Characterization of the Cellular Binding of Exogenous Gangliosides

Abstract

Experiments were performed to characterize the accumulation of different exogenous sialoglycolipids by cells.A biphasic kinetics of accumulation appeared to be characteristic for intact primary and permanent monolayer cells and chicken erythrocytes whereas that of chicken erythrocyte ghosts was monophasic. Saturation levels could be reached with Galbl+3 GalNAcpl+4 Gal[3+2aNeuAc]-, !I1 +4 GlcPl -1'Cer and Galfil-+3 GalNAcfil -4 Ga1[3c2aNeuAc8+2aNeuAc]fil+4 Glc@ -1'Cer with concentrations of 100 and 200 nmol per ml culture medium, respectively. Under these conditions the amount of 1.3 -1.4 nmol of sialoglycolipids per 100 pg of cellular protein were found to be cell-bound after approximately two hours. In case of the ganglioside NeuAccr2+3GalfiI -4Glcfll -1'Cer no saturation could be reached.The intermediate plateau of accumulation was abolished by the agents colchicine and sodium fluoride which on the other hand had no influence on the level of saturation. In contrast the presence of cytochalasin B, 2-deoxy-~-glucose plus sodium azide, and glutardialdehyde as well as low temperature decreased the amount of incorporated gangliosides drastically. The effect of cytochalasin B was limited to monolayer cultures.Mild trypsinisation liberated some 70 -85 % of cell-associated gangliosides even after druginhibited accumulation of gangliosides. Also some 87 -95 % of the cell-bound ganglioside NeuAca2+3 Galpl -4 Glcpl -1'Cer remained accessible to the action of exogenous sialidase, thus excluding gross internalisation Over the last several years reports have been accumulating that glycosphingolipids as constituents of the outer surface of mammalian cell membranes significantly contribute by means of their carbohydrate structures to a variety of cellular events. Since glycolipids are taken up by cells from the culture medium, attempts were reported to supplement surface membrane glycolipids by incorporation of exogenous compounds [I]. Glycolipid uptake was accompanied by a reduction in growth rate and saturation density [2-61.Abbreviations. Abbreviations were used according to principles outlined earlier Wiegandt, H. (1973) Hoppe-Seyler's Z . Physiol. Chetn. 354, 3049-1056. The first name given is that used in the text (for simplification the term NeuAc has been deleted in the text), the third name is designated as recommended by IUPAC-IUB Lipid Nomenclature, 1976, the final designation is according to Svennerholm (1963) J . Neurochem. 10, 613-623. GL,,lNeuAc = (I13NeuAc-Lac-Cer) = NeuAcct2-3 Gdlpl -t4 Glc,!?l-1'Cer = GM3; GGtetlNeuAc = (I13NeuAc-CgOse4-Cer) = Galp1+3 GalNAcfil-4 Gdl[3t2aNeuAc]fi1+4 GlcPl -1'Cer = GMl; Gct,,2b-NeuAc = I13(NeuAc)z-GgOse4-Cer = Galpl-3 GalNAcPl-4 Gal[3+2aNeuAc8t2aNeuAc]pl+4 Glcpl-1'Cer = GDlh.Enzymes. Trypsin (EC 3.4.21.4); galactose oxidase or D-galactose: oxygen 6-oxidoreductase (EC 1.1.3.9).

Roles for UDP-GlcNAc 2-Epimerase/ManNAc 6-Kinase outside of Sialic Acid Biosynthesis

“…However, to address concerns raised by reports that work-up conditions alter cellular and tissue distributions of gangliosides (51) and to ensure that the flow cytometry method provided biologically-relevant results in our hands, we tested the concentration and time dependence of the uptake of endogenous GM3 and GD3. In these tests, we observed ganglioside uptake and incorporation consistent with literature reports (52). Specifically, time-and dose-dependent increases in GM3 (Fig.…”

GM1 ganglioside regulates the proteolysis of amyloid precursor protein

“…Exogenous gangliosides can be incorporated into the plasma membrane in a manner analogous to endogenous gangliosides under appropriate conditions. Wiegandt and co-workers 29,30 have reported that exogenous gangliosides are taken up by cells, but are incorporated in the plasma membrane in a different way than endogenous gangliosides and are not degraded. They also observed that most of the gangliosides were removed by adding serum or serum albumin to the culture medium.…”

Shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins

“…(i) as loosely associated micelles removable by serum proteins; (ii) as a protein-bound serumresistant, but protease-sensitive ganglioside fraction; and (iii) as gangliosides associated in a protease-resistant manner [75][76][77].…”