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Metabolism of Cerebroside Sulfate and Subcellular Distribution of Its Metabolism of Cerebroside Sulfate and Subcellular Distribution of Its Metabolites in Cultured Skin Fibroblasts from Controls, Metachromatic Leukodystrophy, and Globoid Cell Leukodystrophy Koji Inui, Masumi Furukawa, Shintaro Okada, and Hyakuji Yabuuchi Department ofPediatrics, Osaka University Medical School, Osaka 553, Japan Abstract each step result in metachromatic leukodystrophy (MLD), globoid cell leukodystrophy (GLD), and Farber disease. Due With pulse-chase study of 1-['4CJstearic acid-labeled cerebro- to enzyme deficiencies, massive lysosomal storage of lipids is side sulfate ('4C-CS) and subsequent subcellular fractionation demonstrated in MLD (4) and Farber disease (5). In GLD it is by Percoll gradient, the metabolism of CS and translocation of well known that there is no accumulation of galactosylcera- its metabolites in human skin fibroblasts from controls, meta- mide and that the major abnormalities are the presence of a chromatic leukodystrophy (MLD), and globoid cell leukodys- large number of globoid cells, a severe lack of myelin and trophy (GLD) were studied. In control skin fibroblasts, CS was astrogliosis in the nervous system (6). transported to lysosome and metabolized there to galactosyl- The enzymic defects of most lysosomal storage disorders ceramide (GalCer) and ceramide (Cer) within 1 h. During the have been clarified, but the molecular mechanisms that lead to chase period, radioactivity was increased at plasma membrane the clinical and pathological manifestations remain largely plus Golgi as phospholipids and no accumulation of GalCer or obscure. Recent morphological studies of neurons from Cer was found in lysosome. In MLD fibroblasts, 95% of '4C- humans (7), cats (8), and dogs (9) with gangliosidoses have CS taken up was unhydrolyzed at 24 h-chase and accumulated shown meganeurities, inappropriate proliferation ofsecondary at not only lysosome but also plasma membrane. In GLD fibro- neurites, aberrant synaptogenesis, somatic swelling, and ab- blasts, GalCer was accumulated throughout the subcellular normal somatic processes. Biochemically, impaired neuro- fractions and more accumulated mainly at plasma membrane transmitter metabolism was suggested in cerebral cortical and plus Golgi with longer pulse. This translocation of lipid from cerebellar synaptosomes from cats with GM, gangliosidosis lysosome seems to have considerable function, even in lipi- (10) and increased amount of ganglioside in synaptosomal dosis, which may result in an imbalance of the sphingolipid membranes from feline GM, and GM2 gangliosidosis was pre- pattern on the cell surface and these changes might be one of sented (11). These studies suggested that altered membrane causes of neuronal dysfunction in sphingolipidosis. structure could be one of causes of neuronal dysfunction. To understand the mechanism causing neurological dys- Introduction function in sphingolipidoses, clarification of the synthesis, translocation and insertion of sphingolipids in the different Sphingolipids are important membrane lipids and particularly subcellular components in normal and pathological states is abundant in neuronal membranes. As outer surface mem- essential. In the present study, using I-['4C]stearic acid-labeled brane constituents of animal cells, they play important roles, cerebroside sulfate (14C-CS), we studied the metabolism of CS including membrane stability, nerve conduction, receptor ac- and the subcellular distribution of its metabolites in cultured tion, oncogenic transformation and other functions that are skin fibroblasts from normal control, MLD, and GLD. Our necessary for growth, differentiation, and survival ( 1-3). Cere- findings indicated that enzyme-deficient cells stored lipid in broside sulfate (CS),' galactocerebroside with a sulfate esteri- both lysosomes and plasma membranes, suggesting that the fying carbon-3 of the galactosyl moiety, is one of myelin con- altered lipid composition of membrane is a possible pathogen- stituents and has an important role during developmental pe- esis of neuronal dysfunctions in sphingolipidoses. riod encompassing myelination (4). The metabolic pathway of is well known and defects ofthe catabolism at this lipid genetic Methods Address reprint requests to Dr. Inui, Department of Pediatrics, Osaka Materials. Chemicals were purchased from the indicated sources: University Medical School, Fukushima-ku, Osaka 553, Japan. Eagle's minimum essential medium (Nissui Seiyaku Co., Tokyo, Receivedfor publication 25 November 1986 and in revisedform 8 Japan); HRP-wheat germ agglutinin (Seikagaku Co., Tokyo, Japan); September 1987. ovalbumin grade V (Sigma Chemical Co., St. Louis, MO); Percoll and density marker beads (Pharmacia Fine Chemicals, Uppsala, Sweden); 