An accumulation of galactocerebroside in kidney from mouse globoid cell leukodystrophy (twitcher)

The twitcher mouse is genetically determined mutant characterized by a deficiency of galactocerebroside beta-galactosidase. In this study, a significant accumulation of galactocerebroside was demonstrated in twitcher mouse kidney. The data suggest that mouse Krabbe's disease is not only involved in CNS, but also in visceral organs.     

In vitro biosynthesis of sialosylgalactosylceramide (G7) by mouse brain microsomes.

A sialytransferase activity which catalyzes the synthesis of sialosylgalactosylceramide (G7) from added galactocerebroside and CMP-N-acetylneuraminic acid has been demonstrated in mouse brain microsomes. The enzyme reaction shows a pH optimum of 6.3 and requires detergents. Both Mn2+ and Ca2+ inhibited the reaction, whereas Mg2+ had no effect. The apparent Km for galactocerebroside leading to G7 was estimated to be 8.7 X 10(-4) M. The same microsomal preparation also synthesized hematoside when ceramide lactoside was the glycolipid acceptor. The apparent Km for ceramide lactoside was about one-tenth that for galactocerebroside. When the preparations were partially inactivated by heat the synthesis of G7 and of hematoside was reduced at approximately the same rate. Liver appeared to have the highest activity for G7 synthesis (as well as of hematoside), followed by brain. The synthesis of B7 by mouse brain microsomes in vitro demonstrates a new pathway for brain ganglioside synthesis.     

Interaction and fusion of unilamellar vesicles containing cerebrosides and sulfatides induced by myelin basic protein

The effects of myelin basic protein on the aggregation, lipid bilayer merging, intercommunication of aqueous compartments and leakage of small unilamellar vesicles of egg phosphatidylcholine containing different proportions of galactocerebroside and sulfatide were investigated. This was performed employing light scattering, absorbance changes and fluorescence assays (resonance energy transfer, Terbium/dipicolinic acid assay and carboxyfluorescein release). The apposition of membranes rapidly induced by myelin basic protein is enhanced by sulfatide but reduced by galactocerebroside compared to vesicles of egg phosphatidylcholine alone. On the other hand, the presence of either glycosphingolipid in the membrane interferes with the induction by myelin basic protein of lipid bilayer merging, subsequent fusion and changes of the membrane permeability. Our results support an important modulation by sulfatide and galactocerebroside on the interactions among membranes induced by myelin basic protein, depending on the relative proportions of the glycosphingolipids and phosphatidylcholine.     

A Cyclic AMP Analogue Induces Synthesis of a Myelin-Specific Glycoprotein by Cultured Schwann Cells

Neonatal rat Schwann cells, cultured with agents which increase intracellular cyclic AMP, were prompted to resume synthesis of a 170,000 Mr glycoprotein which is specific to peripheral nervous system myelin and is herein referred to as P170K. We have shown previously that similar treatment induces the synthesis by Schwann cells of the myelin lipid, galactocerebroside. In contrast to P170K and galactocerebroside, syntheses of P0 and myelin basic protein were not induced. Intracellular cyclic AMP is thus likely to be a participant in the complex system regulating myelination.     

Galactocerebroside biosynthesis pathways of Mycoplasma species: an antigen triggering Guillain–Barré–Stohl syndrome

Infection by Mycoplasma pneumoniae has been identified as a preceding factor of Guillain–Barré–Stohl syndrome. The Guillain–Barré–Stohl syndrome is triggered by an immune reaction against the major glycolipids and it has been postulated that M. pneumoniae infection triggers this syndrome due to bacterial production of galactocerebroside. Here, we present an extensive comparison of 224 genome sequences from 104 Mycoplasma species to characterize the genetic determinants of galactocerebroside biosynthesis. Hidden Markov models were used to analyse glycosil transferases, leading to identification of a functional protein domain, termed M2000535 that appears in about a third of the studied genomes. This domain appears to be associated with a potential UDP-glucose epimerase, which converts UDP-glucose into UDP-galactose, a main substrate for the biosynthesis of galactocerebroside. These findings clarify the pathogenic mechanisms underlining the triggering of Guillain–Barré–Stohl syndrome by M. pneumoniae infections.     

