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PubMed1990

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts.

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.

Animals↗

PubMed1979

Glycosphingolipids of subcellular fractions from normal rat liver and Morris hepatoma 5123TC.

Neutral glycosphingolipids and gangliosides were quantified in lipid extracts from plasma membranes, mitochondria, microsomes, and nuclei isolated from normal rat liver and Morris hepatoma 5123TC. Results showed a higher content of glycosphingolipids, especially gangliosides, in hepatomas and differences in the distribution of glycosphingolipids among subcellular fractions. Differences in the glycosphingolipid composition of the hepatoma, namely, the absence of trisialogangliosides and an increase in the lower molecular weight gangliosides, reflected an altered metabolism of glycosphingolipids in this tumor. The results indicated that changes in membrane glycosphingolipids were not restricted to the cell surfaces of malignant cells, inasmuch as intracellular membrane fractions also exhibited altered glycosphingolipid profiles.

Animals↗

PubMed2005

The glycosphingolipid, lactosylceramide, regulates beta1-integrin clustering and endocytosis.

Glycosphingolipids are known to play roles in integrin-mediated cell adhesion and migration; however, the mechanisms by which glycosphingolipids affect integrins are unknown. Here, we show that addition of the glycosphingolipid, C8-lactosylceramide (C8-LacCer), or free cholesterol to human fibroblasts at 10 degrees C causes the formation of glycosphingolipid-enriched plasma membrane domains as shown by visualizing a fluorescent glycosphingolipid probe, BODIPY-LacCer, incorporated into the plasma membrane of living cells. Addition of C8-LacCer or cholesterol to cells initiated the clustering of beta1-integrins within these glycosphingolipid-enriched domains and the activation of the beta1-integrins as assessed using a HUTS antibody that only binds activated integrin. On warming to 37 degrees C, beta1-integrins were rapidly internalized via caveolar endocytosis in cells treated with C8-LacCer or cholesterol, whereas little beta1-integrin was endocytosed in untreated fibroblasts. Incubation of cells with C8-LacCer or cholesterol followed by warm-up caused src activation, a reorganization of the actin cytoskeleton, translocation of RhoA GTPase away from the plasma membrane as visualized using total internal reflection fluorescence microscopy, and transient cell detachment. These studies show that LacCer can regulate integrin function both by modulating integrin clustering in microdomains and by regulating integrin endocytosis via caveolae. Our findings suggest the possibility that aberrant levels of glycosphingolipids found in cancer cells may influence cell attachment events by direct effects on integrin clustering and internalization.

Caveolae↗

PubMed1975

Glycosphingolipids of human KB cells grown in monolayer, suspension, and synchronized cultures.

Studies have been carried out on the glycosphingolipids of human KB cells grown in monolayer and suspension culture, and by synchronization of the latter with a double thymidine (2mM) block. Glycosphingolipids were identified tentatively by thin layer chromatography, gas-liquid chromatography, and combined gas-liquid chromatography and mass spectrometry. The predominant gangliosides in the these cells were AcNeu-Gal-Glc-Cer and AcNeu-Gal-GalNAc-Gal-(AcNeu). Glc-Cer. Theprincipal neutral glycosphingolipids were Glc-Cer, Gal-Glc-Cer, Gal-Gal-Glc-Cer, and GalNAc-Gal-Gal-Glc-Cer. Incubation of KB cells (grown in monolayer and subsequently in suspension culture) for 48 hours with D-[1-14Clgalactose resulted in appreciable incorporation of radioactivity into all of the principal glycosphingolipids of these cells. These experiments confirmed that KB cells are capable of synthesizing their constituent glycosphingolipids. KB cells grown in suspension culture showed A 2- to 3-fold increase inthe concentration of Glc-Cer, Gal-Glc-Cer, GalNAc-Gal-Gal-Glc-Cer, and AcNeu-Gal1NAc-Gal-Gal-Glc-Cer, and AcNeu-Gal-Ga1NAcGal-(AcNeu)-Glc-Cer. Thus, the occurrence of tissue culture-dependent changes in the level of glycosphingolipids is demonstrated. Perhaps messages governing the synthesis of glycosphingolipids are translated earlier in thecell cycle under certain conditions of growth and are affected by cell-cell contact and cell adhesion.

Carbon Radioisotopes↗

PubMed2000

Modifications of glycosphingolipid profile and synthesis in normal rat fibroblasts and in syngeneic neoplastic cells at different subculture stages.

