ISOLATION AND STRUCTURAL CHARACTERIZATION OF GLYCOSPHINGOLIPIDS OF IN-VITRO PROPAGATED BOVINE AORTIC ENDOTHELIAL-CELLS

Neutral glycosphingolipids and gangliosides were isolated from 3.7 x 1 0(9) primary bovine aortic endothelial cells and structurally characte rized by immunological and chemical methods, Glucosyl- and lactosylcer amide were detected as the main neutral glycosphingolipids (28% and 40 % of total orcinol stain, respectively); LcOse(3)Cer and nLcOse(4)Cer were expressed to somewhat minor amounts (16% and 10% of total orcinol stain, respectively), and nLcOse(6)Cer occurred only in trace quantit ies, No neutral glycosphingolipids of the ganglio-series (GgOse(3)Cer and GgOse(4)Cer) and the globe-series (GbOse(4)Cer and the Forssman an tigen) have been detected; only traces of GbOse(3)Cer were identified by TLC immunostaining, Positive CD15 bands obtained by TLC overlay wit h anti-Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-R antibody indicated t he presence of lipid bound Lewis(x) antigen, whereas the isomeric Lewi s(a) structure (Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-R) was not de tectable. G(M3) substituted with Neu5Gc and Neu5Ac in a 2:1 ratio was the major ganglioside comprising about 95% within the whole gangliosid e fraction, G(M3)-structures were further characterized by FAB-MS and GC-MS of the native compounds and their permethylated derivatives, C-1 8-sphingosine was the only long chain base, whereas variation occurred due to C-24:0,C-24:1 and C-16 fatty acids, Terminally alpha 2-3 sialy lated neolacto-series gangliosides with nL-cOse(4)- and nLcOse(6)Cer ( <5% of total resorcinol stain) were found in almost equal quantities, whereas no alpha 2-6 sialylated counterparts were detected, Fucosylate d gangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis(x), sialyl Lewis(a), and VIM-2 antigen) and sulfoglucuronylneolacto serie s structures with HNK-1 epitope were not detectable in the acidic glyc osphingolipid fraction by TLC immunostaining, Gangliotetraose-type gan gliosides G(M1) and G(D1a) (<1% of total resorcinol stain) as well as traces of G(D1b) and G(T1b) have been distinctly identified by combine d choleragenoid-TLC-immunostaining and previous neuraminidase treatmen t, The expression of dominant glycosphingolipids lactosylceramide and G(M3)(Neu5Gc) was proved by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma mem brane and subcellular distribution patterns, The results provide the b asis for investigation of the role of glycosphingolipids as cell surfa ce antigens of cellular interaction as well as receptors for blood com ponents and macromolecules of the extracellular matrix.