Ganglioside GD3 traffics from the trans-Golgi network to plasma membrane by a Rab11-independent and brefeldin A-insensitive exocytic pathway

Gangliosides, complex glycosphingolipids containing sialic acids, have been found to reside in glycosphingolipid-enriched microdomains (GEM) at the plasma membrane. They are synthesized in the lumen of the Golgi complex and appear unable to translocate from the lumenal toward the cytosolic surface of Golgi membrane to access the monomeric lipid transport. As a consequence, they can only leave the Golgi complex via the lumenal surface of transport vesicles. In this work we analyzed the exocytic transport of the disialo ganglioside GD3 from trans-Golgi network (TGN) to plasma membrane in CHO-K1 cells by immunodetection of endogenously synthesized GD3. We found that ganglioside GD3, unlike another luminal membrane-bounded lipid (glycosylphosphatidylinositol-anchored protein), did not partition into GEM domains in the Golgi complex and trafficked from TGN to plasma membrane by a brefeldin A-insensitive exocytic pathway. Moreover, a dominant negative form of Rab11, which prevents exit of vesicular stomatitis virus glycoprotein from the Golgi complex, did not influence the capacity of GD3 to reach the cell surface. Our results strongly support the notion that most ganglioside GD3 traffics from the TGN to the plasma membrane by a non-conventional vesicular pathway where lateral membrane segregation of vesicular stomatitis virus glycoprotein (non-GEM resident) and glycosylphosphatidylinositol-anchored proteins (GEM resident) from GD3 is required before exiting TGN.     

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.     

Association of Cellular Prion Protein with Gangliosides in Plasma Membrane Microdomains of Neural and Lymphocytic Cells

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrP^c presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrP^c and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrP^c-enriched microdomains. In lymphoblastoid T cells PrP^c molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrP^c was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrP^c-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrP^c immunoprecipitates. The physical association of PrP^c with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrP^c-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Distribution of major brain gangliosides in olfactory tract of frogs

Gangliosides are major cell-surface determinants in the central nervous system (CNS) of vertebrates, found both in neuronal and glial cell membranes. Together with cholesterol and glycosylphosphatidylinositol (GPI) - anchored proteins, gangliosides are involved in organization of plasma membrane microdomains. Based on biochemical studies, frog brain was previously described as having low quantities of gangliosides and their distribution pattern in specific brain regions was unknown. Using highly specific monoclonal antibodies generated against four major brain gangliosides (GM1, GD1a, GD1b and GT1b), we examined the distribution of these molecules in CNS of four different species of frogs (Rana esculenta, Rana temporaria, Bufo bufo and Bufo viridis). We also studied the distribution of myelin- associated glycoprotein (MAG), an inhibitor of axonal regeneration, which is a ligand for gangliosides GD1a and GT1b. Our results show that ganglioside GDla is expressed in neurons of olfactory bulb in all studied animals. In the brain of Rana sp., GD1a is expressed in the entire olfactory pathway, from olfactory bulbs to amygdala, while in Bufo sp. GD1a is restricted to the main olfactory bulb. Furthermore, we found that most of myelinated pathways in frogs express MAG, but do not express GD1a, which could be one of the reasons for better axon regeneration of neural pathways after CNS injury in amphibians in comparison to mammals.     

Essential Roles of Gangliosides in the Formation and Maintenance of Membrane Microdomains in Brain Tissues

Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Recently, novel roles of gangliosides in the regulation of complement system were reported. Here we summarized roles of gangliosides in the formation and maintenance of membrane microdomains in brain tissues by comparing complement activation, inflammatory reaction and disruption of glycolipid-enriched microdomain (GEM)/rafts among several mutant mice of ganglioside synthases. Depending on the defects in ganglioside compositions, corresponding up-regulation of complement-related genes, proliferation of astrocytes and infiltration of microglia were found with gradual severity. Immunoblotting of fractions separated by sucrose density gradient ultracentrifugation revealed that DAF and NCAM having GPI-anchors tended to disappear from the raft fraction with intensities of DKO > GM2/GD2 synthase KO > GD3 synthase KO > WT. The lipid raft markers tended to disperse from the raft fractions with similar intensities. Phospholipids and cholesterol also tended to decrease in GEM/rafts in GM2/GD2 synthase KO and DKO, although total amounts were almost equivalent. All these results indicate that GEM/rafts architecture is destroyed by ganglioside deficiency with gradual intensity depending on the degree of defects of their compositions. Implication of inflammation caused by deficiency of gangliosides in various neurodegenerative diseases was discussed.     

