Cell surface mobility of a bacterial R-form glycolipid, mR595, after binding to rat cells, and its effect on the mobility of concanavalin A receptors

Binding of small amounts of glycolipid mR595 to rat cells, followed by sequential incubation of cells at 37 °C with rabbit anti-glycolipid mR595 and fluorescein-conjugated sheep anti-rabbit γ-globulin antisera results in the localization of fluorescence at one pole of the cell surface (capping). Binding of higher amounts of glycolipid mR595 to cells not only inhibits formation of glycolipid caps but those of the ConA receptor-fluorescent ConA complex as well. Glycolipid mR595 binding does not alter [³H]ConA binding to cells but cell agglutination by ConA is inhibited in a competitive fashion. Binding of small amounts of ConA to cells does not affect glycolipid capping. Colchicine and cytochalasin B (CB) treatment of cells inhibits glycolipid cap formation.     

Regulation of glycolipid synthesis during differentiation of clonal murine muscle cells

The two clonal murine muscle cell lines G7 and G8, originally derived from the M114 line [20], represent unique models for comparative studies of myogenesis. Glycolipid synthesis was examined during differentiation using [³H]-galactose and [³H]-glucosamine as precursors. Upon G7 contact glucosylceramide labeling increased and nLcOse₅Cer labeling stopped. During membrane fusion, glucosylceramide labeling stopped and lactosylceramide became the major synthetic product. G8 cells presented a different pattern, with increased labeling of GbOse₃Cer during myogenesis. The major ganglioside synthesized by both myoblasts was GM3, and more complex structures were observed following completion of myotube formation. Total glycopeptide labeling increased when G8 myoblasts fused and remained elevated in myotubes, whereas no differences during fusion of G7 cells were noted. Upon comparison of the two clonal lines, the only consistent observation was a significant increase in the synthesis of total gangliosides and neutral glycolipid during cell contact and membrane fusion (p < 0.02). The results suggest that changes in the synthesis of specific glycolipid structures during myogenesis are unique to each muscle cell line examined. However, transient increases in synthesis of total myoblast gangliosides and neutral glycolipids may be a more general phenomenon, possibly by curbing proliferation or by altering myoblast membrane fluidity characteristics during differentiation.Abbreviations MG6 VI³NeuAc-V⁴Gal-IV³GlcNAc-nLcOse₄Cer - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - Gal galactose - GlcNH glucosamine - PBS phosphate buffered saline - CK creatine kinase     

Binding of Salmonella minnesota R-form glycolipid mR595 to rat fibroblasts and its effect on cell metabolism and cell behaviour

Trypsinized normal rat embryo fibroblasts and untrypsinized and trypsinized transformed rat fibroblasts have two orders of binding sites for bacterial glycolipid mR595. The high order sites fix 1–3 μg glycolipid mR595/10⁵ cells and those of the low order fix about 6 μg glycolipid mR595/10⁶ cells. Ca⁺⁺ is required for the low order glycolipid mR595 binding to be trypsinized but not to the untrypsinized transformed rat fibroblasts. The low order binding but not to the untrypsinized transformed rat fibroblasts. The low order binding is temperature dependent with the transition temperature lying between 25 and 37°C. Exogenously added ganglioside and glycoproteins contained in the fetal calf serum do not inhibit fixation of glycolipid mR595. Only β-lipoprotein at high concentrations is slightly inhibitory. Glycolipid mR595 fixation to transformed fibroblast does not alter their morphology and appears to slightly improve cell attachment to substratum. Glycolipid mR595 fixation results in a lengthening of the S-phase of the cell cycle and a reduction in 2-deoxyglucose uptake. Uptake of inorganic phosphate is not affected. Inhibition of phospholipid synthesis is observed in mR595 fixed fibroblasts whereas synthesis of cell surface glycoproteins and the content of cellular gangliosides is not affected.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Exposure of major glycolipids in human Pk and p erythrocytes

The exposure of glycolipids in P^k and p red cells was studied by the galactose oxidase/ NaB²H₄ and galactose oxidase/NaB³H₄ surface labeling techniques. The major glycolipid in P^k cells, ceramide trihexoside was efficiently labeled when high amounts of galactose oxidase were used. In contrast, the major glycolipid in p cells, ceramide dihexoside was not oxidized by galactose oxidase. However, minor components with longer oligosaccharide chains were readily labeled in p cells by the galactose oxidase/NaB³H₄ method.Abbreviations CDH ceramide dihexoside, LacCer - CTH ceramide trihexoside, GbOse₃Cer     