Glycosphingolipids are plasma membrane macromolecules involved in diversified recognition functions on the cell surface resulting in modulation of cell adhesion and differentiation. As the in vitro cellular system of the neoplastic cell line SGS/4A and syngeneic normal fibroblasts (FG) represents a useful tool for studies on molecular mechanisms regulating cell adhesion, neoplastic transformation and cellular ageing, we studied the changes of glycosphingolipid and of the enzymes involved in their metabolism in both cultured cells at different subculture stages. The FG subculture progression induces a drastic decrease of total glycosphingolipid content with consistent alterations in the molecular composition. In particular, a significant decrease of GM(3), a slight increase of GD(1a), the disappearance of 'b'-series gangliosides and the drastic reduction of triosylceramides were observed. On the contrary, the increasing number of SGS/4A subcultures, characterized by a specific and different glycosphingolipid composition as compared with FG cells, does not cause modifications. Although glycosyltransferase activity levels quite well parallel the glycosphingolipid patterns and can account for the noted variations, the mRNA expression analysis of two glycosyltransferases suggests that the in vitro cell ageing of normal rat fibroblasts causes drastic changes in the glycosphingolipid profile through the regulation, at either the transcriptional or post-translational level, of some biosynthetic enzymes.

Animals↗

PubMed1993

Fucosylated glycosphingolipids of human myeloid cells.

Our efforts to determine the carbohydrate binding specificity of two myeloid-specific monoclonal antibodies (VIM-1 and VIM-10) resulted in the purification of three fucosylated glycosphingolipids from human chronic myelogenous leukemia cells. After repeated high-performance liquid chromatographic separations, two forms of fucosylated glycosphingolipids were resolved. VIM-1 and VIM-10 were found to bind to the heptaosylceramide Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer with the Le(x) (Lewis X) epitope, but not to either the hexaosylceramide Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1-Cer or the octaosylceramide Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer, each with the Le(y) (Lewis Y) epitope. The latter two glycosphingolipids are the first Le(y) antigens to be purified from human leukocytes and structurally characterized. Binding studies with a range of glycosphingolipids from myeloid cells and other biological sources demonstrated that VIM-1 and VIM-10 bind to Le(x) glycosphingolipids with five or more sugar residues, but not to a glycosphingolipid (III3Fuc-nLc6Cer) with an internal Le(x) trisaccharide.

Carbohydrate Conformation↗

PubMed2001

Interaction of GM2 activator protein with glycosphingolipids.

GM2 activator protein is a protein cofactor that stimulates the hydrolysis of the GalNAc and the NeuAc in GM2 by beta-hexosaminidase A and sialidase, respectively. To understand the mechanism of action of GM2 activator, the interaction of this protein with GM2 and/or beta-hexosaminidase A has been the subject of interest since the purified GM2 activator became available. Numerous techniques including ultracentrifugation, isoelectric a focusing, polyacrylamide gel electrophoresis, gel filtration, thin layer chromatogram overlay, and fluorescence dequenching assay have been used to investigate the binding and the affinity of GM2 activator to various glycosphingolipids. It has been generally accepted that GM2 activator must have a very weak binding with the enzyme, because they can be easily separated from each other by gel filtration. Therefore, the interaction of GM2 and GM2 activator has been the focus for most of he study. Although preferential association of GM2 activator with GM2 was detected by some methods, GM2 activator was found also to bind other glycosphingolipids. Isolation of the specific complex that consists of only GM2 activator and GM2 from incubation mixture containing the activator protein and mixed glycosphingolipids has not been successfully carried out. Ultracentrifugation and gel-filtration are the mildest methods for the isolation of the complexes. However, these methods do not separate the complexes formed by specific interaction from that formed by non-specific association. In fluorescence dequenching assay, the attempt to isolate the complex of R18 lipid probe with GM2 activator was also not successful. Since GM2 activator and glycosphingolipids contain hydrophobic domains in their molecules, the non-specific hydrophobic interactions between the two components can greatly interfere with the isolation of true functional complexes. Among the reported methods, thin layer chromatography overlay and the assay based on the inhibition of fluorescence dequenching by various glycosphingolipids are more informative than the others on the binding between GM2 activator and the carbohydrate head groups of glycosphingolipids.

Binding Sites↗

PubMed1993

Developmental changes in neutral glycosphingolipids of mouse placenta.

