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.     

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.     

The influence of ganglioside insertion into brain membranes on the rate of ganglioside degradation by membrane-bound sialidase

Microsomal membranes isolated from calf brain contain a sialidase which cleaves ganglioside substrates naturally occurring within these membranes as well as exogenously added [3H]ganglioside GD1a. Micelles of [3H]ganglioside GD1a bind to the microsomal membranes in two steps. The first step, called adsorption, is fast and reversible by treatment with trypsin; the second step, called uptake, is slower and not reversible. The product of the enzymic degradation, [3H]ganglioside GM1, is exclusively located in the ganglioside pool taken up by the sialidase-bearing membranes, and not in the trypsin-releasable pool. Electron spin resonance (ESR) studies using a spin-labelled analogue of ganglioside GD1a indicate that the ganglioside uptake by microsomal membranes is accompanied by the disappearance of the micellar structure and by the 'dilution' of the probe molecules with membrane lipids. These findings suggest that exogenously added ganglioside substrate inserts into the microsomal membrane before it is recognized as substrate by the membrane-bound sialidase. Therefore, the influence of pH, ionic strength and membrane-fluidizing agents on the degradation rate measured with exogenous ganglioside GD1a does not only reflect kinetic parameters of the enzymic reaction itself but also the velocity of ganglioside insertion. Increasing ionic strength reduces the degradation rate. The acceleration of insertion with falling pH values shifts the measured pH optimum of the ganglioside degradation to lower values (pH 3.6) and masks the substantial residual sialidase activity at pH 5-7. The membrane-fluidizing alcohol n-hexanol greatly accelerates ganglioside insertion as well as ganglioside degradation. The latter was clearly demonstrated by studying the hydrolysis of endogenous ganglioside substrates, and is due to a decrease of the apparent Km value and an increase in the Vmax value. The Vmax value was also enhanced by freezing and thawing of the microsomal membranes.     

Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts

To probe the functions of membrane gangliosides, the availability of ganglioside-depleted cells would be a valuable resource. To attempt to identify a useful genetic model of ganglioside depletion, we assessed ganglioside metabolism in murine GM3 synthase (GM3S)-/- knockout primary embryonic fibroblasts (MEF), because normal fibroblast gangliosides (GM3, GM2, GM1, and GD1a), all downstream products of GM3S, should be absent. We found that heterozygote MEF (GM3S+/-) did have a 36% reduced content of qualitatively normal gangliosides (7.0+/-0.8 nmol LBSA/mg cell protein; control: 11+/-1.6 nmol). However, two unexpected findings characterized the homozygous (GM3-/-) MEF. Despite complete knockout of GM3S, (i) GM3-/- MEF retained substantial ganglioside content (21% of normal or 2.3+/-1.1 nmol) and (ii) these gangliosides were entirely different from those of wild type MEF by HPTLC. Mass spectrometry identified them as GM1b, GalNAc-GM1b, and GD1alpha, containing both N-acetyl and N-glycolylneuraminic acid and diverse ceramide structures. All are products of the 0 pathway of ganglioside synthesis, not normally expressed in fibroblasts. The results suggest that complete, but not partial, inhibition of GM3 synthesis results in robust activation of an alternate pathway that may compensate for the complete absence of the products of GM3S.     

Selective ganglioside desialylation in the plasma membrane of human neuroblastoma cells*

