Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes

Abstract

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-Acα2 - 3Galβ1 - 4(Fucα1 - 3)GlcNAcβ1 - 3Galβ1 - 4Glc NAcβ1-3Galβ1-4Glcβ1-1Cer. Furthermore, monosialylated ceramide deca-, undeca-, dodeca- and tridecasaccharides with three (nLcOse8Cer) and four (nLcOse10Cer) linear lactosaminyl repeats were identified, carrying one to three fucoses. The ceramide portions were found to contain C18 sphingosine and predominantly C(16:0) fatty acids. All monosialogangliosides were homogenous concerning their terminal α2-3Neu5Ac-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 sialyl 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.