Role of basal lamina in Schwann cell glycolipid biosynthesis

Schwann cells that are deprived of axonal contact switch their glycolipid metabolic pathway from primarily galactocerebroside (GalCe) synthesis to the formation of glucocerebroside (GlcCe) and its homologs. The removal of axonal influence has a dual effect on Schwann cell phenotype; they lose the ability to assemble both myelin and basement membrane. To determine whether a loss of basement membrane directly affects glycolipid expression, we have examined lipid biosynthesis in Schwann cells which were allowed to interact with axons of dorsal root ganglion neurons but which were deprived of the ability to assemble basal lamina. These Schwann cells resemble those from myelinating nerve in that they synthesize a large amount of galactohydroxycerebroside. This suggests that axon contact, even in the absence of basement membrane, is sufficient to induce the GalCe metabolic pathway.Abbreviations DRG dorsal root ganglia - GalCe galactocerebroside - GalCe-OH galactohydroxycerebroside - GlcCe glucocerebroside - GL-2 lactosylceramide - GL-3 trihexosylceramide - GL-4 tetrahexosylceramide - HPTLC high-performance thin-layer chromatography - MGDG monogalactosyl diacylglycerol - NL non-polar lipids - PC phosphatidylcholine - Su sulfatide - Su-OH hydroxysulfatide     

Accessibility of plasma membrane sialoglycoconjugates of novikoff tumor cells to exogenous neuraminidase and proteases

Plasma membrane glycoconjugates of Novikoff tumor cells were radioactively labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄ Submission of the radioactively labeled glycoconjugates to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by fluorography revealed the presence of at least ten major glycoproteins and a glycolipid fraction. The glycolipid fraction contained 34% of the cell-surface radioactive label. Pretreatment of cells with neuraminidase from Vibrio cholerae reduced radioactive labeling of the glycoproteins by 71% and that of the glycolipids by 39%. Sequential treatment of cells with papain and neuraminidase further reduced radioactive labeling of the glycolipid fraction, indicating that resistance of this fraction to the hydrolytic action of neuraminidase was determined, at least in part, by steric factors. Incubation of cells with papain resulted in extensive degradation of most of the radioactively labeled glycoproteins with the exception of a subset of glycoproteins having apparent molecular weights of $\text{48 000 ± 5000}$. Trypsin was more selective, degrading three glycoproteins having apparent molecular weights of 200 000, 140 000 and 37 000.     

Glucolipid Synthesis in Acanthamoeba castellanii*

SYNOPSIS. Cell-free preparations of Acanthamoeba castellanii trophozoites transfer glucose from UDP-[U-¹⁴C]glucose to a chloroform-soluble form. This radioactive material has been isolated by thin-layer chromatography; it contains an alkali-labile and an alkali-stable (unsaponifiable) component. Treatment of the enzymic product with 0.1 N KOH for 15 min at 0 C or 20 C releases radioactivity into the aqueous phase as glucose. During this treatment, 30–60% of the original glycolipid remains chloroform-soluble. It is considered to be an alkali-stable glycolipid because no further loss of radioactivity occurs during an additional 45-min of treatment with 0.1 N KOH. During incubation with 0.1 N HCI at 100 C glucose is released quantitatively from both the untreated glycolipid and the alkali-stable glycolipid with a half-time of 6 min. Glycolipid formation is inhibited by UDP and is reversible; extracts catalyze the formation of UDP-glucose from the alkali-stable glucolipid and UDP. The chemical and physical properties of the alkali-stable glycolipid are consistent with a glucosyl phosphoryl polyprenol structure. Extracts prepared from cysts catalyze the formation of glycolipids aiso, but the glucosyltransferase activity/cell decreases during the course of encystment. Radioactivity is incorporated into the fraction insoluble in chloroform-methanol-water (1:1:1:) during these incubations when UDP-[U-¹⁴C]glucose or [¹⁴C]glycolipid is the substrate.     

Structural analysis by mass spectrometry and NMR spectroscopy of the glycolipid sulfate from Halobacterium salinarium and a note on its possible function

The major glycolipid sulfate of the extreme halophile Halobacterium salinarium was isolated and characterised mainly by mass spectrometry and NMR spectroscopy. The mass spectrum of the permethylated, desulfated and trimethylsilylated derivative showed the molecule to be a trihexosyl glycerol C₂₀-diether with the sulfate group on the terminal hexose. A 3-position of the sulfate was indicated by the mass spectrum obtained after acetylation and trimethylsilylation (solvolysis of sulfate and replacement by a trimethylsilyl group). The NMR spectrum of the desulfated permethylated glycolipid gave conclusive evidence for the presence of one β and two α anomeric protons. With the knowledge of degradation data it was possible to assign the β signal to galactose (terminal hexone), and the α signals to glucose and mannose. These data together make it likely that the glycolipid sulfate is identical in structure with the glycolipid from Halobacterium cutirubrum characterised previously (M. Kates and P.W. Deroo, J. Lipid Res., 14 (1973) 438).On the basis of a suggested function of cerebroside sulfate of animal origin (identical polar end with the bacterial glycolipid: β-galactopyranose-3-sulfate) and the present knowledge of ion transport in Halobacteria, it is proposed that the bacterial glycolipid may function as a selective K⁺ receptor for the K⁺ transport from a high-Na⁺ and low-K⁺ outside medium.     

