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PubMed1992

Metabolism of GM1 ganglioside in cultured skin fibroblasts: anomalies in gangliosidoses, sialidoses, and sphingolipid activator protein (SAP, saposin) 1 and prosaposin deficient disorders.

Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with GM1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in GM1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2-4 fold relative to controls. Labelled GM2 ganglioside accumulated in 4 variants of GM2 gangliosidosis, whereas labelled GM3 ganglioside accumulated in sialidosis, galactosialidoses and sphingolipid activator protein 1 (SAP-1, saposin B) and prosaposin (saposin A, B, C and D) deficient lipidoses. The reduced degradation of GM3 ganglioside in the SAP-1 and prosaposin deficiencies was attributed to the deficient function of SAP-1. The prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from GM1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in prosaposin deficiency.

Child, Preschool↗
PubMed1985

The gene coding for a sphingolipid activator protein, SAP-1, is on human chromosome 10.

SAP-1 is a sphingolipid activator protein found in human tissues required for the enzymatic hydrolysis of GM1 ganglioside and sulfatide. It appears to be missing in patients who have a genetic lipidosis resembling juvenile metachromatic leukodystrophy. Using rabbit antibodies against human SAP-1 it could be visualized in extracts from cultured human skin fibroblasts after sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose membrane and immunochemical staining (Western blotting). A series of 23 human-Chinese hamster ovary cell hybrids containing different human chromosomes were examined. The parent Chinese hamster ovary cells did not have a reacting protein in the region of human SAP-1. Only in the eight hybrid clones containing human chromosome 10 was a reacting protein identified. Other chromosomes were excluded by this method. Therefore the gene for SAP-1 and the genetic mutation resulting in a fatal lipidosis are located on human chromosome 10.

Animals↗
PubMed1995

Sphingolipid activator proteins in the neuronal ceroid-lipofuscinoses: an immunological study.

The molecular defects underlying neuronal ceroid-lipofuscinoses (NCL) are still unknown. However, more data exist on the composition of the hydrophobic storage material characteristic of NCL. Accumulation of subunit c of the mitochondrial ATP synthase has been shown in most forms of human NCL with the exception of the infantile NCL (INCL) for which we have recently demonstrated storage of sphingolipid activator proteins (SAP). In the present study we raised an antiserum against storage cytosomes purified from INCL brain. Using the anti-INCL antiserum and monospecific SAP antisera, we studied storage material isolated from the brains of patients affected with NCL by Western analysis, and found a 12-kDa protein showing a SAP-like immunoreactivity not only in INCL, but also in all the childhood forms of NCL. Furthermore, using the anti-sap-D antiserum for immunohistochemistry, we observed strong immunoreactivity of the storage cytosomes in all major forms of NCL, and also in tissues of non-neuroectodermal origin. From these data we conclude that the presence of SAP within the storage bodies is a phenomenon common to all major forms of human NCL.

Adolescent↗
PubMed1988

[Sphingolipid storage diseases of the central nervous system: bases of biochemical and clinical heterogeneity].

Lysosomal storage disorders are progredient and often fatal diseases most of which result from a pronounced enzyme deficiency. In the case of sphingolipidoses, usually enzymes of sphingolipid catabolism are missing, or only a few percent of normal activity are detectable. For many sphingolipidoses, damage of the central nervous system is characteristic, but neurological and other symptoms can vary greatly, especially in adult variants. This variability is mainly caused by different allelic mutations of the structural genes, resulting in different levels of residual enzyme activity.

Central Nervous System Diseases↗
PubMed1989

Intestinal sphingolipid excretion associated with feeding of phytohemagglutinin lectin (Phaseolus vulgaris) to germ-free and conventional rats.

Intestinal sphingolipids of feces of germ-free and conventional rats were analyzed during the pair feeding of a complete defined diet containing phytohemagglutinin lectin (PHA) from red kidney beans (Phaseolus vulgaris) as 1% dietary protein in comparison to casein fed controls. Phytohemagglutinin in the diet increased the total fecal excretion of sphingomyelins (18-fold for germ-free and 20-fold for conventional rats), of non-acid glycosphingolipids (3.5-fold for germ-free and 9-fold for conventional rats) and also of the gangliosides (2.5-fold) for the germ-free rats compared to controls. For germ-free rats the increase of non-acid glycolipids was ascribed to an effect of the lectin strictly on the small intestinal mucosa, while for conventional rats an effect was seen also on the large intestinal mucosa. Increase of fecal gangliosides of germ-free rats was due mainly to an increased excretion of N-acetylneuraminosyl-lactosylceramide, a ganglioside species restricted to epithelial cells of duodenum, of upper jejunum and of large intestines. The effects on glycolipid excretion observed in germ-free rats and the rather similar effects seen in conventional animals suggested that the influence of dietary PHA was due directly to effects elicited by PHA binding to the enterocyte brush border membrane and not to secondary effects induced by increase in the luminal microflora.

Animals↗
PubMed1987

Regional localization of the gene coding for sphingolipid activator protein SAP-1 on human chromosome 10.

Sphingolipid activator protein SAP-1 is required for the enzymatic hydrolysis of GMI ganglioside and sulfatide. The gene coding for SAP-1 was previously mapped to human chromosome 10 using monospecific antibodies prepared against SAP-1 in synteny analysis of somatic cell hybrids. In this study, we used a cDNA probe for SAP-1 and in situ hybridization to regionally localize the SAP1 gene to the long arm of chromosome 10, region q21-22. Additional mapping data using cell hybrids containing partial chromosome 10 and skin fibroblasts with trisomy 10p are consistent with the in situ hybridization mapping results.

Chromosome Banding↗
PubMed1982

Effects of sphingolipids on erythroblastic maturation in the mouse.

The changes effected by injection of an extract of phospholipids obtained from the plasma of normal human donors (PLE) or an emulsion of commercially available sphingolipids on erythropoiesis in the mouse were studied. The parameters followed were 59Fe uptake by the erythroid tissue and the number of circulating reticulocytes. It was found that in the 12--24-h period following administration of PLE or purified sphingomyelin a significant increase in 59Fe uptake by circulating RBC an by their hemic fraction takes place. This change was associated with a higher 59Fe utilization by the bone marrow and with an increase in the number of circulating reticulocytes.

Animals↗
PubMed1996

Sphingolipid activator proteins in a human hereditary renal disease with deposition of disialogangliosides.

Congenital nephrotic syndrome of the Finnish type is a recessively inherited renal disease with glomerular deposits of the disialoganglioside O-acetyl-GD3. Sphingolipid activator proteins (saposins) stimulate the degradation of glycosphingolipids by lysosomal enzymes, and defects in saposins cause accumulation of substrate lipids in the affected tissues in lysosomal storage disease. Here we report a study of the role of saposins in the accumulation of O-acetyl-GD3 in kidneys of congenital nephrotic syndrome patients. At the mRNA level, the expression of saposin precursor in diseased kidneys appeared normal, and the nucleotide sequence analysis of cDNA clones did not reveal abnormalities in the prosaposin gene. Immunohistologically, saposins were localized mainly to the epithelial cells of the distal renal tubules or to the parietal epithelial cells of glomeruli. In the nephrotic syndrome kidneys, the staining pattern was highly granular and appeared mostly in the apical part of the epithelial lining, unlike the control kidneys. These results show that a major site of ganglioside metabolism is located in the distal nephron. Furthermore, these results suggest that saposins are not directly involved in the metabolism of the terminal sialic acids of disialogangliosides in the nephrotic syndrome kidneys.

Adult↗
PubMed2004

A sphingolipid and tyrosinase inhibitors from the fruiting body of Phellinus linteus.

This paper for the first time reports the isolation of 5 compounds from Phellinus linteus. A sphingolipid (1) and two tyrosinase inhibitory compounds (2, 3) along with two carboxylic acids (4, 5), were isolated from the fruiting body of Phellinus linteus (Berk & Curt) Aoshima. The structure of compound 1 was identified as 1-omicron-beta-D-glucopyranosyl-(2S, 3R, 4E, 8E)-2-[(2R)-2-hydroxyhexadecanoylamino]-9-methyl-4,8-octadecadiene-1,3-diol, known as cerebroside B, based on spectroscopic methods such as 1 D and 2D NMR as well as by acid hydrolysis. Compounds 2 -5 were identified as protocatechualdehyde (2), 5-hydroxymethyl-2-furaldehyde (HMF) (3), succinic acid (4), and fumaric acid (5) based on the spectroscopic evidence. Compounds 2 and 3 inhibited the oxidation of L-tyrosine catalyzed by mushroom tyrosinase with an IC50 of 0.40 and 90.8 microg/mL, respectively. The inhibitory kinetics, which were analyzed by the Lineweaver-Burk plots, were found to be competitive and noncompetitive inhibitors with a Ki of 1.1 microM and 1.4 mM, respectively.

Acetylation↗
PubMed1992

Distribution of saposins (sphingolipid activator proteins) in tissues of lysosomal storage disease patients.

Saposins are a group of small glycoproteins derived from a single precursor protein, prosaposin. Each of the four saposins are involved in lysosomal hydrolysis of various sphingolipids. Our recent investigations have shown that saposins accumulate in tissues of several lysosomal storage diseases patients, including those with Tay-Sachs disease and Gaucher disease. To obtain insight into the mechanism of accumulation and its pathological role, the subcellular distribution of saposins in brain from Tay-Sachs disease and in spleen of Gaucher disease were compared with that of GM2 ganglioside and glucocerebroside, respectively. In both Tay-Sachs brain and Gaucher spleen, saposins were found predominantly in light-density fractions while most of the GM2 ganglioside and glucocerebroside, respectively, were found in heavy-density fractions. These studies indicate that saposins that accumulate in these pathological tissues are not tightly associated with GM2 ganglioside or glucocerebroside.

Brain↗
PubMed2001

Elevation of endogenous sphingolipid long-chain base phosphates kills Saccharomyces cerevisiae cells.

Sphingolipid long-chain base phosphates (LCBPs) regulate cell proliferation, movement and differentiation in higher eukaryotes. To study the function of LCBPs in Saccharomyces cerevisiae, we inactivated LCBP breakdown pathways. Elimination of both the Dpll lyase and the Lcb3 phosphatase pathways by gene deletion was lethal, indicating that these enzymes regulate LCBP levels to prevent accumulation. Lethality was prevented by eliminating the major LCB kinase. Lcb4p, which synthesizes LCBPs, but not by eliminating the minor LCB kinase, Lcb5p. These data imply that death results from an accumulation of LCBPs made by the Lcb4p kinase. By regulating Lcb4 kinase activity, we found that cell death correlates with LCBP accumulation and that C18 dihydrosphingosine-l-P (DHS-P) and C20 DHS-P are most likely the killing molecules. LCB levels were found to be most elevated in a strain lacking Lcb4 kinase, Dpll lyase and Lcb3 phosphatase activity. Analysis of mutant strains suggests that the C18 and C20 species of LCBPs are preferentially degraded by the Lcb3 phosphate phosphatase, while the Dpll lyase prefers C16 DHS-P as a substrate. These and other data indicate the existence of an unknown mechanism(s) for regulating LCB levels. Our results demonstrate that LCBPs may be used in some circumstances to regulate yeast cell growth.

Cell Survival↗
PubMed2006

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3- 3H]serine and [9,10- 3H]palmitic acid from normal cells in culture.

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-(3)H]sphingosine, L-[3-(3)H]serine and [9,10-(3)H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells.

Animals↗
PubMed2006

Sphingolipids, cholesterol, and HIV-1: a paradigm in viral fusion.

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37 degrees C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.

3T3 Cells↗
PubMed1990

Determination of saposin proteins (sphingolipid activator proteins) in human tissues.

Saposins are small glycoproteins which are required for sphingolipid hydrolysis by lysosomal hydrolases. Each saposin (A, B, C, and D) stimulates a different enzymatic activity. A new simple HPLC method to determine the levels of saposins A, C, and D in tissue was developed. Tissues were homogenized in 20 vol of water, boiled, and centrifuged. The supernatant was lyophilized and redissolved in 5 ml of water. A 1.5-ml sample of the solution was applied to a reverse-phase HPLC column (C4 column) and eluted with an acetonitrile gradient. Most contaminants eluted from the column prior to the saposins, which were eluted later as a cluster of peaks. This cluster was collected and then analyzed by another HPLC system equipped with an AX-300 anion-exchange column using a NaCl gradient. Saposins D, A, and C eluted from the AX-300 column separately and in that order. Quantitation of the saposins was made by measuring the sizes of each peak. Standard curves made from pure saposins showed that quantification was linear over a range from 1 to 5 micrograms. Saposin B was measured by its stimulation activity on pure human liver GM1 ganglioside beta-galactosidase. Stimulation was linear up to 80 micrograms of saposin B. Application of this method to analysis of human tissues for their saposin content is presented.

Chromatography, High Pressure Liquid↗
PubMed1984

Biochemical, immunological, and structural studies on a sphingolipid activator protein (SAP-1).

Sphingolipid activator protein-1 (SAP-1) is a glycoprotein found in human tissue extracts that stimulates the enzymatic hydrolysis of at least two glycosphingolipids, including GM1 ganglioside and sulfatide. The ability of purified SAP-1 to stimulate GM1 ganglioside hydrolysis by extracts of cultured fibroblasts from patients with beta-galactosidase deficiency was examined, and all patients had a pronounced deficiency (under 10% of control). Using monospecific antibodies against SAP-1, the concentration was determined in cultured fibroblasts by rocket immunoelectrophoresis. Extracts from 15 control cell lines were found to have 0.72 +/- 0.24 micrograms cross-reactive material/mg protein, while cell extracts from 8 patients with GM1 gangliosidosis involving mental retardation were found to have 1.08 +/- 0.17, which is significantly elevated. When the fibroblast extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroblotting, multiple bands were observed. Controls were found to have two major bands with estimated molecular weights of 9000 and 9500, and a minor band at 7800. Extracts from patients with GM1 gangliosidosis were found to have multiple bands ranging upward to 13,000. Extracts from patients with the most severe clinical types of GM1 gangliosidosis had almost exclusively high-molecular-weight forms (molecular weights above 10,000). Treatment of SAP-1 from control liver with endoglycosidase D caused a decrease in the Mr 9500 band and increased in the Mr 7800 band. When SAP-1 from GM1 gangliosidosis liver was treated sequentially with neuraminidase, beta-galactosidase, and endoglycosidase D, almost all of it was converted to the forms found in control human liver.

Cells, Cultured↗
PubMed1992

Phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and the phosphoinositol sphingolipids are found in the plasma membrane and stimulate the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

Several plasma membrane phospholipids have been studied for their ability to modulate the activity of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae. We show here that phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2), and/or the phosphatidylinositol and PIP kinases are localized primarily in the plasma membrane. Previous in vivo studies with S. cerevisiae have shown that large, rapid, and reversible changes occur in the levels of PIP and PIP2 congruent with changes in cellular ATP levels. We demonstrate here that isolated plasma membranes exhibit the same changes in PIP and PIP2 content when they are supplied with or washed free of ATP. Using a mixed micellar assay we systematically studied the efficacy of the plasma membrane lipids in sustaining the activity of the plasma membrane H(+)-ATPase. We demonstrate for the first time that a number of plasma membrane glycerophospholipids effectively stimulate the ATPase, including PIP, PIP2, and cardiolipin. Phosphoinositol-containing sphingolipids, major components of the plasma membrane, are also shown to stimulate the ATPase at significantly lower levels than the glycerophospholipids and must also be considered as important effectors in vivo.

Cell Fractionation↗
PubMed1986

Circular dichroism spectra of aqueous dispersions of sphingolipids.

The circular dichroism (CD) spectra of a number of sphingolipids dispersed in water have been studied. The lipids include cerebrosides such as palmitoyl cerebroside, glucocerebroside from the spleen of Gaucher patients, bovine brain galactocerebrosides type I and type II, (BCI and BCII, respectively) and also sphingomyelins such as egg sphingomyelin and bovine brain sphingomyelin. Changes in the CD spectra of the lipids which occur upon heating and cooling and the effects of cholesterol, phosphatidylcholine and the opiate leucine enkephalin were studied.

Animals↗
PubMed1983

Effect of essential fatty acid deficiency on the epidermal sphingolipids of the rat.

The epidermal sphingolipids from rats maintained on either a rat stock diet or a fat-free diet have been analyzed. Thin-layer chromatographic analyses have revealed glucosylceramides, acylglucosylceramides and four fractions of ceramides, one of which proved to be an acylceramide. The relative amounts of the glucosylceramides, acylglucosylceramides and acylceramides were increased in the essential fatty acid-deficient epidermis while one ceramide fraction was diminished. The other two ceramide fractions remained unchanged. The acylceramides and acylglucosylceramides from normal rat epidermis both contained long-chain omega-hydroxy acids in amide linkage to sphingosine bases and high proportions of linoleic acid in ester linkage. The linoleate, which is known to be crucial for the formation and maintenance of the epidermal water barrier, was replaced by oleate in the essential fatty acid-deficient rats.

Animals↗