A sensitive assay of galactocerebroside beta-galactosidase by high-performance liquid chromatography using galactosyl-N-dansyl-sphingosine as substrate

A rapid and sensitive method was devised for determining β-galactosidase activity specific for galactocerebroside. A fluorescent derivative of galactocerebroside, 1-O-galactosyl-2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was used as substrate, and the product, 2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was taken into organic solvent phase. Quantitative analysis of 2-N-dimethylaminonaphthalene-5-sulfonyl-sphingosine was carried out fluorometrically by use of high-performance liquid chromatography on silica gel column.     

Contents

The thermotropic behavior of mixtures of cholesterol with phosphatidylserine (PS) or galactocerebroside was investigated.In PS/cholesterol mixtures at low cholesterol concentrations only one peak in the thermograms is obtained. At about Xchol = 0.3 another peak at higher temperatures is also seen, this peak stems from phase transition of almost pure cholesterol phase. However, increase of Xchol results in further incorporation of cholesterol into the mixture.In galactocerebroside-cholesterol mixtures above Xchol 0.4, at least two domains differing in cholesterol content and thermotropic properties coexist.In the presence of cholesterol even at 1:1 molar ratios the phosphatidylserine or galactocerebroside are still undergoing melting.     

Application of highly purified anti-galactocerebroside antibody to the analysis of lipid-lipid or lipid-protein interactions. Significance of lipid matrix

The significance of the lipid matrix in the reaction of liposomal antigen, antibody and complement (Ag-Ab-C) was analyzed using purified anti-glactocerebroside antibody and synthetic lipids and the following results were obtained. 1. For the optimal Ag-Ab-C reaction it was necesary that galactocerebroside (galxCMH) and lecithin molecules were well dispersed by virtue of cholesterol (Chol) and that the molar ratio of cholesterol to the sum of galactocerebroside and lecithin was more than one. 2. The Ag-Ab-C reactivity changed depending upon the chain length of lecithin, and the maximal reaction was observed in the case of dilauroylphosphatidylcholine. When the fatty acyl chain of lecithin was either shorter or longer than that of dilaurosylphosphatidylcholine, the reactivity was reduced. 3. The Ag-Ab-C reactivity was increased by elongation of the fatty acyl chain of galactocerebroside and an abrupt change was found to be around the carbon number 8 to 10 of the fatty acyl chain. 4. The Ag-Ab-C reactivity was elevated by the increase in unsaturation of fatty acyl moiety. 5. There is a tendency that an increase in the charge of the lipid matrix leads to the reduction of the Ag-Ab-C reactivity. 6. The results suggest that the physicochemical properties of lipids and especially the lipid-lipid interaction in the hydrophobic region of the lipid matrix play an important role in the Ag-Ab-C reaction.     

An improved preparation of bovine brain proteolipid

An improved preparation of proteolipid from bovine brain white matter is described. The product obtained by repeated acetone precipitation is completely soluble in chloroform-methanol and has a fairly constant composition: 35% protein, 40% galactocerebroside, and about 25% phospholipid.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids.

Multilameller liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside GM1, monosialoganglioside GM2, monosialoganglioside GM3, disialoganglioside GD1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distributions of 14C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome preparation. Liver uptake up encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides, regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing GD1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing GD1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids

Multilamellar liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside G_M1, monosialoganglioside G_M2, monosialoganglioside G_M3, disialoganglioside G_D1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distribution of ¹⁴C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome prepartion. Liver uptake of encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from the liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing G_D1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing G_D1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Use of fluorescence-labelled cerebroside as a substrate for galactosylceramidase of human skin fibroblasts

Possible use of the fluorescently labelled cerebroside, 1-O-(beta-D-galactosyl)-2-N-[6-(2-antroyl)hexanoyl]-4-sphingeni n (Gal-A-sphingenin) as a substrate for galactosylceramidase (GC) from human skin fibroblasts was investigated. Studies involving TLC and fluorimetric methods revealed enzymatic splitting of Gal-A-sphingenin whose degree correlated with the amount of the enzyme and incubation time. Some kinetic parameters of GC were determined, using Gal-A-sphingenin as substrate. It was shown that the values of specific activity of GC (1.6 nmol/mg protein/hr) and Km (0.025 mM) for Gal-A-sphingenin agree well with the corresponding values obtained with the use of the galactocerebroside as a natural substrate. In experiments with mixtures of Gal-A-sphingening and galactocerebroside used as substrates in different molar ratios, it was shown that the enzyme splits each of the substrates with equal velocity. The results of experiments with the enzyme samples from skin fibroblasts of healthy individuals and of patients with GM1-gangliosidosis (GM1-beta-D-galactosidase deficiency) suggest that Gal-A-sphingenin is a specific substrate for GC.     

Temporal and Quantitative Expression of the Myelin-Associated Lipids,Ethanolamine Plasmalogen,Galactocerebroside, and Sulfatide,in the Differentiating CG-4 Glial Cell Line

We determined the expression of three myelin-typical lipids in the continuous CG-4 glial cell line of oligodendrocyte progenitor cells, as the cells differentiated into oligodendrocytes. On 6 different days during the first 9 days of oligodendrocyte development, cells were labeled for 24 h with [³H]ethanolamine to label ethanolamine plasmalogens or with [³H]galactose to label the galactocerebroside and sulfogalactocerebroside; and the amount of labeled lipid expressed on each day was determined. Each labeled lipid was expressed with its own specific time course and in a defined amount on each day of differentiation. Increased labeling of plasmalogens and sulfogalactocerebroside started at early developmental stages, and increased labeling of galactocerebroside started at later stages. The results indicate that the differentiating CG-4 cell line provides a valuable system to investigate factors affecting the early time course of myelin-lipid expression and the amounts expressed.     

GALACTOCEREBROSIDE SULFOTRANSFERASE: FURTHER CHARACTERIZATION OF THE ENZYME FROM RAT BRAIN

Abstract— Myelin has an unusual lipid composition, being particularly rich in sulfatide. This lipid is synthesized by the transfer of sulfate from phosphoadenosine phosphosulfate to galactocerebroside, catalyzed by galactocerebroside sulfotransferase. This paper describes a sensitive assay for the sulfotransferase (capable of measuring activity in as little as 10 μg of extracted rat brain protein) so that this enzyme can be readily investigated in isolated cells, or the small amounts of tissue available in developing animals. Both manganase (20 m m ) and thiol reagents were required for optimal activity. This assay was used to monitor the purification of the sulfotransferase from rat brain. Extraction of the enzyme from crude homogenates required the nonionic detergent, Triton X-100, at pH 7–7.5. Removal of Triton X-100 from the extracted enzyme resulted in a soluble but less active enzyme, the activity of which could then be restored with detergents. Stability of the detergent-extracted enzyme was investigated, and even at —40°C there was a 20% loss of activity over 10 days. By standard procedures 500-fold purification of the enzyme has been achieved.     