The autophagy machinery restrains iNKT cell activation through CD1D1 internalization

Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4⁺ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4⁺ T cell stimulation.     

Leb-active glycolipid in human plasma: Measurement by radioimmunoassay

A radioimmunoassay that measures Le^b-active glycolipids in human plasma has been developed using antiserum from a goat immunized with a Le^b blood group hapten, lacto-N-difucohexaose I, conjugated to polylysine. Binding by the antiserum of lacto-N-difucohexaose I conjugated to ¹²⁵I-labeled bovine serum albumin is specifically inhibited by Le^b-active ceramide hexasaccharide. Plasma levels of the glycolipid are quantitated by comparing the inhibitory activity of plasma with that of the purified Le^b-active glycolipid. Plasma samples from 35 blood group O Le(a ? b +) individuals contain Le^b-active ceramide hexasaccharide at an average concentration of 0.9 μg/ml (range: 0.2 to 2.5 μg/ml); no Le^b-active glycolipid (less than 0.02 μg/ml) could be detected in plasma from blood group O Le(a + b?) or O Le(a? b?) individuals. Plasma from A₁ Le(a ? b+) individuals contains less Le^b-active glycolipid than plasma from A₂ Le(a? b+) individuals: its level in 19 samples of A, Le(a? b+) plasma averages 0.2 μg/ml (range: 0.1 to 0.45 μg/ml), and its level in 9 samples of A₂ Le(a? b+) plasma averages 1.1 μg/ml (range 0.8 to 1.3 μg/ml). About one-third of the total Le^b-active glycolipid in whole blood is associated with erythrocytes and the rest is found in plasma.     

Forssman Antigen exposed on Surface Membrane after Viral Transformation

IN the course of transformation by oncogenic agents, cells not only synthesize new surface membrane components but also their membrane structure is altered. This can lead to the exposure of components already existing in the plasma membrane of the parent cell¹. The Forssman antigen, a glycolipid with N-acetylgalactosamine as a terminal determinant², was not found on the surface of baby hamster kidney cells in tissue culture (BHK). Cells transformed by oncogenic viruses such as polyoma virus (Py-BHK) or Rous sarcoma virus displayed Forssman activity on their surfaces^3,4.     

Isolation and characterization of a glycolipid from Dictyostelium discoideum

A glycolipid was isolated from a lipid extract of the cellular slime mold Dictyostelium discoideum and characterized. From the results of analyses by thin-layer chromatography and infrared spectrometry, it was identified as a steryl glycoside. The steryl glycoside was further analyzed by gas-liquid chromatography/mass spectrometry as a trimethylsilyl ether derivative, and its quantitative and qualitative changes during the development of D. discoideum were examined. Δ²²-Stigmastenyl-d-glucoside was the major constituent of the steryl glycoside and comprised more than 90% of the total steryl glycoside fraction in cells at all stages of development. The content of the steryl glycoside was higher in vegetative-stage cells, late aggregation-stage cells, and 1-day sorocarps than in cells of other stages. The glycolipid fraction was often contaminated by a lipid which was also isolated and identified as a ceramide containing 2-hydroxy fatty acids and 4D-hydroxysphinganine.     

Production of crystalline surface-active glycolipids by a strain of torulopsis apicola

The conditions for the production of extracellular glycolipid with Torulopsis apicola IMET 43747 have been investigated. Different culture conditions resulted in the production of either water-soluble or crude crystalline glycolipids. Growth was always accompanied by a strong decrease of the pH-value in the reaction medium. Cultivation at pH-value below 2 yielded the water-soluble product. The addition of either sodium citrate or sodium hydroxide to correct the pH-value to 3, resulted in the formation of large amounts of crystalline glycolipids. Depending on the kind of carbon source and its relative concentration, the product concentration was 5–90 g 1⁻¹ with maximal yields of 0.46 g g⁻¹ (product per substrate) after growth on a mixture of plant oils and glucose. The crude crystalline glycolipid mixture can be separated from the culture medium by filtration. It is composed of 80% of one major crystalline glycolipid and different minor compounds. Purification by liquid chromatography on silica gel yields the pure compound. This main product is a nonionic glycolipid with remarkable interfacial activities.     