1. Abbreviations used in this paper: Cer, ceramide; CS, cerebroside cerebroside sulfate (Supelco, Inc., Bellefonte, PA); phospholipid kit sulfate; '4C-CS, 1-['4C]stearic acid-labeled cerebroside sulfate; FFA, (Serdary Research Laboratories, Inc., Ontario, Canada); choline- stearic acid; GalCer, galactosylceramide; GLD, globoid cell leukodys- methyl-['4C]sphingomyelin, 1-['4C]stearic acid and UDP-['4C(U)]- trophy; MLD, metachromatic leukodystrophy; PC, phosphatidylcho- galactose (New England Nuclear, Boston, MA); 4-methylumbelliferyl line; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, glycosides (Koch-Light Laboratories, Colnbrook, United Kingdom). phosphatidylserine. All other reagents were purchased from standard commercial sup- pliers. J. Clin. Invest. Preparation oflipids. 14C-CS was prepared according to the method © The American Society for Clinical Investigation, Inc. ofDubois et al. (12) and purified by silica gel column chromatography 0021-9738/88/02/0310/08 $2.00 and preparative TLC. The radiopurity was 99% checked by TLC. Volume 81, February 1988, 310-317 Cell culture. Human skin fibroblasts were grown from forearm skin 310 K. Inui, M. Furukawa, S. Okada, and H. Yabuuchi biopsies with Eagle's minimum essential medium supplemented with 5 min and the lower phase was transferred to a small tube. After 10% fetal calf serum, glutamine (5 mM), and fungizone (25 ,ug/ml) evaporation under nitrogen stream, cellular lipid extracts were ana- (complete medium) and maintained in a 5% CO2 incubator at 37°C. lyzed by silica gel TLC (HPTLC; Merck & Co., Rahway, NJ) with The cell lines from two patients with infantile GLD were supplied by chloroform/methanol/water (70:30:4, by volume) and the plate ex- Dr. K. Suzuki, Albert Einstein College (New York) and Dr. D. A. posed to x-ray film (Fuji RX) for 7-14 d as described by Kudoh et al. Wenger, University ofColorado Health Sciences Center (Denver, CO). ( 13). For lipid extract from subcellular fractions, every three fractions The other cell lines from control subjects, patients with late infantile starting from a light density were combined to make eight fractions MLD and infantile GLD were established in this laboratory with in- after the assay of marker enzymes. These eight fractions were ultra- formed consents. centrifuged at 100,000 g for 90 min at 4°C to remove Percoll. The CS feeding experiments. `4C-CS was dissolved in the medium as supernatant was taken into a glass tube and the surface of pellet was described by Kudoh et al. (13) with minor modification (lipid concen- rinsed with small amount of distilled water. Then 4 ml of chloroform- tration changed to 10 nmol/ml). In some CS feeding experiments, FCS methanol (2:1, by volume) was added, well vortexed and kept standing was omitted. For pulse and chase studies, cells were incubated with 4 overnight. Then the upper phase was removed by centrifugation, 2 ml ml of complete medium (40 nmol, specific activity of "4C-CS; 15,000 of the theoretical upper phase was added. After mixing and sitting dpm/nmol) for 1 h and the feeding was terminated by changing the overnight, the lower phase was taken to a small tube and evaporated medium to 10 ml of fresh complete medium without '4C-CS. Sterile under nitrogen stream. The recovery ofradioactivity by this procedure condition was maintained throughout the experiments. After the chase was consistently > 95%. Extracted lipids were analyzed by the method period, the cells were washed three times with PBS, then detached by described above. incubation with 0.25% trypsin for 3 min at 37°C. To neutralize tryp- For the quantification of lipid, each radioactive area on the plate sin, 1% BSA in PBS was added. The cells were harvested by centrifuga- was carefully scraped by tracing the autoradiogram and the radioactiv- tion and washed three times with PBS. Without this procedure, about ity (expressed by disintegrations per minute) was measured using a 1.3 times higher radioactivity was detected in the cells. The increased scintillation counter (Mark III; Tracor Analytic, Chicago, IL) after radioactivity was found to be solely unhydrolyzed 14C-CS detected in addition of 4 ml of ACN II (Amersham Corp., Ontario, Canada). The plasma membrane plus Golgi fraction. This washing procedure effec- amount of each lipid is expressed as a percentage of total radioactivity tively removed the attached `4C-CS from cell surface. taken up by the cells. Subcellular fractionation. Fractionation was conducted using the For the identification of radioactive lipid, each radioactive spot on self-forming gradient medium (Percoll). Cell pellets obtained by the the plate was scraped, eluted from silica gel and cochromatographed method described above were suspended in 0.5 ml of 0.25 M sucrose with standard lipids in two-dimensional
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