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Role of lysosomal acid ceramidase in the metabolism of ceramide in human skin fibroblasts
ARCHIVES
Vol.
OF BIOCHEMISTRY
208, No. 2, May,
AND BIOPHYSICS pp. 444-455, 1981
Role of Lysosomal Acid Ceramidase in the Metabolism Ceramide in Human Skin Fibroblastsl WINSTON
W. CHEN,2 John
F.
ANN
B. MOSER,
AND HUGO
of
W. MOSER
Kennedy Institute Johns
and the Department of Neurology, Hopkins University School of Medicine, Baltimore, Maryland 21205 Received December 3, 1980
A Z&fold accumulation of ceramide was demonstrated in cultured skin fibroblasts from a patient with Farber’s disease, an inborn error of metabolism in which acid ceramidase activity is deficient. To investigate the role of acid ceramidase in the metabolism of ceramide in fibroblasts, we have investigated the lysosomal degradation of ceramide that was taken up by fibroblasts from an exogenous lipid suspension. Fluorescent 4-nitrobenz-2-oxa-1,3-diazole-7-aminododecanoyl-sphingosine (NBD-ceramide) from an exogenous ceramide suspension was incorporated into the intracellular structures of fibroblasts at 37°C. Study of the cellular uptake of exogenous [3H]oleylsphingosine showed that the rate of ceramide accumulation was nearly identical in Farber’s disease and normal fibroblasts. The deficiency of acid ceramidase in Farber’s fibroblasts resulted in the decrease of cellular degradation and uptake of ceramide and the increase of retention time of ceramide in these diseased cells. Studies of subcellular fractionation of these fibroblasts showed that the accumulated ceramide was located in the lysosomal fraction. As a result, the density of the lysosomal fraction of Farber’s fibroblasts was found to be less than that of controls. These results suggest the defect of cellular metabolism in this inherited disease is located within the lysosome.
Previous studies have shown that cultured fibroblasts rapidly incorporated exogenous lipids via uptake processes of liposomes and low-density lipoprotein (l-9). Lysosomes have also been implicated in the catabolism of exogenous lipids, since degradation of lipids in lysosomes is impaired by amphiphilic cationic drugs, such as chloroquine, which are known to affect lysosomal processes (10-18). Moreover, direct evidence that the degradation of cholesterol esters takes place in lysosomes was provided by studies on skin fibroblasts cultured from patients with a genetically determined deficiency of acid cholesterol esterase and in which cholesterol esters accumulated (19, 20). However, little is known about the uptake and metabolism of ceramide in intact fibroblasts. The present ‘This work has been supported in part by Grants 2ROl-NS13513-02 and NS 16955-01 from the National Institutes of Health and PEW Foundation. “To whom requests for reprints should be addressed. 0003-9861/81/060444-12$02.00/O Copyright C 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
studies were undertaken to investigate the incorporation of an exogenous ceramide suspension into normal cultured skin fibroblasts, and to follow the turnover of this compound in these cells. We then compared these activities with those in fibroblasts from patients with Farber’s disease, which have been shown in vitro to be severely deficient in acid ceramidase. Farber’s disease (lipogranulomatosis) is a rare disorder with an autosomal recessive inheritance (21, 22). It is characterized by accumulation of ceramide in the spinal cord, brain stem, and dorsal root ganglia (23,24) and excretion of excessive amounts of ceramide in the urine (25). The accumulation of ceramide in kidney and cerebellum has been shown to correlate with the deficiency of acid ceramidase (26). The deficiency of this enzyme has also been demonstrated in cultured skin fibroblasts (27) and leukocytes (28). These tissues also contain an alkaline ceramidase but this does not appear to be deficient in Farber’s disease (29).