The mammalian placenta is a unique organ for the study of developmental changes. Placentas of laboratory animals such as the mouse allow for the determination of the exact stage of pregnancy, which cannot be achieved with human placenta. In this study, neutral glycosphingolipids were isolated from mouse (inbred strain C57BL/6) placentas, from day 10 to day 18 of gestation, and were separated by high performance thin layer chromatography. Densitometric measurements after orcinol staining showed, at day 10 of gestation, the presence of mono-, tetra-, tri- and dihexosylceramide in decreasing quantities, as well as four unidentified spots. On day 12, the glycosphingolipid composition changed with the disappearance of the unidentified spots and the appearance of an orcinol positive migrating similarly to the Forssman antigen; no further changes occurred between days 12 and 18 of gestation. The identity of the Forssman-like glycosphingolipid with the Forssman antigen was established by binding of 125I labelled Helix pomatia agglutinin (alpha-GalNAc specific) to glycosphingolipids separated on high performance thin layer chromatography plates, and by the reaction of the isolated glycosphingolipid with a monoclonal anti-Forssman antibody. The appearance of the Forssman antigen at day 12 of gestation coincided with the day of final maturation of the mouse placenta and subsequent cessation of growth, suggesting a possible role of the glycosphingolipid during embryonic development.

Animals↗

PubMed1976

[Plasma glycosphingolipids: concentration and distribution in different types of hyperlipoproteinemia (author's transl)].

There are mainly four neutral glycosphingolipids in human blood plasma: Monohexosyl, dihexosyl, trihexosyl and tetrahexosyl ceramide. In patients with hyperlipoproteinemia (hlp) type IIa (n = 10), type IIb (n = 9), type IV (n = 24), and type V (n = 11) all four fractions of plasma glycosphingolipids were elevated compared to healthy subjects (n = 23). In all types of hlp monohexosyl ceramides were significantly augmented (p less than 1%). Trihexosyl and tetrahexosyl ceramides demonstrated only in single types statistically striking elevation. There was no significant elevation of dihexosyl ceramides. In the VLDL the distribution of all glycosphingolipids showed increase in hlp type IV and type IIb. In the LDL the distribution showed increase in type IIa. There exists a strong correlation with the elevated prae-beta-lipoproteins respectively beta-lipoproteins. It is concluded that elevation of glycosphingolipid levels in plasma is a metabolic consequence of hyperlipoproteinemia. It is considered that a part of plasma glycosphingolipids is de novo synthesized by the liver like VLDL. The role of plasma glycosphingolipids in atherogenesis is discussed.

Female↗

PubMed1988

Dexamethasone-induced alterations in the glycosphingolipids of rat kidney.

To determine whether glucocorticoids would influence the glycosphingolipid composition of the rat kidney, male albino rats of the Sherman strain were subcutaneously administered dexamethasone (100 micrograms/100 g body wt/day) or diluent for four days. The compositions of ceramide and of acidic and neutral glycosphingolipids of the kidneys of these animals were then examined and compared. The results demonstrated that dexamethasone administration: 1) increased the content of ceramide and of acidic and neutral glycosphingolipids in kidney; 2) increased the relative percentage of globotriaosyl- and globotetraosyl-ceramide, but decreased the relative percentages of glucosylceramide; 3) decreased the relative percentages of GM3 and increased other gangliosides; 4) increased the relative percentages of N-glycolylneuraminic acid in GM3; 5) did not appear to influence significantly the long-chain bases of the major glycosphingolipids; and 6) altered the relative percentages and chain length of the hydroxy and nonhydroxy fatty acids of the major acidic and neutral glycosphingolipids in this tissue. The data show that dexamethasone administration induces quantitative and qualitative changes in the glycosphingolipids of the rat kidney.

Animals↗

PubMed1985

Thermotropic behavior of binary mixtures of dipalmitoylphosphatidylcholine and glycosphingolipids in aqueous dispersions.

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-GM1, sulfatide, GM3, GM1, GD1a, GT1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1-6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-GM1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.

Calorimetry, Differential Scanning↗

PubMed1976

Localization of neutral glycosphingolipids in human plasma.

1. The localization of the neutral glycosphingolipids glucosylceramide, lactosylceramide, trihexosylceramide and globoside in human plasma was investigated. Glycosphingolipids were isolated and analysed by gas-liquid chromatography. 2. After Sephadex gel chromatography of human plasma, about 75% of the glycosphingolipids were found in the fraction containing most of the lipoproteins. 3. After fractionation of the lipoproteins by ionic precipitation, 15-25% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction, 30-45% in the low density lipoprotein fraction and 40-50% in the high density lipoprotein fraction. 4. After fractionation of lipoproteins by density-gradient ultracentrifugation, 15% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction and 85% in the low density and high density lipoprotein fractions. No glycosphingolipids could be detected in the ultracentrifugal residue which contains the bulk of the albumin.

Ceramides↗

PubMed1997

Blood group B glycosphingolipids in alpha-galactosidase deficiency (Fabry disease): influence of secretor status.

