Effects of neonatal hypoxia/ischemia on ganglioside expression in the rat hippocampus.

Neonatal Hypoxia-Ischemia (HI) triggers a cascade of biochemical events that result in neuronal injury, but the mechanisms underlying these processes are not completely understood, and information regarding the effect of HI on the synthesis of brain glycoconjugates is lacking. The present work evaluates the effects of neonatal HI on hippocampal ganglioside synthesis. Seven-day-old rat pups were exposed to HI for 2.5 h according to the modified Levine model and samples from hyppocampus were obtained at 30 min as well as at 1, 2 and 4 days later. The activity for synthesis of gangliosides was evaluated by determining the incorporation of N-acetyl [3H]neuraminc acid ([3H]NeuAc) into the endogenous gangliosides of Golgi membranes and by determining the activity of Sial-T2 (GD3 synthase) and GalNAc-T (GM2 synthase), the two enzymes acting on sialyllactosylceramide (GM3) at the branching point of synthesis of a- and b-ganglioside pathway. Northern blot experiments were also conducted to determine transcription levels of the mRNAs specific for these transferases. Neonatal HI caused a relative increase of in vitro [3H]NeuAc incorporation into endogenous lactosylceramide, which was most noticeable at 30 min and I day post-event and disappeared by day 2 and 4. The transient accumulation of [3H]GM3 correlated with decreases in the activities of GD3- and GM2 synthase measured at 30 min and at 1 day after the HI insult. No significant variations in the expression of the genes for these enzymes were observed. Results suggest that transient accumulation of GM3 may be due to post-translational events negatively modulating both GD3- and GM2 synthase activities.

Modulation of epidermal growth factor receptor phosphorylation by endogenously expressed gangliosides.

The effect of changing the ganglioside composition of Chinese hamster ovary K1 cells on the function of the epidermal growth factor receptor (EGFr) was examined by studying the signalling pathway generated after the binding of epidermal growth factor (EGF) both in cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity and in cell lines expressing different gangliosides as the result of stable transfection of appropriate ganglioside glycosyltransferases. After stimulation with EGF, cells depleted of glycolipids showed EGFr phosphorylation and extracellular signal-regulated protein kinase 2 (ERK2) activity as parental cells expressing GM3 [ganglioside nomenclature follows Svennerholm (1963) J. Neurochem. 10, 613-623] or as transfected cells expressing mostly GM2 and GD1a as the result of stable transfection of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase. However, cells stably transfected with CMP-NeuAc:GM3 sialyltransferase and expressing GD3 at the cell surface showed both decreased EGFr phosphorylation and ERK2 activation after stimulation with EGF. Results suggest that changes in the ganglioside composition of cell membranes might be important in the regulation of the EGF signal transduction.

Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus.

Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.

GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex.

Glycosyltransferase cDNAs contain a variable number of potential N-glycosylation sites. Here we examined the occupancy and relevance for the activity and intracellular trafficking of the only potential N-glycosylation site of the mouse beta1,3galactosyltransferase (Gal-T2 or GA1/GM1/GD1b synthase) in Gal-T2 cDNA transfected CHO-K1 cells. Transfected cells synthesize a Golgi located active enzyme of 43 kDa whose N-glycan was metabolically labeled from [3H]mannose and was Endo-H sensitive. Inhibition of N-glycosylation by Tunicamycin or by point mutation of the N-glycosylation site resulted in the synthesis of a polypeptide of 40 kDa which lacked enzyme activity and was concentrated in the endoplasmic reticulum (ER). Inhibition of ER glucosidases by Castanospermine impaired the exit of a form of Gal-T2 having reduced enzyme activity from the ER. The N-terminal Gal-T2 domain (aa 1-52) was able to direct and to retain the green fluorescence protein in the Golgi complex. Taken together, these results indicate that Gal-T2 depends on N-glycosylation for its activity and for proper trafficking to, but not its retention in, the Golgi complex.

GM3 alpha2,8-sialyltransferase (GD3 synthase): protein characterization and sub-golgi location in CHO-K1 cells.