444
HUMAN
LYSOSOMAL
ACID CERAMIDASE
Ultrastructural studies of Farber’s subcutaneous and visceral tissues have shown inclusions that contain lamellar, rectilinear or curvilinear structures (30-32). In cultured Farber’s skin fibroblasts, an accumulation of small curvilinear structures has been demonstrated after ceramide was added to the culture medium (33). Although i.t is clear that the deficiency of acid ceramidase is a primary defect in Farber’s disease, and it has been suggested that this deficiency leads to the accumulation of ceramide in pathological tissues (27-29), how the deficiency of ceramidase affects the accumulated ceramide and its subcellular location has not been reported. In this report, we have investigated the metabolic role of acid ceramidase in cultured skin fibroblasts by permitting the cells to take up ceramide and then studying the degradation of the accumulated ceramide in lysosomes. This report describes a comparison of the accumulation and metabolism of ceramide between normal and Farber’s diseased fibroblasts, and shows that ceramide accumulates within the lysosomal fraction, thus significantly lowering its density. EXPERIMENTAL
PROCEDURES
Materials and chromatographic procedure. 9,16[3H]Oleic acid (sp act, 5.66 Ci/mmol) was purchased from New England Nuclear (Boston, Mass.). Eagle’s minimum essential medium, penicillin, streptomycin, glutamine, and fetal calf serum were purchased from Grand Island Biological Company (Grand Island, N. Y.). Sphingosine, oleic acid, sphingomyelin, cerebroside, ceramide, galactoceramide, lactosylceramide, and GM3 gangliaside were obtained from Supelco Inc. (Bellefonte, Pa.). 4-Nitrobenz-2-oxa-1,3-diazole-7aminododecanoic acid (NBD-dodecanoic acid)3 was generously supplied by Drs. D. K. Struck and R. E. Pagano, Carnegie Institution of Washington, Baltimore, Maryland. Triphenylphosphine and 2,2’-dipyridyldisulfide (Aldithiol 2) were purchased from Eastman Kodak (Rochester, N. Y.). Methylene chloride was obtained from Burdick and Jackson Laboratories (Muskegon, Mich.). Thin-layer chromatography was
3 Abbreviations used: NBD-dodecanoic acid, 4-nitrobenz-2-oxa-1,3-diazole-7-aminododecanoic acid; NBD-ceramide, 4-nitrobenz-2-oxa-l,3-diazole-7-aminododecanoyl-sphingosine; LDL, low-density lipoprotein; BSA, bovine serum albumin.
ROLE IN CERAMIDE
METABOLISM
445
performed on precoated plates (Analtech, Newark, Del.) in one of the following solvent systems: Solvent A, CHC13:CH30H:2 N NHIOH in Hz0 (40:1&l); Solvent B, CHC13:CH30H:Hz0 (65:25:4); and Solvent C, CHC13:CH30H:CH&OOH (94:2:5). Two-dimensional thin-layer chromatography was performed in the same plates in Solvent C and then in Solvent D, CHC13:CH30H:Hz0 (65265). The radiochromatogram was obtained by scanning with a Berthold Model LB 2760 radiochromatogram scanner. Lipids and fatty acids were detected by spraying the plate with 0.001% sodium anilinonaphthalsulfanate or by exposing it to Iy vapor. The radioactivity associated with lipids and fatty acids was detected by scraping segments of silica gel into scintillation vials for counting. Cells. The normal human fibroblasts used in this study were the same strains as those used in our previous studies (27,28). The specific activity of acid ceramidase and the ceramide content in these cells were in the same range as those in 10 other normal fibroblasts derived from skin biopsies of healthy adults and children (Table I). The fibroblasts from the patient with Farber’s disease were derived from a Syear-old girl with typical clinical manifestations, whose skin fibroblasts have been previously shown to be severely deficient in acid ceramidase (27). All cells were grown in monolayers and studied between the 3rd and 12th passages. Cells were maintained in a humidified 5% COZ-95% air atmosphere at 37°C in 60-mm petri dishes containing 5 ml of growth medium which consisted of Eagle’s