Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes.
The isolation and structural characterization of fucosylated neolacto-series gangliosides with linear poly-N-acetyllactosaminyl chains from normal human granulocytes is described. Gangliosides were purified by consecutive use of anion exchange HPLC on Fractogel TMAE-650(S), adsorption and reversed phase HPLC on Nucleosil 50-7 and Nucleosil 7C18 columns, respectively. TLC immunostaining with carbohydrate specific monoclonal antibodies, fast atom bombardment-mass spectrometry (FAB-MS) of the permethylated derivatives and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates were used for structure elucidations. One ganglioside was identified as sialyl Lewis(x) antigen with nLcOse6Cer core, Neu5-Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc NAc beta 1-3 Gal beta 1-4Glc beta 1-1Cer. Furthermore, monosialylated ceramide deca-, undeca-, dodeca- and tridecasaccharides with three (nLcOse8Cer) and four (nLcOse10Cer) linear lactosaminyl repeats were identified, carring one to three fucoses. The ceramide portions were found to contain C18 sphingosine and predominantly C16:0 fatty acids. All monosialogangliosides were homogenous concerning their terminal alpha 2-3 Neu5Ac-sialylation, but different in their fucosylation status. Beside VI3Neu5Ac, V3Fuc-nLcOse6Cer, in two of the fucosylated polylactosaminyl ganglioside fractions the sialyl Lewis(x) epitope was found, whereas five species expressed the terminal VIM-2 motif. The role of protein linked sialy Lewis(x) epitope of human granulocytes as a ligand for endothelial leukocyte adhesion molecule-1 (ELAM-1; E-selectin) and platelet activation-dependent granule external membrane protein (PADGEM; P-selectin) is well documented. However, the involvement of endothelial cells E-and/or P-selectin mediated cell-cell adhesion via lipid bound sialyl Lewis(x) and/or VIM-2 epitopes on human granulocytes has to be proved in further investigations.
Müthing
J
J
Institute of Cell Culture Technology, University of Bielefeld, Germany.
Spanbroek
R
R
Peter-Katalinić
J
J
Hanisch
F G
FG
Hanski
C
C
Hasegawa
A
A
Unland
F
F
Lehmann
J
J
Tschesche
H
H
Egge
H
H
D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005643:Fucose / Q000737:chemistry*; D005732:Gangliosides / Q000737:chemistry* / Q000302:isolation & purification*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006098:Granulocytes / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D016339:Spectrometry, Mass, Fast Atom Bombardment
D005732:Gangliosides; D011134:Polysaccharides; D005643:Fucose; C037199:poly-N-acetyllactosamine