Glucosylceramide synthesized in vitro from endogenous ceramide is uncoupled from synthesis of lactosylceramide in Golgi membranes from chicken embryo neural retina cells.

It is known that ceramide (Cer), the precursor of sphingoglycolipids and of sphingomyelin, participates in events leading to activation of the apoptotic pathway, and per se or through conversion to glucosylceramide (GlcCer) modulates formation of neuritic processes in developing neurons. To learn about the fate of de novo synthesized Cer and GlcCer we examined, in Golgi membranes from chicken embryo neural retina cells, the metabolic relationships of endogenous Cer, GlcCer and lactosylceramide (LacCer). Incubation of the membranes with UDP-[3H]Glc revealed a pool of endogenous Cer useful for synthesis of GlcCer. Most of the GlcCer synthesized, however, was not used for synthesis of LacCer, indicating that it was functionally uncoupled from LacCer synthase. On the other hand, incubation with UDP-[3H]Gal revealed a pool of endogenous GlcCer that depending of the integrity of the membranes was functionally coupled to LacCer and ganglioside synthesis. These results indicate that most GlcCer formed in vitro from Cer is topologically segregated from the synthesis of LacCer. However, subfractionation in sucrose gradients of Golgi membranes labeled with both precursors failed to separate membranes enriched in [3H]GlcCer from those enriched in [3H]Gal-labeled LacCer. It is concluded that despite both transfer steps co-localize in the Golgi membranes, coupling of GlcCer synthesis to LacCer synthesis requires conditions not present in our in vitro assay. This suggests that a coupling activity exists that could be relevant for regulation of the cytoplasmic levels of Cer and GlcCer.

Compartmental organization of the synthesis of GM3, GD3, and GM2 in golgi membranes from neural retina cells.

The relationship among lactosylceramide-(LacCer), GD3- and GM2-synthases and between the two last transferases and their common GM3 acceptor was investigated in intact Golgi membrane from chick embryo neural retina cells at early (8-days) and late (14 days) stages of the embryonic development. [3H]Gal was incorporated into endogenous glucosylceramide by incubation of Golgi membranes with UDP-[3H]Gal. Conversion of the synthesized [3H]Gal-LacCer into GM3, and of the latter into GD3, GM2 and GD2 was examined after a second incubation step with unlabeled CMP-NeuAc and/or UDP-GalNAc. With CMP-NeuAc, most [3H]Gal-LacCer was converted into GM3 in either 8- or 14- day membranes. However, while about 90% of GM3 was converted into GD3 in 8-day membranes, only about 25% followed this route in 14-day membranes. With CMP-NeuAc and UDP-GalNAc, about 90% of GM3 was used for synthesis of GM2 in 14-day membranes, while in 8-day membranes about 80% followed the route to GD3, and a part to GD2. Performing the second incubation step in the presence of increasing detergent concentrations showed that conversion of GM3 to GM2 was inhibited at concentrations lower than those required for inhibition of LacCer to GM3 conversion. Taken together, results indicate that transfer steps leading to synthesis of GM3, GD3, GM2 and GD2 from LacCer are functionally coupled in the Golgi membranes, and that GD3- and GM2-synthases compete in a common compartment for using a fraction of GM3 as substrate. In this competition, the relative activities of the transferases and their relative saturation with the respective donor sugar nucleotides, are important factors influencing conversion of GM3 toward either GD3 or GM2.

GT3 synthesis in the proximal Golgi occurs in a compartment different from those for GD3 and GM3 synthesis.

Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [3H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[3H]Gal. In (a), 1 microM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to approximately 50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of > 90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited approximately 30 and 70%, respectively, in 7- and 10-day cells. In (b), > 80% of the [3H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [3H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, < 5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[3H]NeuAc incorporated approximately 20% of [3H]NeuAc into endogenous GT3, and this percentage was not affected by 1 microM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.

Functional coupling of glycosyl transfer steps for synthesis of gangliosides in Golgi membranes from neural retina cells.

The synthesis of the oligosaccharide of gangliosides is carried out in the Golgi complex by successive sugar transfers to proper glycolipid acceptors. To examine how the product of one glycosylation step couples with the next transfer step, the endogenous gangliosides of Golgi membranes from 14-day-old chick embryo retina were labeled from CMP-[3H]NeuAc or UDP-[3H]GalNAc or UDP-[3H]Gal in conditions which do not allow vesicular intercompartmental transport. After saturation of the endogenous acceptor capacity, labeling was mostly in the immediate acceptors of the corresponding labeled sugars. However, some labeled intermediates progressed to more glycosylated gangliosides if the membranes were incubated in a second step in the presence of the necessary unlabeled sugar nucleotides. This was particularly evident in the case of membranes incubated with UDP-[3H]Gal, in which most of the [3H]Gal-labeled lactosylceramide synthesized in the first step was converted to GM3 and GD3, or to GM2 or to GD1a in a second incubation step in the presence of unlabeled CMP-NeuAc alone, or together with UDP-GalNAc, or together with UDP-Gal plus UDP-GalNAc, respectively. Conversion was time dependent and dilution-independent. Since prior reports using brefeldin A indicate that transfer steps catalyzed by GalNAc-T, Gal-T2, and Sial-T4 localize in the trans-Golgi network (TGN), our results lead to the following major conclusions: (a) transfer steps catalyzed by GalNAc-T, Gal-T2, and Sial-T4 colocalize and are functionally coupled in the TGN; (b) proximal Golgi Gal-T1, Sial-T1, and Sial-T2, and their corresponding glycolipid acceptors, extend their presence to the TGN, and (c), GalNAc-T and Sial-T2 compete for a common pool of acceptor GM3 in the synthesis of GM2 and GD3.