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-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.

E-selectin receptors on human leukocytes

“…In prior studies, we demonstrated that polar lipids extracted from human neutrophils that were more than 98% pure supported E-selectin-but not P-selectin-mediated cell adhesion. 33 In this and prior studies, 42,56,57 GSLs from primary human leukocytes were found to include monosialylated poly-LacNAc glycosphingolipids with 5 or more LacNAc repeats and 2 or more fucose residues. Together, these data indicate that human neutrophils express monosialylated fucosylated poly-LacNAc GSLs (myeloglycans) with E-selectin-binding capacity.…”

Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment

“…TLC immunostainings were done as described in several previous publications ( 62,63,70,72 ). In short, Stx1a-, Stx2a-, or Stx2e-containing supernatants (see Stx1a, Stx2a, Stx2e, anti-Stx, anti-GSL, and secondary antibodies above) were employed using the "microscale method" ( 39,77 ). The anti-Stx1 and anti-Stx2 antibodies were used in 1:1,000 dilutions, respectively, and primary anti-Gb3Cer and anti-Gb4Cer antibodies, as well as secondary alkaline phosphatase-conjugated antibodies, were applied in 1:2,000 dilutions each.…”

Tumor-associated CD75s- and iso-CD75s-gangliosides are potential targets for adjuvant therapy in pancreatic cancer

“…Resulting 50 AL volumes were sprinkled on silica-fixed chromatograms of reference gangliosides (see Reference Gangliosides). This ''microscale method'' (because only microliter volumes of primary anti-GSL antibodies containing sera are applied in the first step of the overlay assay to strips of 1.5 Â 10 cm of size) has been described previously by Mü thing et al (25). Binding of serum antibodies was detected with secondary alkaline phosphatase -labeled anti-human IgG plus IgM antibodies.…”