Metabolic Processing of Gangliosides by Human Fibroblasts in Culture — Formation and Recycling of Separate Pools of Sphingosine

Abstract

Human cultured fibroblasts were fed with G,, ganglioside species isotopically labeled at C3 of C1 Ssphingosine, or at C3 of C18-sphinganine, or at the sialic acid acetyl group, and with C18-sphingosine and CIS-sphinganine, both labeled at C1. After a lipid pulse the cells were subjected until 7-day chase; measurements were then made of the radioactive products resulting from the administered long-chain base and ganglioside species catabolism and the salvage processes of catabolic fragments. From the data we drew the following conclusions.The G,, species differing in the long-chain base structure were taken up by the cells and metabolized. About 80% of the total catabolic CIS-sphingosine and C18-sphinganine were recycled for the biosynthesis of complex sphingolipids, the rest being degraded.Results obtained by administering ganglioside species of G,, containing radioactive sphingosine or the free radioactive sphingosine to fibroblasts suggested the existence in the cells of two quite separate pools of sphingosine. One pool was the direct result of either the catabolism of radioactive G,, highdensity microdomains or the diffusion of exogenous sphingosine into the cell; this pool was mainly used for the biosynthesis of the G,, species that contain palmitic and stearic acids. The other pool of sphingosine, the cell basal pool, came from the catabolism of radioactive sphingolipids in the recycling of sphingosine, and was used for the biosynthesis of the G,, species that mainly contain very long fatty acid chains, the main fibroblast endogenous species of GD3. Administration of the ganglioside species of G,, containing sphinganine or free sphinganine to fibroblasts yielded the G,, species containing mainly very long-chain fatty acids and sphingosine.These results show the possible existence of a pool of ganglioside-derived sphingosine, quite separate from the cell basal pool of sphingosine, suggesting that sphingosine derived from sphingolipid catabolism is reduced to sphinganine before entering the sphingolipid biosynthetic pathway.Keywords: ganglioside ; biosynthesis ; sphingosine ; sphinganine ; recycling.Gangliosides, sialic acid containing glycosphingolipids that are normal components of plasma membranes [I], are biosynthesized in the Golgi apparatus and degraded in the lysosomes [2] to fragments that are recycled for biosynthetic purposes [3-61. Among the ganglioside catabolic fragments, sphingosine has attracted scientific interest and increasing attention. It has been shown to act as an intracellular effector through the modulation of protein kinase C and other enzyme activities [7-91. Most of our present information arises from the administration of sphingosine to cells, but little is known about a potential role of a pool of sphingosine originated from ganglioside catabolism in modulating cell function.Catabolic sphingosine is partly recycled, but much of it is degraded [6, ; thus it is difficult to determine to what degree this recycling affects the overall sphingolipid turnover. Moreover, the de novo biosy...

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts

“…We metabolically labeled Clone 15 cells, using uniformly 13 C-labeled fatty acids and the 15 N-sphingolipid precursors, 15 N-sphingosine and 15 N-sphinganine, such that the resulting 15 N-sphingolipids (i.e., sphingomyelin, ceramide, and glycosphingolipids) reached a steady-state distribution in the plasma membrane without 15 N-incorporation into proteins (20). The uniformly 13 C-labeled fatty acids varied in chain length and degree of saturation to promote their incorporation into all sphingolipid and glycerolipid species, presumably producing an even distribution of 13 C-lipids in the cell membrane.…”

Sphingosine 1-Phosphate (S1P) Lyase Deficiency Increases Sphingolipid Formation via Recycling at the Expense of de Novo Biosynthesis in Neurons

“…Ceramide synthases probably trap free sphingosine released from lysosomes at the surface of the ER or in ER-associated membranes. In cell culture models, exogenous sphingosine is taken up by the cells and is largely utilized for the biosynthesis of complex sphingolipids (25)(26)(27)(28). Note that sphingolipids have a turnover of several hours in neuronal cells (29).…”

The Plasma Membrane-associated Sialidase MmNEU3 Modifies the Ganglioside Pattern of Adjacent Cells Supporting Its Involvement in Cell-to-Cell Interactions

“…Under these conditions, because of the slow ganglioside turnover, only a minor portion of the radioactive GD1a reaches the lysosomes and is catabolized (43). COS-7 cells expressing MmNEU3 showed the conversion of GD1a to the corresponding GM1 in the presence of NH 4 Cl, a condition in which the activity of the lysosomal compartment is strongly reduced (Fig.…”

“…MmNEU3 Activity in Living Cells-Administration of [1-3 H]sphingosine to cultured cells at 3 ϫ 10 Ϫ8 M final concentration led to an extensive labeling of the sphingolipids, namely gangliosides, neutral glycosphingolipids, sphingomyelin, and ceramide (43). In addition, a portion of the [1-3 H]sphingosine was catabolized by the cells to radioactive ethanolamine and then recycled for the biosynthesis of radioactive phosphatidylethanolamine.…”