minimum essential medium supplemented with penicillin (100 units/ml), streptomycin (100 rg/ml), glutamine (34 mM), and 13% fetal calf serum. Confluent monolayers of cells from 60-mm petri dishes were dissociated with 0.2% trypsin/0.02% EDTA solution and seeded at 1 X 105 cells per dish into 60-mm dishes containing 5 ml of growth medium with 13% fetal calf serum. On Day 3 the medium was replaced with 5 ml of fresh growth medium with 13% fetal calf serum. On Day 6, when the cells were not yet confluent, each monolayer was washed with 3 ml of Dulbecco’s phosphate-buffered saline. Studies of the incorporation of [3H]oleylsphingosine and NBD-ceramide were carried out with these cells (see below). Larger quantities of cells were grown in roller bottles (Corning, 143.9 x 108.72 mm). Dissociated cells from confluent monolayers were seeded at 1 X 106 cells per bottle into a roller bottle and incubated with 100 ml of growth medium supplemented with 13% fetal calf serum in a humidified incubator at 37°C under 5% CO,-95% air atmosphere. Cells were harvested by the procedure described above on Day 7 when the cells were not yet confluent (density, 30 x 106 cells per bottle). The determinations of acid ceramidase and of ceramide cellular content and the subcellular localization of lysosmal hydrolases were carried out with these cells (see below).
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of [Hloleylsphingosine and NBDmoved and the cells were treated with 0.02% try[3H]01eylsphingosine and NBD-ceramide spin/O.028 EDTA for 30 s. After removal of the trypwere prepared by a modification of the procedure of sin solution from the culture dishes, the cells were Kishimoto (34). Radioactively labeled 9,1@[3H]oleic washed twice with 2-ml portions of Dulbecco’s phosacid 0.5 mg (1 mCi), 3.5 mg of triphenylphosphine, and phate-buffered saline. The coverslips were removed 2.9 mg of 2,2’-dipyridyldisulfide were dissolved in 80 from the culture dishes and examined in a fluorescence ~1 of methylene chloride and added to 40 ~1 of a methmicroscope (Ortholux 2, Leitz Wetzlar). The light ylene chloride solution containing 1 mg of sphingosine. source from a 200-W mercury lamp was passed through a heat filter and a G, filter for uv excitation, The reaction was carried out for 18 h at room temperaand fluorescence emission was examined through a ture with vigorous stirring. The reaction was stopped by evaporation of the methylene chloride under Ne gas suppression filter K-490. Cellular incorporation and turnover of [3H]oleylat room temperature, and 1 ml of CHCl, : 0.21 N NaOH in CH,OH (2: 1) was added. The oleate ester derivasphingosine in jihroblasts. A [3H]oleylsphingosine tive was removed by mild alkaline methanolysis in 1 suspension was prepared by sonication of 200 pg of ml of CHCl,:0.21 N methanolic NaOH (2: 1) (35). After [3H]oleylsphingosine (about 36 X 106 cpm) in 2 ml of termination of methanolysis by the addition of 0.2 ml Eagle’s minimum essential medium at 0°C for 15 min. of 0.35 N acetic acid, the product [3H]oleylsphingosine Farber’s and normal fibroblasts (about 600,000 cells) was isolated by the Folch procedure (36) and purified in 60-mm Petri dishes were incubated with [3H]oleylby thin-layer chromatography in Solvent A. sphingosine suspension in a humidified incubator at 37°C for 6, 12, and 24 h. The uptake of ceramide in [3H]01eylsphingosine with R, 0.63 was recovered. The these cells was measured at these times. To measure [3H]oleylsphingosine was analyzed by thin-layer chromatography in Solvents B and C. More than 99% of the degradation of ceramide, Farber’s and normal the radioactivity comigrated with both internal and cells were incubated with [3H]oleylsphingosine suspension for 24 h and then the [3H]oleylsphingosine was external authentic oleylsphingosine. Unlabeled oleylsphingosine was prepared by the same procedure. removed. Cells were