GD3 synthase (Sial-T2) is a key enzyme of ganglioside synthesis that, in concert with GM2 synthase (GalNAc-T), regulates the ratio of a- and b-pathway gangliosides. In this work, we study the sub-Golgi location of an epitope-tagged version of chicken Sial-T2 transfected to CHO-K1 cells. The expressed protein was enzymatically active both in vitro and in vivo and showed a molecular mass of approximately 47 or approximately 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of, respectively, beta-mercaptoethanol. The 95-kDa form of Sial-T2 was also detected if the protein was retained in the endoplasmic reticulum (ER) due to impaired glycosylation, indicating that it was formed in the ER. Confocal immunofluorescence microscopy showed Sial-T2 localized to the Golgi complex and, within the organelle, partially co-localizing with the mannose-6-phosphate receptor, a marker of the trans-Golgi network (TGN). In cells treated with brefeldin A, a major fraction of Sial-T2 redistributed to the ER, even under controlled expression to control for mislocalization due to protein overloading. In experiments of incorporation of sugars into endogenous acceptors of Golgi membranes in vitro, GD3 molecules formed by incubation with CMP-NeuAc were converted to GD2 upon incubation with UDP-GalNAc. These results indicate that Sial-T2 localizes mainly to the proximal Golgi, although a fraction is located in the TGN functionally coupled to GalNAc-T. Consistent with this, most of the enzyme was in an endoglycosidase H (Endo-H)-sensitive, neuraminidase (NANase)-insensitive form. A minor secreted form lacking approximately 40 amino acids was Endo-H-resistant and NANase-sensitive, indicating that the cells were able to process N-glycans to Endo-H-resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO-K1 cells, most Sial-T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN.

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.

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.

Mouse beta 1,3-galactosyltransferase (GA1/GM1/GD1b synthase): protein characterization, tissue expression, and developmental regulation in neural retina.

The composition of cell surface gangliosides is largely dependent on the relative activities of Golgi resident glycosyltransferases. In the brain of birds and mammals, complex gangliosides (GM2, GM1, GD1a, GD1b, GT1b) abound at late stages of development and in the adult, due to the relatively high activities of the UDP-GalNAc:LacCer/GM3/GD3 beta 1,4-N-acetylgalactosaminyltransferase (GalNAc-T) and the UDP-Gal: GA2/GM2/GD2 beta 1, 3-galactosyltransferase (Gal-T2) relative to that of CMP-NeuAc:GM3 alpha 2,8-sialyltransferase (Sial-T2). Unlike brain, the mature mammalian neural retina abundantly expresses the simple ganglioside GD3, in relation to complex gangliosides, due to the low activity of GalNAc-T and Gal-T2 relative to Sial-T2. Here we describe the isolation and characterization of a mouse Gal-T2 cDNA that drives the synthesis of an epitope-tagged protein of molecular mass 43 kDa, which was enzymatically active and localized to the Golgi complex in transfected cell lines. Using this cDNA as a probe, it was found that Gal-T2 is coded by a single gene located in chromosome 17, and that the coding sequence is contained in a single exon. The expression of the specific Gal-T2 mRNA (approximately 1.8 kb) was highest in testis, which also showed elevated Gal-T2 activity. In the postnatal neural retina, Gal-T2 mRNA increased after day 3, maintained high levels of expression by days 4-7, and then decreased to initial values by day 10. The developmental pattern of mRNA expression was temporally coincident with that of Gal-T2 activity expression, indicating that this enzyme is under transcriptional control in the neural retina.

Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase.

GD3 synthase (ST8Sia I) transfers a sialic acid in alpha-2-->8 linkage to the sialic acid moiety of GM3 to form the ganglioside GD3. The cDNAs of GD3 synthases predict several putative N-glycosylation sites. In this work we have examined the occupancy of these sites in a chicken GD3 synthase and how they affect its activity and intracellular traffic. COS-7 cells were transfected with an influenza virus hemagglutinin (HA) epitope-tagged form of GD3 synthase (GD3 synthase-HA). Cells acquired GD3 synthase activity, cell surface GD3 immunoexpression, and GD3 synthase-HA immunoreactivity in the Golgi complex. In Western blots, a main GD3 synthase-HA band of 47 kDa was detected, which was radioactive upon metabolic labeling with [2-3H] mannose. Tunicamycin prevented the incorporation of [2-3H]mannose into GD3 synthase-HA, blocked the enzyme activity, and promoted a reduction of the enzyme molecular mass of 6-7 kDa. Timed deglycosylation with N-glycosidase F showed that all three potential N-glycosylation sites of GD3 synthase-HA were glycosylated. The deglycosylated forms were enzymatically more unstable than the native form. Tunicamycin treatment of cells led to retention of GD3 synthase-HA immunoreactivity in the endoplasmic reticulum (ER). Castanospermine and deoxynojirimycin, inhibitors of the ER-processing enzymes alpha-glucosidases I and II, also prevented the exit from the ER but did not essentially affect the enzyme specific activity. 1-Deoxymannojirimycin and swainsonine, inhibitors of mannosidases, did not affect either the enzyme activity or the Golgi localization. Results indicate that (a) N-glycosylation is necessary for GD3 synthase to attain and to maintain a catalytically active folding, and for exiting the ER; and (b) N-glycan trimming in the ER, while not required for enzyme activity, is necessary for proper trafficking of GD3 synthase to the Golgi complex.