Isolation and purification of Lea blood-group active and related glycolipids from human plasma of blood-group A Lea individuals

From 8 1 of human plasma of blood-group A Le^a nonsecretors three different Le^a blood-group active ceramide pentasaccharides (a total of 4.65 mg) have been isolated, all revealing glucose, galactose, N-acetylglucosamine and fucose in molar ratios of 1 : 2 : 1 : 1 as determined by gas liquid chromatography. A fourth blood-group active fraction (0.72 mg) represents a mixture of a Le^a active ceramide pentasaccharide and an A active ceramide hexasaccharide (molar ratio 7.7 : 2.3 as calculated from the content of different aminosugars). Additionally, two different globosides, two different hematosides and a new N-acetylglucosamine containing ceramide tetrasaccharide were obtained. All 9 glycolipid fractions demonstrated homogeneity in analytical high performance thin layer chromatography (HPTLC) using 4 different solvent systems. 0.2 μg of each Le^a active glycolipid completely inhibited the agglutination of O Le(a + b ?) erythrocytes by 50 μl of 4 hemagglutinating units of caprine anti Le^a serum. At least 0.04 μg of each Le^a antigen are sufficient for incubation to convert 9 × 10⁷ O Le(a?b?) erythrocytes into Le^a-positive cells. Mainly due to the relatively low content of the blood-group A glycolipid in plasma (0.17 mg/8 1), previously negative erythrocytes readily become agglutinable by anti Le^a sera and not by anti A sera after incubation with appropriate plasma.     

Meiothermus rufus sp. nov., a new slightly thermophilic red-pigmented species and emended description of the genus Meiothermus

Four red-pigmented isolates, with optimum growth temperatures of approximately 55–60 °C and an optimum pH for growth between 7.5 and 8.5, were recovered from hot springs in Central France. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented a new species of the genus Meiothermus. The new isolates could be distinguished from other strains of the species of the genus Meiothermus primarily by the glycolipid profile and fatty acid composition because these organisms lacked the hydroxy fatty acids and the glycolipid variant GL-1a found in all other isolates of the species of Meiothermus examined. On the basis of the results presented here we propose the name Meiothermus rufus for the new species, which is represented by strains CAL-4^T (=DSM 22234^T=LMG 24878^T) and CAL-12 (=DSM 22235=LMG 24879). We also propose emending the genus Meiothermus to include strains that have only one glycolipid instead of two glycolipid variants.     

Characterization of a Lactobacillus gasseri JCM 1131T Lipoteichoic Acid with a Novel Glycolipid Anchor Structure

We determined the chemical structure of lipoteichoic acid (LTA) from Lactobacillus gasseri JCM 1131^T. The repeating unit was comprised of glycerolphosphate and 2-alanylglycerolphosphate. The glycolipid anchor was tetrahexosylglycerol with two or three acyl groups. To our knowledge, this is the first demonstration of a tetrahexose structure in an LTA glycolipid anchor.     

Improving Mycobacterium bovis Bacillus Calmette-Guèrin as a Vaccine Delivery Vector for Viral Antigens by Incorporation of Glycolipid Activators of NKT Cells

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8⁺ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8⁺ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8⁺ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8⁺ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8⁺ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.     

GROWTH OF NORMAL AND TRANSFORMED RAT EMBRYO FIBROBLASTS : Effects of Glycolipids from Salmonella minnesota R Mutants

Addition of glycolipids obtained from Salmonella minnesota R mutants to normal, spontaneously transformed, and SV40-transformed rat embryo fibroblasts in culture results in an inhibition of growth of transformed cells but not of normal cells. In the presence of the glycolipid with the smallest carbohydrate chain length, spontaneously transformed cells stop growing when they reach confluency. Inhibition of growth of transformed cells is inversely related to the chain length of the core sugars. Glycolipid mR595 is shown to bind with the cell membrane of transformed cells and elicits an augmentation in the intracellular level of cyclic AMP. Normal cells bind relatively less glycolipid mR595 and show a lower percent of increase in cyclic AMP due to glycolipid mR595 than do transformed cells.     

A new type of blood group B active glycosphingolipid in rat bone marrow cells. Occurrence of the glycolipid in rat immunocytes and ascites hepatoma

A major glycosphingolipid in rat bone marrow cells was purified, and its structure was studied. The glycolipid was found to exhibit blood group B activity by the hemagglutination inhibition test. The structure was determined to be (formula; see text) by studies of nuclear magnetic resonance, sequential hydrolysis by exoglycosidases, linkage analysis of methylated sugars by gas chromatography-mass spectrometry, and immunological tests. The blood group B active glycolipid was detected not only in the bone marrow cells but also in spleen, thymus, and rat ascites hepatoma AH 7974F cells. Besides the glycolipid, gangliotriaosylceramide, gangliotetraosylceramide, and fucogangliotetraosylceramide were commonly detected in these cells. The similarity between the glycolipid species on the cell surfaces of the immunocytes and the tumor cells is discussed with the respect to an escape mechanism of the tumor cells from the immunosurveillance system.