Incorporation of sugars into glycosphingolipids (GSL) occurs predominantly by recycling pathways

Incorporation of sugars into GSL is diminished in SW13 cells that lack a vimentin intermediate filament (IF) network (vim-) compared to vim+ cells. Sugars can be added to ceramide synthesized de novo by acylation of sphinganine (pathway 1), to ceramide synthesized from sphingosine that is derived from catabolism of sphingolipids (pathway 2), and into GSL that recycle through the Golgi apparatus from endosomes (pathway 3). Gal and GlcNH₂ incorporated into GSL in these three pathways was analyzed by the use of two inhibitors of sphingolipid biosynthesis. β-Chloroalanine inhibits the de novo synthesis of sphinganine (pathway 1), and fumonisin B1 inhibits the acylation of sphinganine and sphingosine (pathways 1 and 2) In both vim+ and vim- cells only 20-40% of sugar incorporation into GSL took place in pathway 1, and 60-80% of sugar incorporation took place in the recycling pathways. NH4CI and chloroquin, inhibitors of endsomal/ lysosomal processing, inhibit sugar incorporation into GSL by as much as 80%. The defect in sugar incorporation in vim- SW13 cells is predominantly in pathways 2 and 3. The relative importance of the 3 pathways was also examined in C6 glioma, NB41A3 neuroblastoma and C2C12 myoblast cells, and in non-transformed human fibroblasts. In all of these cells, the recycling pathways 2 + 3 predominated over pathway 1, as in SW13 cells. Although it is generally believed that virtually all GSL are synthesized in the de novo pathway, these data indicate that the recycling pathways predominate in the incorporation of sugars into GSL in a variety of cell types.