Defect in degradation of blood group B-immunoactive glycosphingolipids in Fabry disease (deficiency of lysosomal alpha-galactosidase EC 3.2.1.22) has been studied using highly sensitive and specific TLC-immunostaining analysis of urinary sediments and tonsillar tissues of blood group B patients and healthy controls, secretors and nonsecretors. The B glycolipid antigens with hexasaccharide chains were consistently found increased (25- to 100-fold) in the urinary sediments of three Fabry patients, blood group B or AB secretors. Conversely, they were absent in the urinary sediment of one blood group B nonsecretor patient. In normal secretors, B glycosphingolipids were present only in traces. Moreover, significant increase in B glycolipid antigens (8-fold) was found in the tonsillar tissue of a Fabry patient blood group B secretor. We conclude that the secretor status is responsible for increased concentration of blood group B glycosphingolipids in both urinary cells and tonsils in alpha-galactosidase deficiency. The quantity of stored B-immunoactive glycosphingolipids, however, is much lower than that of the mainly accumulated glycosphingolipid Gb(3)Cer. The results clearly indicate that active or silent Se gene, which controls synthesis of B-antigen precursors, is responsible for notable difference in B-glycosphingolipids expression in Fabry patients - secretors and nonsecretors. Whether this novel aspect may be of prognostic significance, remains to be established.

ABO Blood-Group System↗

PubMed1986

Human hepatocellular carcinoma-associated changes of glycosphingolipids detected by two monoclonal antibodies FH2 and IB9.

The changes of minor glycosphingolipids associated with human hepatocellular carcinomas were studied using monoclonal antibodies. Glycosphingolipids of twelve patients were isolated from the tumor tissues and their adjacent cirrhotic liver tissues. The immunoreactivities of glycosphingolipids with two monoclonal antibodies FH2 and IB9, that define x-structure (Gal beta 1-4[Fuc alpha 1-3]GlcNAc) and sialosyl alpha 2-6galactosyl residue, respectively, were studied by a solid phase enzyme-linked immunosorbent assay and a chromatogram immunobinding assay. X-structure was detected in the upper-phase neutral glycosphingolipids from both the cirrhotic liver tissues and the tumor tissues, but was generally expressed more strongly in the latter tissues. The thin-layer chromatography patterns of x-active glycosphingolipids were more complex in the tumor tissues. Sialosyl alpha 2-6galactosyl residue was detected in two ganglioside fractions. One ganglioside with faster migration on a thin-layer chromatogram was found in both the non-tumorous tissues and the tumor tissues. Another ganglioside with slower migration was more often detected in the tumor tissues. It was concluded that some of minor glycosphingolipids detected by monoclonal antibodies FH2 and IB9 could be the 'tumor-associated' antigens.

Antibodies, Monoclonal↗

PubMed1991

Degradation of dilauroylphosphatidylcholine by phospholipase A2 in monolayers containing glycosphingolipids.

The ability of phospholipase A2 from porcine pancreas to degrade all of the available dilauroylphosphatidylcholine in mixed monolayers with galactocerebroside, sulfatide, or ganglioside GM1 was investigated at different constant surface pressures. Under the conditions used the interfacial glycosphingolipid composition was continuously enriched as the enzyme action proceeded. The total percentage of phospholipid degradation depends on the surface pressure and on the type of glycosphingolipid. The presence of sulfatide activates the enzyme while galactocerebroside and ganglioside GM1 are inhibitory. The extent of phospholipid hydrolysis is independent of the effect of glycosphingolipids on the enzyme velocity. This is so when the latter is measured either in conditions of constant glycosphingolipid composition and zero-order kinetics [Bianco, I.D., Fidelio, G.D., & Maggio, B. (1989) Biochem. J. 258, 95-99] or under variable surface composition as in the present work. The modulation of phospholipase A2 activity by glycosphingolipids operates at two independent levels. One controls the rate of enzyme activity, and the other modulates the total extent of substrate degradation. This depends on the initial interaction of the enzyme with the interface. The glycosphingolipid effect on the activity is different depending on whether the enzyme has access to the substrate from the subphase or is already adsorbed to the lipid interface.

Animals↗

PubMed1985

Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts.