Gangliosides of the plasma membrane are important modulatorsof cellular functions. Previous work from our laboratory hadsuggested that a plasma membrane sialidase was involved in growthcontrol and differentiation in cultured human neuroblastomacells (SK-N-MC), but its substrates had remained obscure. Wenow performed sialidase specificity studies in subcellular fractionsand found ganglioside GM3 desialylating activity in presenceof Triton X-100 to be associated with the plasma membrane, butabsent in lysosomes. This Triton-activated plasma membrane enzymedesialylated also gangliosides GDla, GD1b, and GT1b, therebyforming GM1; cleavage of GM1 and GM2, however, was not observed.Sialidase activity towards the glycoprotein fetuin with modifiedC-7 sialic acids and towards 4-methylumbelliferyl neuraminatewas solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in ganglioside desialylationof living cells was examined by following the fate of [³H]galactose-labelledindividual gangliosides in pulse-chase experiments in absenceand presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminicacid. When the plasma membrane sialidase was inhibited, radioactivityof all gangliosides chased at the same rate. In the absenceof inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degradedat a considerably faster rate in confluent cultures, whereasthe GM1-pool seemed to be filled by the desialylation of highergangliosides. The results thus suggest that the plasma membranesialidase causes selective ganglioside desialylation, and thatsuch surface glycolipid modification triggers growth controland differentiation in human neuroblastoma cells. ganglioside neuroblastoma cells plasma membrane sialidase     

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.     

Substrate specificity of GM2 and GD3 synthase of Golgi vesicles derived from rat liver

Several GM3 derivatives have been synthesized. Among them were lyso-GM3 derivatives and GM3 analogues with modifications in the sialic acid moiety. They were used as glycolipid acceptors in assays for GM2 and GD3 synthase of rat liver Golgi. Analysis of the resulting enzyme activities and of the reaction products revealed different substrate specificities for GM2 and GD3 synthase although the normal glycolipid acceptor for both transferases is ganglioside GM3. Specificity of GD3 synthase is strongly determined by the substrate's negative charge and the acyl residue in amide bond to the amino group of neuraminic acid, while GM2 synthase reacts quite indifferently to these changes in the sialic moiety of the substrate. Both enzymes seem to be sensitive to the spatial extension at the neuraminic acid's carboxylic group.     

Selective shedding of gangliosides by cells of mouse ascitic sarcoma-37

The profile and content of gangliosides associated with tumour cells of mouse sarcoma-37 and exfoliated from the cell surface were determined. It was shown that 20% of tumour gangliosides shed from the cell surface and only about 7% of all the shed gangliosides were contained in plasma membrane fragments. When incubated in vitro at 37 degrees C for 1 hour, the tumour cells were found to release less than 2% of cellular gangliosides. The main components of cellular gangliosides corresponded in their chromatographic behaviour to GM2 (31-32% of the total ganglioside content), GD3 (17-18%), GD1a (9-11%), GD2 (16-17%) and hematosides (19-21%). The profiles of in vivo and in vitro shed gangliosides were similar but strongly differed from those of cellular gangliosides: the relative content of GM2 was 70-75% and the other gangliosides were found in negligible amounts. The data show that GM2 accumulation in extracellular spaces is rather the result of its selective shedding than the shedding of products of "incomplete" cellular ganglioside synthesis.     

Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression

In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density.GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation.Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase. Published in 2003.     

Induction of GM1a/GD1b synthase triggers complex ganglioside expression and alters neuroblastoma cell behavior; a new tumor cell model of ganglioside function

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.     

