Abstract
After uptake by various cells (human skin fibroblasts, rat neuroblastoma B 104, human neuroblastoma SHSY5Y, murine cerebellar cells), a radioactive and a fluorescent analog of a nondegradable glucosylceramide with sulfur in the glycosidic link were glycosylated to a cell-specific pattern of glycolipid analogs. These results, for the first time, show that a glucosylceramide analog can be conveyed from the plasma membrane of cultured cells to those Golgi compartments that function in the early glycosylation steps of glycolipids. This observation is further confirmed by the fact that the cationic ionophore monensin, known to impede membrane flow from proximal to distal Golgi cisternae, inhibited the formation of complex ganglioside analogs but not those of lactosylceramide, sialyl lactosylceramide (GM3), and disialyl lactosylceramide (GD3).
Highlights
On the cell surface of vertebrate cells, glycosphingolipids (GSL)1 form cell-specific patterns which change with cell differentiation, morphogenesis, and oncogenic transformation
Rat neuroblastoma B 104, human neuroblastoma SHSY5Y, and murine cerebellar cells were incubated with a 10 M lipid1⁄7BSA complex of [14C]C8-Glc-S-Cer and NBD-C8-Glc-S-Cer, respectively, as described under “Experimental Procedures.”
In this paper we present, for the first time, an unambiguous proof for direct glycosylation of glucosylceramide analogs in various cultured cell types
Summary
Materials—SHSY5Y cells were kindly provided by Dr H. -Hexosaminidases were prepared from human placenta [7], and GM2-activator protein was obtained as described [8]. Cell Culture—Monolayer cultures of human skin fibroblasts obtained from biopsy of a male infant and rat neuroblastoma cells B 104 were cultured as described [11]. Human neuroblastoma-derived SHSY5Y cells were grown as described [12]. 105 SHSY5Y cells were seeded and grown for 4 days prior to use. Murine cerebellar cells were prepared essentially as described [13] and were plated onto poly(L-lysine)-coated 35-mm-diameter Petri.
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