Proteomic analysis of ganglioside‐associated membrane molecules: Substantial basis for molecular clustering

Ganglioside GD3 is specifically expressed in human melanomas, and plays a role in the enhancement of malignant phenotypes of melanoma cells. To analyze the mechanisms by which GD3 enhances malignant properties and signals in melanomas, it is essential to clarify how GD3 interacts with membrane molecules on the cell membrane. In this study, we performed proteomics analysis of glycolipid‐enriched microdomains (GEM) with current sucrose density gradient ultracentrifugation of Triton X‐100 extracts and MS. We also examined GD3‐associated molecules using enzyme‐mediated activation of radical sources (EMARS) reaction combined with MS. Comparison of molecules identified as residents in GEM/rafts and those detected by EMARS reaction using an anti‐GD3 antibody revealed that a relatively low number of molecules is recruited around GD3, while a number of membrane and secreted molecules was defined in GEM/rafts. These results suggested that EMARS reaction is useful to identify actually interacting molecules with gangliosides such as GD3 on the cell membrane, and many other microdomains than GD3‐associating rafts exist. Representative examples of GD3‐associated molecules such as neogenin and MCAM were shown.     

Gangliosides act as co-receptors for Salmonella enteritidis FliC and promote FliC induction of human beta-defensin-2 expression in Caco-2 cells

Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces. We reported previously that Salmonella enteritidis flagellin (FliC) induced human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 cells via NF-kappaB activation (Ogushi, K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi, A., Asakura, H., Makino, S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) J. Biol. Chem. 276, 30521-30526). In this study, we examined the role of ganglioside as co-receptors with Toll-like receptor 5 (TLR5) on FliC induction of hBD-2 expression in Caco-2 cells. Exogenous gangliosides suppressed FliC induction of hBD-2 promoter activity and binding of FliC to Caco-2 cells. Incorporation of exogenous ganglioside GD1a into Caco-2 cell membranes increased the effect of FliC on hBD-2 promoter activity. In support of a role for endogenous gangliosides, incubation of Caco-2 cells with dl-threo-2-hexadecanoylamino-3-morpholino-1-phenylpropanol, a glucosylceramide synthase inhibitor, reduced FliC induction of hBD-2 promoter activity. GD1a-loaded CHO-K1-expressing TLR5 cells had a higher potential for hBD-2 induction following FliC stimulation than GD1a-loaded CHO-K1 cells not expressing TLR5. FliC increased phosphorylation of mitogen-activated protein kinase, p38, and ERK1/2. Exogenous gangliosides GD1a, GD1b, and GT1b each suppressed FliC induction of p38 and ERK1/2 phosphorylation. Furthermore, FliC did not enhance luciferase activity in Caco-2 cells transfected with a plasmid containing a mutated activator protein 1-binding site. These results suggest that gangliosides act as co-receptors with TLR5 for FliC and promote hBD-2 expression via mitogen-activated protein kinase.     

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     

The effects of development on activity, specificity and endogenous substrates of synaptic membrane sialidase

Synaptic plasma membranes were prepared from cortices of rats varying in post-natal age between 4 and 30 days. Sialic acid associated with synaptic plasma membrane glycoproteins and gangliosides increased 75% and 50%, respectively, between 4 and 30 days. The amount of sialic acid released from these membrane constituents by intrinsic synaptic sialidase increased 2-4-fold over the same period. Incubation of synaptic plasma membranes with exogenous gangliosides or glycopeptides demonstrated a 2-3-fold increase in sialidase activity during development. The major gangliosides present in synaptic plasma membranes at all ages were GT1, GD1a, GD1b and GM1. Intrinsic sialidase hydrolyzed 50-70% of endogenous GT1 and GD1a gangliosides at all ages. Endogenous GD1b ganglioside was poorly hydrolyzed in young rats and its susceptibility to enzymic hydrolysis increased during development. When exogenous GD1a and GD1b were used as substrates a preferential increase in activity against GD1b occurred during development, the ratio of activity (GD1a/GD1b) decreasing from 3.6 to 1.6 between 7 and 30 days. 10- and 30-day-old synaptic plasma membranes contained complex mixtures of sialoglycoproteins, an increase in the relative concentrations of lower molecular weight sialoglycoproteins occurring during development. Intrinsic sialidase present in 10- and 30-day-old synaptic plasma membranes acted upon all molecular weight classes of sialoglycoproteins.     

Shed gangliosides provide detergent-independent evidence for type-3 glycosynapses

Membrane microdomains, or rafts, at the plasma membrane have been invoked to explain many cellular processes. Protein-protein interactions within such microdomains including, for example, the tetraspanin web are reported to provide a scaffold for signal transduction. However, the nature of such protein-protein interactions is not fully elucidated. Hakomori [S.I. Hakomori, The glycosynapse, Proc. Natl. Acad. Sci. USA 99 (2002) 225-232] has advanced the concept that glycosphingolipids, particularly gangliosides, provide the intermediary link between transmembrane receptors and signal transducers and has redefined membrane rafts as Type-1, -2 or -3 glycosynapses. Here, using simple immunofluorescent analysis of the ganglioside complexes naturally released from cellular microprocesses (termed "footprints") we show that the ganglioside can determine the nature of protein-protein associations. Specifically, we demonstrate that CD36 and the tetraspanin CD151, both of which interact with beta1 integrins, associate together only in the presence of the gangliosides GD2/GD3. These results substantiate the glycosynapse hypothesis and suggest that the nature of the tetraspanin web may be determined by gangliosides.     

Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.     

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.     

Disialyl gangliosides enhance tumor phenotypes with differential modalities

Sialic acid-containing glycosphingolipids, gangliosides are highly expressed in human cancer cells and regulate cell signals transduced via membrane microdomains. Generally, disialyl gangliosides enhance tumor phenotypes, while monosialyl gangliosides suppress them. In particular, gangliosides GD3 and GD2 are highly expressed in melanomas and small cell lung cancer cells, and their expression cause increased cell growth and invasion. In osteosarcomas, expression of GD3 and GD2 also enhanced cell invasion and motility, and caused increased phosphorylation of focal adhesion kinase and paxillin. In addition to focal adhesion kinase, Lyn kinase was also activated by GD3/GD2 expression, leading to the phosphorylation of paxillin. In contrast with melanoma cells, osteosarcomas showed reduced cell adhesion with increased phosphorylation of paxillin. Thus, increased expression of GD3/GD2 caused enhanced activation of signaling molecules, leading to distinct phenotypes between melanomas and osteosarcomas, i.e. increased and decreased adhesion activity. Thus, whole features of glycolipid-enriched microdomain/rafts formed in the individual cancer types seem to determine the main signaling pathway and biological outcome.     

Role of gangliosides in the association of ErbB2 with lipid rafts in mammary epithelial HC11 cells

We analyzed the role of gangliosides in the association of the ErbB2 receptor tyrosine-kinase (RTK) with lipid rafts in mammary epithelial HC11 cells. Scanning confocal microscopy experiments revealed a strict ErbB2-GM3 colocalization in wild-type cells. In addition, analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2, epidermal growth factor receptor (EGFR) and Shc-p66 (proteins correlated with the ErbB2 signal transduction pathway) were preferentially enriched in lipid rafts together with gangliosides. Blocking of endogenous ganglioside synthesis by (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP) induced a drastic cell-surface redistribution of ErbB2, EGFR and Shc-p66, within the Triton-soluble fractions, as revealed by linear sucrose-gradient analysis. This redistribution was partially reverted when exogenous GM3 was added to ganglioside-depleted HC11 cells. The results point out the key role of ganglioside GM3 in retaining ErbB2 and signal-transduction-correlated proteins in lipid rafts.     

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.     

Differential distribution of gangliosides in adult rat ovary during the oestrous cycle

Gangliosides are ubiquitous membrane components in mammaliancells and are suggested to play important roles in various cellfunctions, such as cell-cell recognition, differentiation andtransmembrane signalling. Rat ovary contained GM3, GD3 and GD1aas major gangliosides, and GM1 as a minor one. In order to studytheir distribution in the rat ovary and its possible changesduring the oestrous cycle, frozen sections were stained withspecific monoclonal antibodies against 11 ganglio-series gangliosidesincluding those mentioned above. GM3, GM1 and GD1a were expressedin a spatiotemporally different manner during the oestrous cycle,but GD3 and other gangliosides were not immunohistochemicallydetected. In primary and secondary follicles, GM3, GM1 and GDlawere expressed in theca cells, but not in granulosa cells. Theoocyte in primary, but not secondary, follicles was positiveto the anti-GD1a antibody. In Graafian follicles, GM1 and GD1awere similarly expressed as in secondary follicles, however,the expression of GM3 spread gradually from theca cells to granulosacells. In early Graafian follicles, only GM3 was expressed toa detectable extent from the outer part of the granulosa layerto the inner part Shortly before ovulation, all granulosa cellsand cumulus cells became positive to anti-GM3 antibody. Afterovulation, differential distribution of GM3, GM1 and GD1a wasalso observed in luteal cells. GD1a was localized in thread-likestructures, while GM3 was distributed throughout the cytoplasm,but not in the nucleus. GM1 was localized only in the plasmamembrane and/or its close vicinity. Other ganglio-series gangliosides,including GD3, were not detected to an appreciable extent inthe ovaries by immunohistochemistry ganglioside oestrous cycle rat ovary     

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.     

In vivo modulation of epidermal growth factor receptor phosphorylation in mice expressing different gangliosides

We studied in this work the in vivo phosphorylation of the epidermal growth factor receptor (EGFr) in skin from knockout mice lacking different ganglioside glycosyltransferases. Results show an enhancement of EGFr phosphorylation, after EGF stimulation, in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice as compared with wild-type and Sial-T1 knockout mice. Qualitative analysis of ganglioside composition in mice skin suggest that the increase of EGFr phosphorylation observed in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice in response to EGF might not be primary attributed to the expression of GD3 or a-series gangliosides in mice skin. These studies provide, for the first time, an approach for studying the molecular mechanisms involved in the in vivo regulation of EGFr function by gangliosides.