Subcellular distribution and biosynthesis of rat liver gangliosides

Gangliosides have generally been assumed to be localized primarily in the plasma membrane. Analysis of gangliosides from isolated subcellular membrane fractions of rat liver indicated that 76% of the total ganglioside sialic acid was present in the plasma membrane. Mitochondria and endoplasmic reticulum fractions, while containing only low levels of gangliosides on a protein basis, each contained approx. 10% of total ganglioside sialic acid. Gangliosides also were present in the Golgi apparatus and nuclear membrane fractions, and soluble gangliosides were in the supernatant. Individual gangliosides were non-homogeneously distributed and each membrane fraction was characterized by a unique ganglioside composition. Plasma membrane contained only 14 and 28% of the total GD1a and GD3, respectively, but 80-90% of the GM1, GD1b, GT1b and GQ1b. Endoplasmic reticulum, when corrected for plasma membrane contamination, contained only trace amounts of GM1, GD1b, GT1b and GQ1b, but 11 and 5% of the total GD1a and GD3, respectively. The ganglioside composition of highly purified endoplasmic reticulum was similar. Ganglioside biosynthetic enzymes were concentrated in the Golgi apparatus. However, low levels of these enzymes were present in the highly purified endoplasmic reticulum fractions. Pulse-chase experiments with [3H]galactose revealed that total gangliosides were labeled first in the Golgi apparatus, mitochondria and supernatant within 10 min. Labeled gangliosides were next observed at 30 min in the endoplasmic reticulum, plasma membrane and nuclear membrane fractions. Analysis of the individual gangliosides also revealed that GM3, GM1, GD1a and GD1b were labeled first in the Golgi apparatus at 10 min. These studies indicate that gangliosides synthesized in the Golgi apparatus may be transported not only to the plasma membrane, but to the endoplasmic reticulum and to other internal endomembranes as well.     

Effect of gangliosides on the distribution of a glycosylphosphatidylinositol-anchored protein in plasma membrane from Chinese hamster ovary-K1 cells

Glycosylphosphatidylinositol (GPI)-anchored proteins are clustered mainly in sphingolipid-cholesterol microdomains of the plasma membrane. The distribution of GPI-anchored fusion yellow fluorescent protein (GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1 cells with different glycolipid compositions was investigated. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity or cell lines expressing different gangliosides caused by stable transfection of appropriate ganglioside glycosyltransferases or exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The distribution of GPI-YFP fusion protein expressed at the plasma membrane was studied using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms clusters at the surface of cells expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1 and GD1a, or mostly GM2, or highly expressing GM1. However, no significant changes in membrane microdomains of GPI-YFP were detected in the different glycolipid environments provided by the membranes of the cell lines under study. On the other hand, wild type CHO-K1 cells exposed to 100 microm GM1 before cross-linking with bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the amount of GPI-YFP clusters. These findings clearly indicate that manipulating the glycolipid content of the cellular membrane, just by changing the ganglioside biosynthetic activity of the cell, did not significantly affect the association of GPI-YFP on the cell surface of CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP plasma membrane distribution might be a consequence of the ganglioside level reached in plasma membrane and/or the effect of particular ganglioside species (micelles) that lead to membrane architecture and/or dynamic modifications.     

Ganglioside-protein interactions: spin-label electron spin resonance studies with (Na+,K+)-ATPase membranes

Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from Squalus acanthias have been studied using spin-labeled derivatives of the mono- and disialogangliosides GM1, GM2, GM3, and GD1b, in conjunction with electron spin resonance (ESR) spectroscopy. Ganglioside-protein interactions are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component that corresponds closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled gangliosides whose chain motion is significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A small selectively for the motionally restricted component associated with the protein is found in the order GD1b greater than GM1 approximately equal to GM2 approximately equal to GM3. Comparison with previous results from spin-labeled phospholipids in the same system [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] shows that the spin-labeled monosialogangliosides GM1, GM2, and GM3 display little selectivity in the lipid-protein interaction relative to spin-labeled phosphatidylcholine. The spectral characteristics of both the fluid and motionally restricted spin-labeled components differ very significantly, however, between the gangliosides and the phospholipids. The outer hyperfine splitting of the motionally restricted component is smaller for the gangliosides than for the phospholipids, indicating a smaller degree of motional restriction on interaction of the ganglioside lipid chains with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of gangliosides in primary cultures of rat hepatocytes. Determination of the net synthesis of individual gangliosides by incorporation of labeled N-acetylmannosamine