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.

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.

UDP-sugar pyrophosphatase of rat retina: subcellular localization and topography.

Rat retinal tissue possesses as a developmentally regulated, highly active pyrophosphatase activity that hydrolyzes UDP-GalNAc and UDP-Gal but not CMP-NeuAc (Martina et al.: J Neurochem 62:1274-1280, 1995). We show here that this activity, measured with UDP-[3H]GalNAc as substrate, is associated to the membrane fraction of rat retinal homogenates and, upon subfractionation by isopycnic centrifugation in sucrose density gradients, is concentrated in fractions enriched in light Golgi membranes. We examined also the topographic disposition of the catalytic site of the enzyme in the transverse plane of the membranes by measuring the effect of protease treatment and of added EDTA on its activity. Pronase inhibited 50% of the translocation of UDP-[3H]GalNAc to the lumen of the Golgi vesicles but did not affect the enzyme activity either in the absence or in the presence of detergent. EDTA, a membrane-impermeant molecule, inhibited 90% of the activity of the enzyme but did not affect translocation of UDP-[3H]GalNAc and inhibited only 25% the incorporation of [3H]GalNAc into endogenous glycoconjugates. These results indicate that the translocation of UDP-[3H]GalNAc was not necessary for hydrolysis to occur and strongly suggest that the catalytic site of the UDP-sugar pyrophosphatase is oriented toward the cytosolic side of the Golgi vesicles. We speculate that this activity limits the availability of UDP-GalNAc to its specific translocator and, consequently, the luminal concentration of the nucleotide in the Golgi vesicles. In this way, by limiting the availability of UDP-GalNAc for the conversion of GM3 to GM2 by the GM3:N-acetyl-galactosaminyl transferase, it would contribute to the preferential use of GM3 for synthesis of GD3 and other "b" pathway gangliosides that are characteristic of the rat retina.

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.

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.

A UDP-sugar pyrophosphatase is developmentally regulated in the rat retina.

Rat retina tissue contains relatively high amounts of GD3 in relation to ganglio-series gangliosides even in the adult stages. This was attributed in part to an activity ratio between the enzyme that converts GM3 to GD3 [sialytransferase II (ST-II)] and the enzyme that converts GM3 to GM2 [N-acetylgalactosaminyltransferase (GalNAc-T)] favorable to ST-II. Here we report the presence in the rat retina tissue of an activity that hydrolyzes one of the substrates of GalNAc-T, the donor sugar nucleotide UDP-GalNAc. Chromatographic analyses of the products of degradation indicate that the activity corresponds to a UDP-sugar pyrophosphatase/phosphodiesterase I. The activity is developmentally regulated, increasing after day 4 of postnatal development to reach values approximately 10-fold higher in the adult tissue. The activity sediments with the microsomal membranes, also hydrolyzes UDP-Gal, does not hydrolyze CMP-NeuNAc, requires Mn2+, and does not require detergent. Kinetic data showed that the same activity hydrolyzes UDP-GalNAc and UDP-Gal, each one acting as competitive inhibitor for the hydrolysis of the other (Km and Ki for UDP-GalNAc, 48 and 33 microM, respectively; Km and Ki for UDP-Gal, 5 and 12 microM, respectively). In another set of experiments, it was found that the activities of the GalNAc-T and the enzyme that converts GM2 to GM1 [galactosyltransferase II (Gal T-II)] increased about threefold from birth to day 4 and then decreased to stabilize by day 6 in values that were similar to those at birth and about one-half those of ST-II.(ABSTRACT TRUNCATED AT 250 WORDS)