We have reported [Saito, M., Saito, M., & Rosenberg, A. (1984) Biochemistry 23, 1043-1046] that the monovalent cationic ionophore monensin reduced the incorporation of labeled galactose into oligosaccharidyl glycosphingolipids (globotriaosylceramide, globotetraosylceramide, and gangliosides) and induced a cellular accumulation of glucosyl- and lactosylceramide in cultured diploid human fibroblasts. We have undertaken further studies on the effects of monensin and made comparison with the effects of related monovalent cation transporters on plasma membrane glycosphingolipid anabolism in human fibroblasts. Our results demonstrate that ionic flux can markedly influence glycosphingolipid synthesis, and they indicate that, like glycoprotein, the sites of glycosylation of the initial, precursor glycosphingolipids are different from the sites of higher glycosylation. At a concentration of 10(-7) M, monensin induced the maximum inhibition of incorporation of labeled galactose into polyglycosyl sphingolipids: globotriaosylceramide, globotetraosylceramide, and gangliosides; increased incorporation of labeled galactose into glucosyl- and lactosylceramide was clearly evident, and their content rose measurably in the cell at concentrations of monensin as low as 10(-8) M. These effects of monensin were reversible. Incorporation of labeled galactose into higher glycosylated neutral glycosphingolipids and gangliosides slowly resumed, and the accumulated glycosylceramide diminished after removal of monensin from the culture medium. Ouabain (plasma membrane Na+,K+-ATPase inhibitor) and A23187 (Ca2+ ionophore) also caused a rapid increase in incorporation of labeled hexose into glucosylceramide and decreased its incorporation into higher neutral glycosphingolipids and into gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport, Active↗

PubMed1981

Isolation and structural characterization of human lymphocyte neutral glycosphingolipids.

The neutral glycosphingolipids of human peripheral blood lymphocytes and of the lymphoid cells from a patient with B cell chronic lymphocytic leukemia were chemically analyzed. Four neutral glycosphingolipids were chemically analyzed. Four neutral glycosphingolipids were isolated from each of these two sources and studied by gas chromatography, methylation analysis, and electron impact--desorption mass spectrometry. The results of these studies indicate that the compounds have the following structures: Glc1 leads to 1Cer Gal1 leads to 4Glc1 leads to 1Cer Gal1 leads to 4Gal1 leads to 4Glc1 leads to 1Cer GalNAc1 leads to 3Gal1 leads to 4Gal1 leads to 4Glc1 leads to 1Cer These compounds, belonging to the globo series, were the only neutral glycosphingolipids found in the lymphoid cells. The ceramide (Cer) moiety of all these compounds contained 4-sphingenine with C16:0, C24:0, and C24:1 as the major fatty acid species. There were no structural differences in the neutral glycosphingolipids of peripheral blood lymphocytes compared to those of chronic lymphocytic leukemia cells. Peripheral blood lymphocytes contained more di- than monohexosylceramide whereas the reverse was true of the chronic lymphocytic leukemia cells. The proportion of tri- and tetrahexosylceramide was less than 10% for both types of cells. The results of our analyses did not support the existence of any differences in the major neutral glycosphingolipids among T, B, and chronic lymphocytic leukemia cells.

Carbohydrate Sequence↗

PubMed2001

Differential effects of glycosphingolipids on the detergent-insolubility of the glycosylphosphatidylinositol-anchored membrane dipeptidase.

The insolubility of glycosylphosphatidylinositol (GPI)-anchored proteins in certain detergents appears to be an intrinsic property of their association with sphingolipids and cholesterol in lipid rafts. We show that the GPI-anchored protein membrane dipeptidase is localized in detergent-insoluble lipid rafts isolated from porcine kidney microvillar membranes, and that these rafts, which lack caveolin, are enriched not only in sphingomyelin and cholesterol, but also in the glycosphingolipid lactosylceramide (LacCer). Dipeptidase purified from porcine kidney was reconstituted into artificial liposomes in order to investigate the relationship between glycosphingolipids and GPI-anchored protein detergent-insolubility. Dipeptidase was insoluble in liposomes containing extremely low concentrations of LacCer. In contrast, identical concentrations of glucosylceramide or galactosylceramide failed to promote significant detergent-insolubility. Cholesterol was shown to enhance the detergent-insoluble effect of LacCer. GC-MS analysis revealed dramatic differences between the fatty acyl compositions of LacCer and those of the other glycosphingolipids. However, despite these differences, we show that the unusually marked effect of LacCer to promote the detergent-insolubility of dipeptidase cannot be singularly attributed to the fatty acyl composition of this glycosphingolipid molecule. Instead, we suggest that the ability of LacCer to confer detergent-insolubility on this GPI-anchored protein is dependent on the structure of the lipid molecule in its entirety, and that this glycosphingolipid may have an important role to play in the stabilization of lipid rafts, particularly the caveolin-free glycosphingolipid signalling domains.

Animals↗