The ganglioside content of rat hepatocytes increases several-fold during the first 6 days in monolayer culture. To correlate increased levels with rates of de novo synthesis, the incorporation of N-acetyl-[6-3H]D-mannosamine into individual gangliosides was determined. The calculation of synthetic rates was made possible by the simultaneous measurement of the specific radioactivity of the immediate sialic-acid donor, CMP-Neu5Ac. The CMP-Neu5Ac content of hepatocytes was found by HPLC analysis to be 30.5 nmol/g of plated cells. The specific radioactivity of this precursor pool reached a constant plateau 5 h after addition of the labeled N-acetyl-mannosamine and remained constant for at least 70 h. The incorporation into individual gangliosides was measured in primary cultures of rat hepatocytes between 72 and 144 h after seeding. During this period, the increase in ganglioside levels was greatest. The highest rates of incorporation were seen in GD1a followed by GM3, GM1, GD3 and the polysialylated compounds. The following rates of synthesis (nmol per 60 h and mg of protein) were calculated: GD1a 0.68, GM3 0.59, GM1 0.36, GD3 0.13 and GT1 0.02. These values are compared with the net increase of the gangliosides as measured by the resorcinol reaction.     

Induction of axonal differentiation by silencing plasma membrane-associated sialidase Neu3 in neuroblastoma cells

A reduction of 70% of the plasma membrane-associated sialidase Neu3 activity, due to a corresponding reduction of the enzyme expression by transducing cells with a short hairpin RNA encoding a sequence target (complementary messenger of mouse Neu3), caused neurite elongation in Neuro2a murine neuroblastoma cells. The differentiation process was accompanied in parallel by an increase of the acetylcholinesterase activity, a moderate increase of the c-Src expression and by the presence of the axonal marker tau protein on the neurites. The sphingolipid pattern and turnover in transduced and control cells were characterized by thin layer chromatography, mass spectrometry and metabolic radiolabeling after feeding cells with tritiated sphingosine. Control cells contained about 2 nmol of gangliosides/mg cell protein. GM2 was the main compound, followed by GD1a, GM3 and GM1. In Neu3 silenced cells, the total ganglioside content remained quite similar, but GM2 increased by 54%, GM3 remain constant, and GM1 and GD1a decreased by 66% and 50%, respectively. Within the organic phase sphingolipids, ceramide decreased by 50%, whereas the sphingomyelin content did not change in Neu3 silenced cells.     

A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts

A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GDla isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [3H]GM1 was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4-5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM1, using as low as 10 micrograms of fibroblast homogenate as protein. The detection limit could be lowered to 0.02-0.03 nmol, adopting conditions that, however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm2 plastic flasks was 5.8 +/- 2.5 (SD) nmol liberated GM1 h-1 mg protein-1. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-D-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate. All this supports the hypothesis that human fibroblasts contain sialidases with different subcellular location and substrate specificity. Particularly, the sialidase acting on gangliosides seems to have two sites of subcellular location, a major one at the level of plasma membranes and/or intracellular organelles functionally related with the plasma membranes and a minor one in the lysosomes.     

Ganglioside depletion and EGF responses of human GM3 synthase-deficient fibroblasts

Recognition of important roles of gangliosides in normal and abnormal cell function has motivated pharmacological modification of cellular ganglioside content. However, constitutive depletion of gangliosides in untransformed human cells has not been reported. In this context, the recent identification of a kindred carrying a point mutation in the GM3 synthase [ST3Gal5, Siat9] gene (Simpson MA, Cross H, Proukakis C, Priestman DA, Neville DC, Reinkensmeier G, Wang H, Wiznitzer M, Gurtz K, Verganelaki A, Pryde A, Patton MA, Dwek RA, Butters TD, Platt FM, Crosby AH. 2004. Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet. 36:1225-1229) provided an opportunity to explore this possibility. We established primary cultures of skin fibroblasts of three patients homozygous for this autosomal recessive defect. They exhibited a 93% reduction in ganglioside content (0.8 +/- 0.2 nmol lipid-bound sialic acid per 10(7) cells versus 12.7 +/- 1.3 nmol per 10(7) normal fibroblasts). Importantly, this marked reduction was not compensated by the activation of an alternate pathway of ganglioside synthesis, as occurs in murine GM3 synthase knockout fibroblasts. Cell morphology appeared unaffected, but under stringent conditions EGF-induced proliferation and migration of the mutant fibroblasts were reduced by 80% and 60%, respectively. Probing potential explanations, we found that EGF binding (effective membrane EGF receptor (EGFR) number) was reduced by 52% (to 6.2 +/- 1.9 from 12.8 +/- 2.0 pmol/10(8) normal fibroblasts, P < 0.01), despite normal total EGFR protein. EGFR activation was likewise reduced as was EGF-induced Rho/Rac1 phosphorylation, which is associated with cell migration. We conclude that this GM3 synthase point mutation almost completely depletes human fibroblast cellular gangliosides, dampens membrane EGFR activation, and modulates related critical cell functions such as proliferation and migration. These cells offer a valuable model for the study of ganglioside modulation of cell function.     

