Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells

To investigate in detail the expression of glycosphingolipids (GSLs) on end othelial cells, 4.85 x 10(9) human umbilical vein endothelial cells (HUVECs ) were cultivated in a 21 bioreactor using microcarriers as a support for a nchorage dependent growing cells. Neutral GSLs and gangliosides were isolat ed and their structures were determined by TLC immunostaining, fast atom bo mbardment-mass spectrometry (FAB-MS) of the native GSLs, and gas chromatogr aphy-electron impact mass spectrometry (GC-EIMS) of partially methylated al ditol acetates. GbOse(4)Cer, GbOse(3)Cer, and LacCer, all carrying mainly C 24- and C16-fatty acid beside C18-sphingosine, were detected as the major n eutral GSLs (36%, 23%, and 15% of the total orcinol stain, respectively); G lcCer, nLcOse(4)Cer, and nLcOse(6)Cer were expressed to substantial minor a mounts (9%, 12%, and 5% of the total orcinol stain, respectively), TLC immu nostaining revealed the presence of lipid bound Lewis(x) antigen, whereas t he isomeric Lewis(x) structure was detectable only in very low quantities. G(M3)(Neu5Ac) with C18-sphingosine was the major ganglioside constituting a bout 90 % of the whole ganglioside fraction, The fatty acid composition was determined by GC-MS of fatty acid methyl esters, indicating the predominan ce of C24- and C16-substituted G(M3)(Neu5Ac), followed by C18- and C22-subs tituted species. Terminally alpha 2-3 sialylated neolacto-series gangliosid e IV(3)Neu5Ac-nLcOse(4)Cer was the second most abundant ganglioside in HUVE Cs (8% of the total resorcinol stain), and IV(6)Neu5Ac-nLcOse(4)Cer and VI( 3)Neu5Ac-nLcOse(6)Cer (together less than 2% of total resorcinol stain) wer e found in minor quantities, Lipid bound sialyl Lewis(x) antigen with poly- N-acetyllactosaminyl chains, and traces of gangliotetraose-type ganglioside s G(M1) and G(D1a) were identified by TLC immunostaining, The expression of dominant neutral GSLs LacCer, GbOse(3)Cer, and GbOse4Cer, and of gangliosi de G(M3)(Neu5Ac) was assayed by indirect immunofluorescence microscopy of c ell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns, The complete structural characteriza tion of GSLs from HUVECs contributes to our understanding about their funct ional role, not only of the carbohydrate but also of the lipid moiety, as r eceptors for bacterial toxins, as cell surface antigens of cellular interac tion and as receptors for blood components and macromolecules of the extrac ellular matrix.