washed twice with 2 ml each of Fluorescent NBD-ceramide was prepared by using Eagle’s minimum essential medium and incubated 5 mg of NBD-dodecanoic acid under the conditions dewith 5 ml of growth medium supplemented with 13% scribed above, and the methanolysis, purification, and fetal calf serum for an additional 6, 12, and 24 h. At the analysis also were carried out by the same procedure. indicated times, cells were harvested by the following Both [3H]oleylspingosine and NBD-ceramide were procedure. The growth medium was removed, and the cells were washed with 1 ml of 0.05% trypsin/0.02% found to be hydrolyzed to labeled fatty acid by the acid ceramidase in human postmortem kidney according to EDTA solution and dissociated with 1 ml of 0.2% the procedure previously described (29). trypsin/0.02% EDTA for 1 to 2 min. One milliliter of Measurement of cemmide and of acid ceramidase growth medium (0°C) was added to stop the trypsin activity in Farber’s and normal skin jbroblasts. The reaction. Dissociated cells were transferred into a 15 ceramide in fibroblasts (30 x 106 cells) was extracted ml conical tube and centrifuged at 6009 for 10 min. Cell with 2 ml of chloroform :methanol 2 : 1 (v/v) according pellets were suspended in 5 ml of Dulbecco’s phosto the Folch procedure (36). The extracted lipids were phate-buffered saline and centrifuged, and this washsubjected to mild alkaline methanolysis in 1 ml of chlo- ing procedure was repeated twice. roform:0.21 M methanolic NaOH to remove phosphoThe protein content of the cell pellet was deterlipids (35). The isolation and analysis of ceramides mined by the method of Lowry et al. (37). Total lipids were carried out according to the method of Sugita et were extracted by the Folch procedure (36). The al. (25). Determination of the acid ceramidase activity ester-linked fatty acids were hydrolyzed by mild alkain Farber’s and normal fibroblasts was carried out on line methanolysis (35). After addition of a mixture of 30 x 108 cells, according to the method previously de- standards including oleylsphingosine, methyl fatty scribed (27). acid ester, free fatty acid, sphingomyelin, galactocereIncorporation of NBD-cemmide into jbroblasts. broside, lactosylceramide, and GM, ganglioside (10 pg Farber’s and normal fibroblasts were seeded in 60-mm of each), the [3H]oleylsphingosine was isolated by twopetri dishes containing two coverslips and incubated dimensional thin-layer chromatography in Solvents C with 5 ml of growth medium for 2 days to 30 to 40% and D as described above. confluency. NBD-ceramide suspension was prepared Subcellular localization of lysosomal fraction in by sonic treatment of 500 pg ceramide containing 50 human jibroblasts. Large quantities of Farber’s and mg of NBD-ceramide in 5 ml of Dulbecco’s phosphatenormal fibroblasts were harvested from roller bottles buffered saline at 0°C for 15 min. Farber’s and normal as described above. The cells (30 X 10s cells) were susfibroblasts in the petri dishes were incubated with 1 pended in 4 ml of 25 mM Tris-HCl buffer, pH 7.4, conml of NBD-ceramide suspension in a humidified incutaining 0.25 M sucrose, 1 mM CaCl,, and 0.01 mg/ml of bator at 37°C for 30,60, and 90 min. At these indicated BSA” (designated Tris-HCl buffer) and kept at room time intervals, the NBD-ceramide suspension was retemperature for 15 min before homogenization. FibroPreparation cemmide.
HUMAN
LYSOSOMAL TABLE
ACID
CERAMIDASE
ROLE
IN CERAMIDE
METABOLISM
447
RESULTS
I
CONCENTRATION OF CERAMIDE CONTAINING NONHYDROXY FATTY ACID AND SPECIFIC ACTIVITY OF ACID CERAMIDASE IN HUMAN SKIN FIBROBLASTS
Fibroblast line
Specific activity of acid ceramidase (nmol/h/mg protein)
Concentration of ceramide (wdmg protein)
Normal skin fibroblasts Farber’s skin fibroblasts
1.23 0.039
5.27 14.8
Note. Ceramide and acid ceramidase activity were measured as described under Experimental Procedures.
