Analysis of 3- and 6-linked sialic acids in mixtures of gangliosides using blotting to polyvinylidene difluoride membranes, binding assays, and various mass spectrometry techniques with application to recognition by Helicobacter pylori.
A convenient approach to analyze 3- and 6-linked sialic acids in mixtures of biologically active gangliosides was developed. The procedure was adapted to work on small amounts of material and included parallel tests, which allowed direct analysis of structure and activity. The initial step in the procedure was separation of a mixture of gangliosides by thin-layer chromatography (TLC) and blotting to a polyvinylidene difluoride membrane. The gangliosides were then analyzed (a) by direct desorption from the membrane and fast atom bombardment mass spectrometry (FAB MS), (b) by membrane-binding assay using the NeuAcalpha3- and NeuAcalpha6-specific lectins from Maackia amurensis and Sambucus nigra, respectively, and (c) by TLC binding assay with radiolabeled bacteria. All experiments were performed on a mixture of gangliosides from human leucocytes, which contained species with affinity for the human gastric pathogen Helicobacter pylori. The procedure was used with good results for gangliosides with up to seven sugars per ceramide. A three-sugar ganglioside was identified as GM3 with ceramides composed of sphingosine (d18:1) and 20:0, h20:0, and 24:0 fatty acids. The sequences of four bands in the five-sugar region were consistent with sialylparagloboside (NeuAcalpha3/6Galbeta4GlcNAcbeta3Galbeta4GlcbetaCer). The ceramides were composed of d18:1 and 24:1 fatty acid in the first and third bands, and d18:1 and 16:0 fatty acid in the second and fourth bands from the top. The sialic acid was shown to be 3-linked in the upper two bands and 6-linked in the lower two bands. The same distribution of sialic acid and ceramides but the sequence elongated with one N-acetyllactosamine unit was observed in the less resolved interval containing seven-sugar glycosphingolipids. The direct comparison of binding of lectins and radiolabeled bacteria showed that H. pylori recognized 3-linked sialic acid only. These results were supported by a novel technique of analysis of the sialic acid linkage position by trifluoroacetolysis and gas chromatography/MS. Direct membrane/FAB MS was ineffective for species migrating below the seven-sugar region on the TLC. In this case, the membranes were instead cut in bands and the gangliosides extracted by methanol before analysis by FAB MS.
Johansson
L
L
Institute of Medical Biochemistry, Göteborg University, Göteborg, SE-405 30, Sweden. lena.johansson@medkem.gu.se
Miller-Podraza
H
H
D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D005732:Gangliosides / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry; D016480:Helicobacter pylori / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D011145:Polyvinyls; D016339:Spectrometry, Mass, Fast Atom Bombardment; D005465:Fluorocarbon Polymers
D005732:Gangliosides; D008567:Membranes, Artificial; D019158:N-Acetylneuraminic Acid; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers