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PubMed1986

Stimulatory effect of certain plant sphingolipids on fruiting of Schizophyllum commune.

We reported previously that certain cerebrosides and ceramides from fungi were active upon fruiting of Schizophyllum commune (Kawai, G., and Ikeda, Y. (1982) Biochim. Biophys. Acta 719, 612-618; Kawai, G., and Ikeda, Y. (1983) (Biochim. Biophys. Acta 754, 243-248). This work was undertaken to extend our study to sphingolipids in wheat grain. The cerebrosides from wheat grain were fractionated by high-performance liquid chromatography into at least 40 components with and without the fruiting-inducing activity. Four major active fractions were characterized by thin-layer chromatography, infrared spectroscopy, gas-liquid chromatography, gas-liquid chromatography-mass spectroscopy, and 1H and 13C nuclear magnetic resonance spectroscopy. The active cerebrosides consist of glucose, 2-hydroxyhexadecanoic acid or 2-hydroxyoctadecanoic acid acid, and (4E,8Z)-sphingadienine or (8Z)-sphingenine. The cerebroside with (8Z)-sphingenine became inactive when the double bond was hydrogenated. Diglycosylceramides were as active as the monoglycosylceramides, but triglycosylceramides were only about 10% as active. The relationship between the structure and the activity is discussed.

Basidiomycota↗
PubMed1979

Conversion by rat brain preparation of lignoceric acid to ceramides and cerebrosides containing both alpha-hydroxy and nonhydroxy fatty acids. Effects of compounds which affect sphingolipid metabolism.

Three synthetic compounds which affect sphingolipid metabolism in vitro were examined for their effects on the synthesis in a rat brain system of nonhydroxyceramide, hydroxyceramide, nonhydroxycerebroside, and hydroxycerebroside from [1-14C]ignoceric acid. These compounds are N-octanoyl-D-threo-p-nitrophenylaminopropanediol (Compound I), N-(2,3-epoxydecanoyl)-norephedrine (Compound II), and N-decyl-N'-glucosylthiourea (Compound III). In the presence of up to 0.5 mM Compound I, only the hydroxyceramide fromation increased, while the synthesis of the other three lipids decreased. Compound II strongly inhibited the formation of all four lipids at all concentrations tested (0.1 to 1 mM). Compound III increased the synthesis of both ceramides approximately 2-fold at 1 mM, but the conversion of lignoceric acid to both cerebrosides remained relatively unchanged. Several significant conclusions from these observations are discussed.

Animals↗
PubMed1984

Temperature-induced changes in the hydroxy and non-hydroxy fatty acid-containing sphingolipids abundant in the surface membrane of Tetrahymena pyriformis NT-1.

Sphingolipids make up 30 to 40 mole % of the phospholipids found in the surface membrane of Tetrahymena pyriformis NT-1. We have identified the two major classes as non-hydroxy fatty acid-containing ceramide-2-aminoethylphosphonate (NCAEP) and alpha-hydroxy fatty acid-containing ceramide-2-aminoethylphosphonate (HCAEP). Both classes were well represented in cells grown at 39 degrees C. At this temperature their principal long chain bases were n-hexadeca-4-sphingenine and n-nonadeca-4-sphingenine. The major fatty acid of NCAEP from 39 degrees C-grown cells was palmitic acid and that of HCAEP was alpha-hydroxypalmitic acid. Cells grown at 15 degrees C contained NCAEP, but only traces of HCAEP. By analyzing the incorporation of [1-14C]palmitic acid into cells growing isothermally or shifted from 15 degrees C to 39 degrees C, we obtained evidence favoring a direct conversion of NCAEP to HCAEP. This conversion was blocked in cells grown at 15 degrees C, causing an accumulation of NCAEP. Tetrahymena is a useful model system for studying the poorly understood alpha-hydroxylation process that is of critical importance in myelination of animal nervous tissues.

Animals↗
PubMed1981

Effect of suramin on the activities of degradative enzymes of sphingolipids in rats.

Three to nine days after administration of suramin, 500 mg/kg intravenously in rats, a small amount of the drug (about 0.25 micromoles/g tissue) was retained by the liver and spleen, and a larger amount (about 1.2 micromoles/g tissue) was retained by the kidneys. The activities of the sphingolipid hydrolases beta-hexosaminidase and GM3-sialidase were strongly inhibited by suramin in vitro. The activity of beta-hexosaminidase was inhibited 70% by 10(-5M) and 85% by 10(-4M) suramin, and the activity of GM3-sialidase was inhibited 80% by 10(-4M) suramin. The activities of sphingomyelinase and beta-galactosidase were also inhibited by suramin but at higher concentrations of the drug. Suramin, in vitro is a weak inhibitor of glucocerebrosidase, galactocerebrosidase, alpha-galactosidase and arylsulfatase A (less than 50% inhibition at 10(-3M) concentration of the drug). The inhibition of beta-hexosaminidase by suramin was non-competitive. Inhibition of beta-hexosaminidase and GM3-sialidase may explain the accumulation of GM2 and GM3 gangliosides in the brains of rats treated intracerebrally with suramin (Constantopoulos et al, 1980).

Animals↗
PubMed1980

A lysosomal storage disorder in mice characterized by the accumulation of several sphingolipids.

A strain of BALB/c mice with an autosomal recessive neurologic disorder has been reported previously [1, 2]. The tissues of affected animals have been further examined and the activities of varius lysosomal hydrolases and levels of sphingolipids were compared to those in control mice. There was a substantial diminution of sphingomyelinase and glucocerebrosidase activities in liver, spleen, lung, thymus, and kidney of affected mice. There was a corresponding accumulation of sphingomyelin and glucocerebroside in these tissues. The activity of several other lysosomal hydrolases was elevated. Heterozygotes did not show any of the enzymatic alterations. The brain of affected animals showed substantial accumulation of the gangliosides GM3 and GM2.

Animals↗
PubMed1993

Sphingolipids with inositolphosphate-containing head groups.

InsPCers have been characterized in many plants, fungi, and protozoans but not in animals. There are no well-documented reports of the absence of InsPCers in organisms of these categories and one might possibly consider these lipids to be ubiquitous in plants, fungi, and protozoans. The polar headgroups of these lipids display quite heterogeneous structures depending on the source, including attachment to proteins as possible membrane anchors. The ceramides are with some exceptions composed of phytosphingosine and a very long-chain, usually hydroxylated, fatty acid. The vital nature of such sphingolipids in the plasma membrane is indicated in S. cerevisiae. Clearly, much remains to be discovered about the structure, metabolism, and function of the InsPCers.

Amino Acid Sequence↗
PubMed1995

[Lysosomal enzymes, sphingolipid activator proteins, and protective protein].

The lysosome is an intracytoplasmic acidic vacuole containing more than 60 hydrolytic enzymes for digestion of macromolecules, such as nucleic acids, proteins, lipids and complex carbohydrates. Expression of lysosomal enzyme activities is regulated by various intracellular environmental factors. Mutation of a gene coding for a lysosomal enzyme results in a specific genetic disease, often involving the central nervous system in children. Three groups of functional proteins are known at present for regulation of the expressed enzyme activity in lysosomes. Targeting of a newly synthesized protein is achieved by the mannose 6-phosphate receptor system, which was revealed in the course of I -cell disease research. Many lysosomal enzymes are excessively secreted in the extracellular compartment in the absence of this regulatory system in patients with this disease. Intralysosomal stability of beta-galactosidase is regulated by a multifunctional protein that interacts with two lysosomal enzymes, beta-galactosidase and sialidase, and also exerts catalytic activities as carboxypeptidase, esterase and deamidase under various pH conditions. It is encoded by a gene on chromosome 20, and its mutation results in a neurodegenerative disease in children and adults (galactosialidosis). For digestion of lipid substrates, lysosomal enzymes need specific activator proteins as natural detergents for molecular interaction with these nonpolar compounds. Two different groups of proteins have been revealed. A protein encoded by a gene on chromosome 5 interacts with ganglioside GM2 and its asialo derivative, for their catalytic hydrolysis by beta-hexosaminidase A. Another protein encoded by a gene on chromosome 10 is expressed as a precursor (prosaposin) which is then processed to four small proteins (saposins) with heterogeneous functions. They are essential for hydrolysis of sphingolipid substrates, and genetic deficiency of each protein results in various lipid storage diseases.

Adult↗
PubMed1997

Dietary fumonisins disrupt sphingolipid metabolism in mink and increase the free sphinganine to sphingosine ratio in urine but not in hair.

This study was conducted to investigate the effects of dietary Fusarium moniliforme culture material (M-1325) containing known concentrations of fumonisins B1, B2 and B3 on sphingolipids in urine and hair of mink (Mustela vison) for use as potential, non-invasive biomarkers of exposure to fumonisins in this species. Feeding mink diets containing 86, 22, and 7 ppm or 200, 42, and 12 ppm of fumonisins B1, B2 and B3, respectively, yielded marked increases in urinary free sphinganine (Sa) and free sphingosine (So) concentrations, and free Sa/free So ratios (2 to 11-fold) within 7 d, compared to controls. Free Sa and free So concentrations and Sa/So ratios in hair samples from mink fed the control or high dose fumonisin diets for 100 days were similar and were not apparently altered by exposure to these mycotoxins. These results suggest that Sa/So ratios in urine, but not in hair of mink can serve as an early indicator of exposure to fumonisins in this species.

Animals↗
PubMed1985

Assignment of the gene for human sphingolipid activator protein-2 (SAP-2) to chromosome 10.

Sphingolipid activator protein-2 (SAP-2) has been found to stimulate the enzymatic hydrolysis of glucosylceramide, galactosylceramide, and sphingomyelin. When human skin fibroblast extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by electroblotting and immunochemical staining using monospecific antibodies against SAP-2, two or three major bands with estimated mol. wts. of 9,000-10,000 were found. These antibodies did not crossreact with purified SAP-1, another activating protein, or with extracts of CHO-K1 cells. A series of 22 human/Chinese hamster ovary cell hybrids containing different human chromosomes were examined by this method. All eight hybrid clones containing human chromosome 10 were found to have crossreacting protein in this region. Other chromosomes could be excluded by this method. From these results, we conclude that the gene coding for human SAP-2 is located on chromosome 10.

Animals↗
PubMed1998

Distinct roles for sphingolipids and glycosphingolipids at different stages of neuronal development.

Studies on the roles of sphingolipids (SLs) and glycosphingolipids (GSLs) at distinct stages of neuronal development have been performed using primary cultures of hippocampal neurons, which are unique among neuronal cultures inasmuch as they develop by a well-characterized and stereotypic sequence of events that gives rise to fully differentiated axons and dendrites. Our data demonstrate that SLs and GSLs play at least three distinct roles in regulating neuronal development, namely: (i) ceramide enhances the formation of minor neuronal processes from lamellipodia and the subsequent stage of axonogenesis; (ii) glucosylceramide synthesis, but not the synthesis of higher-order GSLs, is required for normal axon growth and for accelerated axonal growth upon stimulation by growth factors; and (iii) at both of these stages, ceramide at high concentrations can induce apoptotic cell death. Together, these observations are consistent with the possibility that minor process formation and apoptosis are regulated by ceramide-dependent signaling pathways, whereas axonal growth requires glucosylceramide synthesis, perhaps to support an intracellular transport pathway.

Animals↗
PubMed1987

Gaucher disease: accurate identification of asymptomatic French-Canadian carrier using nonlabeled authentic sphingolipid substrate N-palmitoyl dihydroglucocerebroside.

Gaucher disease is an autosomal recessive sphingolipidosis associated with deficient glucocerebroside beta-glucosidase activity. It is a panethnic metabolic disorder, but the carrier frequency is particularly high among Ashkenazi Jews (estimated between 1:12-1:25). In order to establish a reliable and convenient biochemical assay method for differentiating asymptomatic Gaucher carriers from normal individuals, glucocerebroside beta-glucosidase activity was determined in peripheral blood lymphocytes and cultured skin fibroblasts of 11 Gaucher obligate heterozygotes using the authentic nonlabeled sphingolipid substrate N-palmitoyl dihydroglucocerebroside and the artificial fluorogenic substrate 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGP). The level of lymphocyte beta-glucosidase activity on the glucocerebroside substrate was observed to range from 42-65% of that of the control mean, and there was no overlap of enzyme activity between the Gaucher heterozygotes and controls. However, when the artificial fluorogenic substrate 4MUGP was used, the level of beta-glucosidase activity in 2 of the Gaucher obligate heterozygotes was noted to overlap with that of the control individuals. Contrary to findings in the lymphocytes, cultured skin fibroblasts appear to be a reliable enzyme source for Gaucher carrier detection even when the artificial fluorogenic 4MUGP substrate was used, as the level of beta-glucosidase activity in all of the Gaucher obligate heterozygotes tested was intermediate and distinctly separated from that of the control persons. Using the lymphocyte glucocerebroside beta-glucosidase assay and fibroblast 4MUGP beta-glucosidase assay methods, we identified the carrier status in 3 other relatives and ruled it out in 4 others. These data suggest that nonlabeled glucocerebroside is a reliable and highly specific substrate for either lymphocyte or fibroblast beta-glucosidase activity assay in identifying asymptomatic Gaucher carriers. Use of the 4MUGP substrate for differentiating Gaucher heterozygotes from control persons, on the other hand, should be restricted to the fibroblast enzyme assay method, as considerable overlap of enzyme activity was noted in lymphocytes.

Adult↗
PubMed1997

Lysosomal proteolysis of prosaposin, the precursor of saposins (sphingolipid activator proteins): its mechanism and inhibition by ganglioside.

Saposins A, B, C, and D, which are required for the enzymatic hydrolysis of sphingolipids by specific lysosomal hydrolases, are produced by proteolytic processing of their common precursor protein, prosaposin. Our previous observation suggested that lysosomal cathepsin D may be involved in the proteolysis of prosaposin. Herein we report the involvement of cathepsin D in the proteolytic processing of prosaposin. An antibody against human placental cathepsin D blocked the proteolytic activity toward prosaposin in a human testicular lysosomal protease mixture (glycoprotein fraction). On immunoblot analysis using a monoclonal antibody against human saposin C, cathepsin D showed a similar proteolytic pattern as that of a human testicular glycoprotein fraction and hydrolyzed prosaposin into products of 48 and 29 kDa. The Km and Vmax values were 0.9 microM and 167 nmol/h/mg, respectively. N-Terminal sequence analysis indicated that the 48-kDa band was a mixture of two trisaposins, including domains for saposins A, B, and C and saposins B, C, and D, respectively. A similar study also showed that the 29-kDa band contained two disaposins, including domains for saposins A and B and saposins C and D, respectively. By longer treatment with cathepsin D, disaposins were further processed into mature saposin A and small fragments (14.5-17.5 kDa) containing individual saposins and portions of interdomain sequences. These small fragments were no longer processed by cathepsin D, but trimmed to fragments having similar molecular sizes (10.5-11.5 kDa) to those of mature saposins by a rat lysosome preparation. These findings indicated that cathepsin D is involved in the maturation of saposins but that, in addition to cathepsin D, other proteases appear to be involved in the maturation of saposin B, C, and D in lysosomes. Gangliosides, which specifically form complexes with prosaposin and saposins, inhibit proteolysis of prosaposin by cathepsin D. This finding indicates that prosaposin may be protected from lysosomal proteolysis by forming a complex with gangliosides in vivo.

Animals↗
PubMed1997

[14C]ceramide synthesis by sphingolipid ceramide N-deacylase: new assay for ceramidase activity detection.

Sphingolipid ceramide N-deacylase (SCDase) is an enzyme which hydrolyzes the N-acyl linkage between fatty acid and sphingosine in ceramide of various glycosphingolipids and sphingomyelin. Recently the enzyme was found to catalyze the hydrolysis and its reverse reaction under different conditions. We report here an innovative method for synthesis of radioisotope-labeled ceramide with high specific activity using the reverse hydrolysis reaction of SCDase. More than 80% of free fatty acid was transferred to sphingosine when 1 nmol [14C]stearic acid and 2 nmol sphingosine were incubated with 5 microU SCDase at 37 degrees C for 20 h in 10 microliters of 25 mM phosphate buffer, pH 7.0, containing 0.3% Triton X-100. Free [14C]fatty acid and sphingosine were easily separated from synthesized [14C]ceramide by using a Sep-Pak Plus Silica and a Sep-Pak CM cartridge, respectively. We also developed a sensitive assay method for ceramidase using the [14C]ceramide prepared. The method consists of separation of the [14C]fatty acid released from [14C]ceramide by the action of enzyme on thin-layer chromatography followed by analysis and quantification with an imaging analyzer (BAS1000). This method was sensitive and qualitative and enabled the detection of ceramidase activity in invertebrates for the first time as well as in several human cancer cell lines.

Amidohydrolases↗
PubMed2000

1,25 dihydroxyvitamin D(3) activates sphingomyelin turnover in ROS17/2.8 osteosarcoma cells without sphingolipid-induced changes in cytosolic Ca(2+).

