improve the identification of the binding epitope, a new technique was developed to analyze the ketosidic linkage position between a terminal sialic acid and the consecutive monosaccharide. Permethylation and reduction with LiAlH4 followed by trifluoroacetolysis in 1000:1 trifluoroacetic anhydride:trifluoroacetic acid (24 h, 100 degrees C) results in the cleavage of glycosidic but not ketosidic bonds. The disaccharide products were analyzed by gas chromatography-mass spectrometry and sialyl-3 or -6 position and NeuAc or NeuGc are identified by their separate retention times and mass spectra. The method was worked out on model saccharides and applied on five-sugar gangliosides (sialylparaglobosides) of human leucocytes. Radiolabeled Helicobacter pylori was shown to bind to the upper part, but not to the lower part, of the five-sugar interval of a mixture of gangliosides separated on a thin-layer chromatogram. Using a membrane blotting procedure the active and inactive bands were isolated and shown to be NeuAcalpha2-3- and NeuAcalpha2-6-paraglobosides, respectively.
Johansson
L
L
Institute of Medical Biochemistry, Göteborg University, Sweden. lena.johansson@medkem.gu.se
Karlsson
K A
KA
D001422:Bacterial Adhesion; D001665:Binding Sites; D002240:Carbohydrate Sequence; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005732:Gangliosides; D019158:N-Acetylneuraminic Acid