Large scale biosynthesis of ganglioside analogues by RERF-LC-AI cells cultured in HYPERFlask

The efficient production of ganglioside analogues was accomplished using RERF-LC-AI cells cultured in HYPERFlask (High Yield PERformance Flask). Eight kinds of ganglioside analogues (GM3, GM2, sialylparagloboside, GD3, di-sialylated lacto-N-tetraose, and another three kinds of analogues with intricate structures) were synthesized by the saccharide primer method using lung squamous-cell carcinoma line RERF-LC-AI and 12-azidododecyl β-lactoside primer. The yield for each analogue obtained using HYPERFlask was higher than yields obtained from 100-mm dishes.     

Ganglioside composition of astrocytes

Short-term and long-term (greater than 7 months) cultured astrocytes from 14-day-old rat brain were analyzed for ganglioside content. Analysis of the extracted gangliosides by HPTLC revealed that ganglioside GM1 was absent in 35 days and 235 days cultured astrocytes, and that the predominant ganglioside was GM3, showing a double band in both cases. A small amount of the disialogangliosides (GD3, GD1a) was also detected. More than 70% of radioactivities into ganglioside fractions by cultured astrocytes, in the presence of N-[3H]-acetylmannosamine, appeared in ganglioside GM3. The upper band component of GM3 increased 60% in long-term astrocyte cultures compared to 35-day-old cultures. Also, an increased GD3 content in long-term astrocyte cultures was detected. These results suggest that the increase of GD3 and upper band GM3 in long-term cultured astrocytes might be related to the appearance of small processes showing strong reactivity against GFAP and vimentin during astrocyte-subculture.     

The adhesion protein TAG-1 has a ganglioside environment in the sphingolipid-enriched membrane domains of neuronal cells in culture

We studied the interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains by ganglioside photolabeling combined with cell surface biotin labeling. After cell photolabeling with radioactive photoactivable derivatives of GM3, GM1 and GD1b gangliosides, followed by cell surface biotin labeling, sphingolipid-enriched domains were prepared and immunoprecipitated with streptavidin-coupled beads, under experimental conditions preserving the integrity of the lipid domain. About 50% of the total radioactivity linked to proteins was associated with acylated tubulin, about 10% with a 135-kDa protein present as a series of species with pI ranging from 6.5 to 8.0, about 5% with a protein of about 70 kDa and with pI near to 6.5. By immunoprecipitation with streptavidin-coupled beads under conditions disrupting the integrity of the lipid domain, the 135 kDa protein was recovered in the immunoprecipitate, that did not contain tubulin. Thus, the 135 kDa protein has an exoplasmic domain, and it was then identified as the GPI-anchored neural cell adhesion molecule TAG-1. Remarkably, TAG-1 was cross-linked in a similar extent by the photoactivated ganglioside GM3, GM1 and GD1b. The three gangliosides bear different oligosaccharide chains, suggesting that the ganglioside/TAG-1 interaction is not specifically associated with the ganglioside oligosaccharide structure.