GA2/GM2/GD2 synthase localizes to the trans-golgi network of CHO-K1 cells.

UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T) transforms its acceptors into the gangliosides GA2, GM2 and GD2. It is well established that it is a Golgi-located glycosyltransferase, but its sub-Golgi localization is still unclear. We addressed this question in Chinese hamster ovary K1 cell clones stably transfected with a c-myc-tagged version of GalNAc-T which express the enzyme at different levels of activity. In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and on the sub-Golgi localization of the GalNAc-T (by immunocytochemistry). We found that in cell clones expressing moderate levels of activity, GalNAc-T immunoreactivity behaved as the trans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA completely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in cell clones expressing high levels of activity and treated with BFA, most GalNAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did the medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and GD1a was not completely blocked. These results indicate that GalNAc-T is a TGN-located enzyme and that the mechanism that localizes it to this compartment involves steps that, when saturated, lead to its mislocalization to the cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by modification of local glycolipid composition due to the activity of the expressed enzyme were not the main cause of mislocalization, since it persists when glycolipid synthesis is inhibited with d, l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl.

Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase.

GM3-positive Chinese hamster ovary cells (CHO-K1 cells) lack the ability to synthesize GM2 and the complex gangliosides GM1 and GD1a from [3H]Gal added to the culture medium. However, they acquire the ability to synthesize GM2 and to synthesize and immunoexpress complex gangliosides upon transient transfection with a cDNA encoding the human GM3:N-acetylgalactosaminyl transferase (GM2 synthase). The activities of endogenous GM1- and GD1a-synthases in the parental cell line and in cells transfected with the plasmid with or without the GM2 synthase cDNA were essentially identical and comparable in terms of specific activity with the endogenous GM3 synthase. Results indicate that glycosyltransferases acting on GM2 to produce GM1 and GD1a are constitutively present in CHO-K1 cells, and that the expression of their activities depend on the supply of the acceptor GM2. In addition, these results lend support to the notion that GM2 synthase is a key regulatory enzyme influencing the balance between simple and complex gangliosides.

Cloning, characterization and developmental expression of alpha2,8 sialyltransferase (GD3 synthase, ST8Sia I) gene in chick brain and retina.

GD3 and GM2 synthases act on ganglioside GM3 at the branching point of the pathway of synthesis of gangliosides in which the "a", "b" and "c" families are produced. The relative activities of these enzymes are important for regulating the ganglioside composition of a given tissue. In the present work, we report the cloning and characterization of a chick GD3 synthase cDNA. The cloned cDNA directed the synthesis of a functionally active enzyme in transiently transfected CHO-K1 cells and was highly homologous to mammalian GD3 synthases. In Northern blot experiments the cDNA detected a single specific GD3 synthase mRNA of about 9.0 kb both in the chicken brain and retina. The abundance of the specific mRNA transcript declined steadily from E7-E9 to very low values around PN2. The levels of enzyme activities measured at the same developmental stages roughly followed the changes of specific mRNA levels in both tissues. In situ hybridization of embryonic neural retina cells in culture showed that both glial- and neuron-like cells expressed the specific GD3 synthase mRNA, although with different intensities. Results indicate that transcription and/or stability of the specific GD3 synthase mRNA constitute a level of control of the expression of GD3 synthase and indirectly of the ganglioside composition in the developing chicken central nervous system (CNS).

Expression of beta 1-4 N-acetylgalactosaminyltransferase gene in the developing rat brain and retina: mRNA, protein immunoreactivity and enzyme activity.

The developmental pattern of expression of the UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (GalNAc-T) gene was examined in the rat brain and retina. A GalNAc-T cDNA cloned from a rat olfactory bulb cDNA library was used as a probe for Northern blot and in situ hybridization experiments and a rabbit polyclonal antibody to rat GalNAc-T peptide was used for Western blot analysis. In Northern blot experiments, a single approximately 3 kb transcript was detected both in brain and retina. In brain, the abundance of this transcript increased from E15 to PN1-5 and then declined while, in retina, it increased steadily from PN1 to PN13-24. The developmental trends of GalNAc-T mRNA expression, GalNAc-T immunoreactive protein and GalNAc-T activity were comparable in brain. In retina, however, GalNAc-T activity and GalNAc-T peptide immunoreactivity followed developmental patterns that were similar between them and different from that of the specific mRNA. Results suggest that post-transcriptional controls of the GalNAc-T gene expression operate in the rat CNS, which are particularly evident in retina. The expression of the GalNAc-T gene in glial and neuronal cells was examined in rat retina cell cultures by in situ hybridization. The GalNAc-T mRNA was abundant in GM1+/GD3+ neurons and almost absent in the flat, GM1-/GD3+ Müller glia-derived cells.

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.

Effects of brefeldin A on synthesis and intracellular transport of ganglioside GT3 by chick embryo retina cells.

Ganglioside GT3 is the precursor of c-series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub-Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10-day-old chick embryo were cultured with [3H]galactose in the absence (control cells) or in the presence of 1 micrograms/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C. perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC-fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA-treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a-, b-, and c- series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi-synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.