Inhibitory effect of liposomes containing sulfatide or cholesterol sulfate on syncytium formation induced by bovine immunodeficiency virus-infected cells

The effect of galactocerebroside 3'-sulfate (sulfatide) or cholesterol sulfate on syncytium formation induced by bovine immunodeficiency virus (BIV)-infected cells was investigated in vitro. Sulfatide was purified from bovine brain and incorporated in liposomes which were composed of egg phosphatidylcholine (PC), cholesterol (Chol), and dipalmitoylphosphatidic acid (DPPA). Either sulfatide- or cholesterol sulfate-containing liposomes effectively prevented syncytium formation induced by BIV-infected cells, but the inhibitory effect of sulfatide alone on syncytium formation was low. On the other hand, neither liposomes containing galactocerebroside nor liposomes composed of egg PC, Chol, and DPPA had any effect on syncytium formation induced by BIV-infected cells. These results suggest that liposomes containing sulfatide or cholesterol sulfate are an efficient agent to inhibit syncytium formation induced by BIV-infected cells, and that sulfate residue might play an important role in the inhibition of syncytium formation.     

Effects of phospholipids on substrate specificity of β-galactosidase purified from Aspergillus oryzae

When entrapped into liposomes composed of phosphatidylcholine and other lipids, β-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) purified from Aspergillus oryzae could cleave the β-galactosidic bond of the terminal galactose of galactocerebroside and GM₁-ganglioside (II³NeuAc-GgOse₄Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosylglucosylceramide), while the free enzyme could not. The products of the hydrolysis of galactocerebroside were found to be β-galactose and ceramide, which was confirmed by using a fluorescent analog of galactocerebroside, 1-O-galactosyl-2-N-(1-dimethylaminonaphthalene-5-sulfonyl)-sphingosine, as substrate. The formation of GM₂-ganglioside (II³NeuAc-GgOse₃Cer, N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosylglucosylceramide) by the hydrolysis of GM₁-ganglioside was also demonstrated. The lipid composition of the liposomes influenced the amount of the enzyme entrapped and the activity of the trapped enzyme. A large amount of the enzyme was entrapped into the liposomes composed of phosphatidylcholine-cholesterol-stearoylamine (molar ratio, 7:2:1). The enzyme trapped in the liposomes and that in those of phosphatidylcholine-cholesterol-sulfatide (molar ratio, 7:2:1) had higher activity on galactocerebroside and GM₁-ganglioside than that in other liposomes. The activity of β-galactosidase trapped in liposomes was increased in the presence of detergent, while that of the free enzyme was not changed.By a similar procedure to introduce enzymes into hydrophobic environments, enzymes other than β-galactosidase might come to possess different substrate specificities.     

Astrocytes support incomplete differentiation of an oligodendrocyte precursor cell.

Glial fibrillary acidic protein-positive astrocytes, but not neurons or fibroblasts, support the differentiation of an oligodendroglial precursor cell expressing O4 antigen and vimentin into an O4 antigen-positive, but vimentin-negative oligodendrocyte. Further maturation into galactocerebroside (O1)-positive oligodendrocytes is, however, not achieved under the culture conditions used, neither in the presence of astrocytes nor neurons.     

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     

Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayers.

The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition region of the enzyme.     

Lipid microdomain formation: characterization by infrared spectroscopy and ultrasonic velocimetry

We demonstrate the use of vibrational infrared spectroscopy applied to characterize lipid microdomain sizes derived from a model raft-like system consisting of nonhydroxy galactocerebroside, cholesterol, and dipalmitoylphosphatidylcholine components. The resulting spectroscopic correlation field components of the lipid acyl chain CH₂ methylene deformation modes, observed when lipid multilamellar assemblies are rapidly frozen from the liquid crystalline state to the gel phase, indicate the existence of lipid microdomains on a scale of several nanometers. The addition of cholesterol disrupts the glycosphingolipid selectively but perturbs the di-saturated chain phospholipid matrix. Complementary acoustic velocimetry measurements indicate that the microdomain formation decreases the total volume adiabatic compressibilities of the multilamellar vesicle assemblies. The addition of cholesterol, however, disrupts the galactocerebroside domains, resulting in a slight increase in the lipid assemblies’ total adiabatic compressibility. The combination of these two physical approaches offers new insight into microdomain formation and their properties in model bilayer systems.