A photoreactive derivative of radiolabeled GM1 ganglioside: preparation and use to establish the involvement of specific proteins in GM1 uptake by human fibroblasts in culture

A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with [3H]acetic anhydride of very high specific radioactivity. The fluorenyl group removed by ammonia treatment was substituted by a nitrophenyl azide group. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 and cold natural GM1 (1:10 by mol) for different times and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive: one, of about 30 kDa, interacted with the ganglioside molecules inserted into the outer membrane layer; three, in the region of 46 kDa, interacted with the portion of associated ganglioside able to be released by trypsin treatment. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of the sulfatide/GM1 activator protein (SAP-1) in control and mutant cultured skin fibroblasts

Sphingolipid activator proteins (SAP) are relatively low-molecular-mass proteins that stimulate the hydrolysis of specific sphingolipids by the required lysosomal enzymes. SAP-1 or sulfatide/GM1 ganglioside activator protein has previously been demonstrated to stimulate the enzymatic hydrolysis of sulfatide, GM1 ganglioside and globotriaosylceramide. Using monospecific rabbit antibodies against human liver sulfatide/GM1 activator, the biosynthesis and processing of this activator were studied in cultured skin fibroblasts from controls and patients with GM1 gangliosidosis and a variant form of metachromatic leukodystrophy. When [35S]methionine was presented in the medium to control human fibroblasts for 4 h, the majority of the immunoprecipitable radiolabeling was confined to bands within three regions of apparent molecular mass 65-70, 35-52 and 8-13 kDa. The only immunoprecipitable radiolabeled species excreted into the medium when NH4Cl was present had an apparent molecular mass of 70 kDa. When the excretion products were given to fresh cells followed by incubation for up to 24 h there was production of the mature species. Treatment of the 70 kDa form with endoglycosidase F resulted in production of a 53 kDa molecular mass form. Pulse-chase experiments indicated that the initial immunoprecipitable translation product was 65 kDa which increased to 70 kDa over the next hour. The 65 kDa species must result from co-translational glycosylation of the polypeptide chain. Apparently, intralysosomal processing converts the 13 kDa form to the 8-11 kDa species. The cells from the patient with GM1 gangliosidosis could not process to the smallest species found in controls due to the deficiency of acid beta-galactosidase. Patients who have a variant form of metachromatic leukodystrophy do not make any immunoprecipitable radiolabeled products in the cells or in the media. This indicates a severe mutation in the gene coding for this activator protein. The production of such small mature species from a relatively large precursor form may regulate the production of this interesting protein.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

Precursor-product relationship between GM1 and GD1a biosynthesized from exogenous GM2 ganglioside in rat liver

The demonstration of a precursor-product relationship in the course of GM1 and GD1a biosynthesis is described in the present paper. We injected rats with GM2 gangliosides [GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1'Cer] of brain origin, which were isotopically radiolabeled on the GalNAc ([GalNAc-3H]GM2) or sphingosine ([Sph-3H]GM2) residue. We then compared the time-courses of GM1 and GD1a biosynthesis in the liver after the administration of each radiolabeled GM2 derivative. After the administration of [GalNAc-3H]GM2, GM1, and GD1a were both present as doublets, that could be easily resolved on TLC. The lower spot of each doublet was identified as a species having the typical rat brain ceramide moiety and represented gangliosides formed through direct glycosylation of the injected GM2. The upper spot of each doublet was identified as a species having the typical rat liver ceramide moiety and represented gangliosides formed through recycling of the [3H]GalNAc residue, released during ganglioside catabolism. After the administration of [Sph-3H]GM2, only ganglioside with the rat brain ceramide moiety were found, that represented the sum of ganglioside formed through direct glycosylation and those formed through recycling of some sphingosine-containing fragments. In each case, the time-course of GM1 and GD1a biosynthesis exhibited a precursor-product relationship. The curve obtained from the direct glycosylation showed a timing delay with respect to those obtained from recycling of GM2 fragments. These results are consistent with the hypothesis that the sequential addition of activated sugars to a sphingolipid precursor is a dissociative process, catalyzed by physically independent enzymatic activities.     

Interaction of the thyrotropin receptor on rat FRTL-5 thyroid cells with thyrotropin and a thyrotropin-stimulating autoantibody from Graves' patients

FRTL-5 rat thyroid cells were either surface-labeled with 125I or biosynthetically labeled with [3H]N-acetylglucosamine, solubilized by lithium diiodosalicylate and immunoprecipitated after sequential exposure to bovine thyrotropin and anti-bovine thyrotropin. Autoradiography of polyacrylamide gels run under denaturing conditions and in the presence of a reducing agent revealed two prominent bands with approximate molecular weights of 66-70 kDa and 47 kDa. Immunoprecipitation of the same radiolabeled and solubilized membrane preparations with a Graves' disease IgG having thyroid stimulating but no thyrotropin-binding inhibiting activity revealed only one major band, migrating near the 47 kDa component reactive with thyrotropin. No bands were immunoprecipitated in control incubations using normal human IgG or substituting radiolabeled, solubilized membranes from a rat thyroid cell line with no thyrotropin receptor activity. Thin layer chromatography of Folch extracts of the [3H]-N-acetylglucosamine-labeled immunoprecipitates obtained by either procedure indicated that a specific thyroid ganglioside was coprecipitated with the immunoprecipitated proteins in both cases.     