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] initiates the hydrolysis of sphingomyelin in ROS 17/2.8 osteosarcoma cells with the resultant generation of cell-associated ceramide. Increases in ceramide levels were detectable at 15 min and maximal one hour after exposure of cells to 1,25(OH)(2)D(3). Neither 1,25(OH)(2)D(3) nor exogenous ceramide elicited a change in cytosolic free Ca(2+) ([Ca(2+)](i)). Transient elevations in [Ca(2+)](i) were observed when cells were exposed to exogenous sphingosine, but there was no detectable conversion of ceramide to sphingosine in 1, 25(OH)(2)D(3)-treated cells. Ceramide also did not stimulate Ca(2+) uptake across ROS 17/2.8 cell plasma membranes. Collectively, these results suggest that 1,25(OH)(2)D(3) activates sphingomyelin turnover in ROS 17/2.8 osteosarcoma cells but that the sphingolipid metabolite ceramide is not responsible for 1,25(OH)(2)D(3)-induced activation of plasma membrane Ca(2+) channels.

Animals↗
PubMed2000

Chimeras of Delta6-fatty acid and Delta8-sphingolipid desaturases.

The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids. However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6. These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site. This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets.

Fatty Acid Desaturases↗
PubMed2000

Sphingolipid biosynthesis by L-PDMP after rat MCA occlusion.

L-PDMP (L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) exhibits stimulatory effects on glycosphingolipid biosynthesis and its neurotrophic actions in cultured neuron. The effects of intraperitoneal administration of L-PDMP on sphingolipid metabolism and behavioral changes in the rat following permanent occlusion of the left middle cerebral artery (MCA) were investigated. The L-PDMP treatment induced increases in glucosylceramide (ganglioside precursor) and sphingomyelin (SM) levels in the ischemic cerebral cortex, and improved acquisition of memory and learning in the Morris water maze task. The pharmacological effects of L-PDMP have been proposed to have a significant activity on promoting cell survival and improving neural functions.

Animals↗
PubMed1976

[Sphingolipid storage disease as an example of a molecular neuropathology (author's transl)].

A short survey on the sphingolipid storage diseases is presented. The chemical nature of the accumulated substances is related to the genetically induced enzymic blocks on their biodegradation. Two disorders are stressed with alter the nervous system: metachromatic leukodystrophy and familiar infantile amaurotic idiocy (GM2-gangliosidosis). The difficulties in the causal interpretation of three variants of the latter disease due to the involvement of isoenzymes are dealt with. The relationship between the enzyme defect in these disorders and their time of clinical onset is discussed. Finally, the diagnostic possibilities are presented which are a prerequisite for preventing a further dissemination of these therapy-resistent inborn errors of metabolism.

Child, Preschool↗
PubMed1993

Synthesis and characterization of a bioactive 82-residue sphingolipid activator protein, saposin C.

The sphingolipid activator protein, saposin C (also termed SAP 2), was chemically synthesized, purified, and characterized. The fully protected 82-residue protein was synthesized by automated solid-phase methods, with multiple recoupling steps resulting in a high average coupling efficiency of 98.8%. The overall yield was estimated to be approx 40%. Deprotection and cleavage of the peptide from the resin was followed by folding in the absence of chaotropic agents at pH 8.5. The protein was purified by reversed-phase high pressure liquid chromatography (HPLC) and its purity determined by capillary electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The composition of the synthetic saposin C was determined by amino acid analysis. Its sequence was verified by Edman sequence analysis of overlapping peptide fragments generated by chymotryptic and Staphylococcus aureus V8 digestions. The sequence at the C-terminus was determined by digestion with carboxypeptidase P, followed by phenylthiohydantoin (PTH) derivitization and HPLC analysis of the released amino acid residues. Deglycosylated native saposin C appeared as a lower molecular-weight species than synthetic saposin C on SDS-PAGE. This has been explained by amino acid and C-terminal analysis showing native saposin C to be two amino acids shorter at the C terminus than a deduced sequence (from cDNA) previously published. Synthetic saposin C displayed 85% of full biological activity as determined by its ability to stimulate glucocerebrosidase activity in vitro: Synthetic and native saposin C increased glucocerebrosidase catalyzed hydrolysis of 4-methylumbelliferyl beta-D-glucoside by factors of 6.0 and 7.1, respectively. Furthermore, synthetic and native saposin C share similar K(act) values (0.5 and 1.5 microM respectively) indicating that they bind to glucocerebrosidase with similar affinities.

Amino Acid Sequence↗