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-³H]galactose or [³H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [³H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.     

Ganglioside glycosyltransferase assay using ion-exchange chromatography

A rapid method for determination of ganglioside glycosyltransferase activity has been developed employing ion-exchange chromatography. Using 13-day chick brain as a source of uridine diphospho-N-acetyl-D-galactosamine: ganglioside GM3 N-acetylgalactosaminyltransferase (ganglioside GM2 synthetase), we were able to accurately measure transfer of N-[3H]acetylgalactosamine (GalNAc) from UDP-[3H]GalNAc to GM3 by application of the reaction mixture to small columns of DEAE-Sepharose and elution of radiolabeled GM2 reaction product with 10 mM potassium acetate in methanol. This method proved to be as reliable and sensitive as previously published assays but requires less time and fewer manipulations.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Formation of a cytosolic ganglioside-protein complex following administration of photoreactive ganglioside GM1 to human fibroblasts in culture

A GM 1 ganglioside derivative bearing a photoreactive nitrophenyl azide group and tritium labeled at the acetyl group of N-acetylneuraminic acid, has been administered to cultured human fibroblasts. With photolabeling experiments we found that a portion of the ganglioside in the cell cytosol was associated with a soluble protein of about 30 kDa.     

Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification

Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine.     

Formation of free sphingosine and ceramide from exogenous ganglioside GM1 by cerebellar granule cells in culture.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GM1 in order to have it inserted into the plasma membrane and metabolized. Among the formed metabolites radioactive sphingosine and ceramide were identified. [3H]Ceramide started to be measurable after 10 min of incubation (pulse), and [3H]sphingosine after 15 min. Their concentrations increased with pulse time, and, after a 1-hour pulse, with chase time. After a 1-hour pulse with 2 x 10(-6) M [3H-Sph]GM1 followed by a 4-hour chase, the amount of [3H]sphingosine and [3H]ceramide formed were 0.04 and 0.4 pmol/10(6) cells, respectively. Particularly the ability to produce sphingosine was higher in differentiated than in undifferentiated cells. It is concluded that ganglioside turnover contributes to the maintenance of the intracellular levels of free sphingosine and ceramide.     

Soluble gangliosides in cultured neurotumor cells

Abstract: The biosynthesis and degradation of glycosphingolipids were studied in cytosolic and membrane fractions obtained from rat glioma C6 cells. Both pools had a similar composition of neutral glycosphingolipids but the soluble pool contained only a few percent of the total. The major ganglioside in C6 cells was GM3, of which only 2% was soluble. Whereas the bulk of the membrane GM3 was accessible to surface labeling procedures, the soluble GM3 was not. Mouse neuroblastoma N18 cells also contained small amounts of cytoplasmic gangliosides corresponding to GM3, GM2, GM1, and GDla. When C6 cells were incubated with medium containing [³H]galactose at 37°C, the specific activity of soluble GM3 initially increased more rapidly than that of membrane GM3; by 4 h, the specific activities in both pools became equal. Total incorporation into the membrane pool, however, was always several-fold greater even at the shortest incubation times examined. The labeling pattern of neutral glycosphingolipids in both soluble and membrane fractions indicated the existence of a precursor-product relationship between glucosylceramide and other glycosphingolipids. When labeled cells were transferred to nonradioactive medium, glucosylceramide disappeared the most rapidly, with a 50% loss within <6 h. The turnover rates of other glycosphingolipids were much slower. Although cytosolic GM3 was degraded more rapidly (t_1/2= 26 h) than membrane-bound GM3 (t_1/2= 44 h), its turnover rate was much slower than the time required for transport of GM3 to the cell surface (20–30 min). Our results are consistent with the existence of a small intracellular pool of soluble gangliosides and neutral glycosphingolipids that is stable and independent of the main membrane-bound pool. Although the role of these cytosolic glycolipids is unknown, they do not appear to represent a transport pool between the site of synthesis and the plasma membrane.     

Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture.

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.     

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.     

Metabolic activities in human skin fibroblasts preloaded with labeled GM2-ganglioside

Confluent cultures of human skin fibroblasts were maintained for 10 days with sphingosine labeled [3H]GM2. Labeled medium was then replaced with normal medium and the cells maintained for 42 days with weekly medium changes. Cells were harvested at regular intervals and cells, medium, and trypsin digest supernatant analyzed for [3H]GM2 and its metabolic products. The ganglioside can be membrane associated and removed by trypsin, or membrane incorporated and trypsin insensitive. The membrane incorporated material is apparently transported to the lysosomes slowly by membrane flow, where 80% of the cellular GM2 can be metabolized by day 42. [3H]GM2 as well as its metabolic products in control cells is continuously released into the medium, during which it can also become associated with the cell surface membrane. There is no detectable metabolism of the [3H]GM2 in GM2 gangliosidosis cell lines over the extended post-labeling period, indicating that there is no residual enzyme activity in these cells. Undegraded GM2 is continuously released into the medium and remains associated with the cell surface membrane as well.     

Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study

The uptake of ganglioside analogues by a permanent mouse fibroblast cell line has been studied by radio-tracer techniques and ESR spectroscopy with 3H- and nitroxide-labeled compounds. Analogues of GM1, GM2, and GM3 monosialogangliosides and of GD1a and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9-, or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the ganglioside associated with the cells could be removed by trypsin treatment and was shown to consist of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogues at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile, and temperature dependence displayed by the spectra were similar to those found for fluid phospholipid bilayer model membranes, and the high effective order parameters suggested a location in the cell plasma membrane. Similar results were obtained for all the different ganglioside analogues, indicating a common anchoring region in the hydrophobic interior of the membrane. Under the incubation conditions used the amount of trypsin-resistant ganglioside analogue taken up by the cells was about 15 nmol/mg of cellular protein, irrespective of the nature of the oligosaccharide moiety. By use of the natural ganglioside [3H]GM3, the trypsin-resistant uptake was about 19 nmol/mg of cellular protein. Although these amounts are quite similar, the uptake kinetics differed between the true ganglioside GM3 and the ganglioside analogues.     

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphocytes. Saturation binding by monosialylated [3H]-GM1 to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (KD) of 2.2 +/- 1.4 microM and a binding capacity near 2 fmoles/cell. Competitive inhibition of [3H]-GM1 binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM1-congeneric gangliosides. A comparison between the results of these binding studies and ganglioside-induced decrease of CD4 expression demonstrated that every aspect of [3H]-GM1 binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of ganglioside binding to specific sites on CD4+ T-helper lymphocytes.     

Synthesis of a photoreactive, radiolabelled derivative of the oligosaccharide of GM1 ganglioside.

A photoreactive, radioiodinatable derivative of the oligosaccharide (GM1OS) of ganglioside GM1 was synthesized as follows: GM1OS was generated from GM1 by ozonolysis and alkaline fragmentation, and reductively aminated to GM1OSNH2 (1-amino-1-deoxymonosialogangliotetraitol). The latter compound was then reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) to form GM1OSNH-ASA [1-(4-azidosalicoylamido)-1-deoxymonosialogangliotetraitol], which was radioiodinated and further purified. To test the [125I]GM1OSNH-IASA [1-(4-iodoazidosalicoylamido)-1-deoxymonosialogangliotetraitol+ ++] as a probe for ganglioside-binding proteins, the derivative was incubated with cholera toxin, which specifically binds GM1, followed by photolysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The probe only labelled the B or binding subunit of cholera toxin, but not the A or adenylyl cyclase activating subunit. Labelling was inhibited by excess GM1OS, but not by the oligosaccharides from gangliosides GD1a and GD1b. [125I]GM1OSNH-IASA and analogous oligosaccharide derivatives may be valuable probes for detecting ganglioside-binding proteins.     

Disposition of exogenous tritium-labelled GM1lactone in the rat.

The disposition of labelled [3H]GM1lactone, the inner ester of ganglioside GM1, was studied in the rat. After i.v. administration [3H]GM1lactone was quickly converted to its corresponding open form most likely by plasma esterases, and then displayed a pharmacokinetic profile identical to [3H]GM1. Following intramuscular administration of [3H]GM1lactone [3H]GM1 levels in plasma and in tissues were higher than those obtained after the administration of an equivalent dose of [3H]GM1. This increased bioavailability means that GM1lactone can be considered as a potential prodrug of GM1.     

Uncoupling of ganglioside biosynthesis by Brefeldin A

We have studied the effect of Brefeldin A (BFA), an antiviral antibiotic, on glycosphingolipid metabolism in primary cultured cerebellar cells. Cells were labeled metabolically with [14C]galactose, or pulse-labeled with precursors of glycosphingolipid biosynthesis; i.e., [14]serine, [3H]palmitic acid or [3H]sphingosine. In all cases BFA (1 microgram/ml) strongly inhibited (75-95%) ganglioside biosynthesis beyond the stage of GM3 and GD3, that is the formation of GM1, GD1a, GT1b and GQ1b. Simultaneously an accumulation of GlcCer, LacCer, GM3 and GD3 was observed (up to 2000%). These effects could be reversed fully by removal of the BFA from the culture medium. These results indicate that the LacCer-, GM3- and GD3-synthases of murine cerebellar cells are localized together on the proximal site of the Golgi apparatus, probably in the cis-Golgi compartment. It is probable that sphingomyelin synthase and some of the other glycosyltransferases involved in ganglioside biosynthesis are localized in distinct compartments beyond the cis Golgi.