Acid Ceramidase Activity and Ceramide Content in Cultured Farber’s and Normal Skin Fibroblasts Table I shows that there was a 2.8-fold accumulation of ceramide in Farber’s fibroblasts compared with normal fibroblasts. As anticipated, acid ceramidase activity was deficient in Farber’s fibroblasts, while the activity of another lysosomal enzyme, P-N-acetylglucosaminidase, was normal. Cellular Incorporation of NBD-Ceramide into Farber’s and Normal Fibroblasts
To investigate the effect of acid ceramidase on ceramide metabolism in intact cells, we studied the incorporation of exogfluorescent NBD-ceramide into blasts were homogenized by 30 strokes at room tem- enous perature in a 5-ml glass homogenizer equipped with a Farber’s and normal fibroblast cells. MonoTeflon-coated pestle. The cell homogenate was then layers of Farber’s and normal fibroblasts immediately cooled to 0°C and centrifuged at 6009 for were incubated with NBD-ceramide sus10 min at 4°C. The pellet of unbroken cells was reho- pension at 37°C as described under Experimogenized by the same procedure and centrifuged at mental Procedures and examined by fluo600g for 10 min at 4°C. The two supernatant fractions rescence microscope. After incubation for were combined and centrifuged at 100,OOOgfor 1 hr. 30 min at 37°C a large quantity of fluoresThe membrane fraction which was recovered from the cent NBD-ceramide was observed inside pellet was suspended in 0.5 ml of 25 mM Tris-HCl Farber’s and normal cells. The intensity buffer, pH 7.4, containing 22% sucrose, 1 mM CaCly, and 0.01 mg/ml BSA and transferred on top of a 4-ml and pattern of fluorescence were similar in normal and Farber’s cells, although the inlinear sucrose gradient, from 22 to 50% sucrose, in Tris-HCl buffer. This gradient was centrifuged at tensity of fluorescence was found to be in100,OOOg for 12 h. Ten fractions of 0.45 ml were col- creased with time of incubation. Figures lected from the gradient with a Densi Flow II 1A and B, which were obtained with (Buchler). The protein content of each fraction was Farber’s cells, show that the fluorescent measured by the method of Lowry et al. (37). LysoNBD-ceramide is located mainly in the insomal b-glucosidase, /3-N-acetylglucosaminidase, and tracellular structures of cells in the vicinity arylsulfatase A were measured according to the of nuclei. A similar fluorescent NBD-ceramethods previously described (38). mide pattern was observed in normal cells Subcellular localization of [3H]oleylsphingosine in (data not shown, see Fig. 1). Farber’s Jibroblasts. To determine the localization of In a duplicate experiment, to determine accumulated [3H]oleylsphingosine in Farber’s fibrothat remaining NBD molecules were assoblasts, these cells were incubated with [3H]oleylsphingosine suspension for 24 h at 37°C and then with fresh ciated with ceramide, Farber’s and normal medium supplemented with 13% fetal calf serum for cells were incubated with NBD-ceramide an additional 24 h as described under “Incorporation suspension for 90 min at 3’7°C. Total lipids and Turnover of [“HH]Oleylsphingosine in Fibroblasts.” were extracted by Folch’s extraction proThese cells, which have accumulated [3H]oleylsphincedure (36) and analyzed by two-dimengosine, were mixed with a large quantity of nonrasional thin-layer chromatography. It was dioactive Farber’s cells (30 x 106 cells) and subjected found that the NBD fluorescence was assoto subcellular fractionation (see above). Lysosomal hyciated exclusively with the NBD-ceramide drolases, P-glucosidase, P-N-acetylglucosaminidase, fraction. and arylsulfatase A, were determined in each fraction To determine the localization of the (38). Aliquots (0.1 ml) of each fraction were used to measure radioactivity. NBD-ceramide in cell suspensions, both
448
CHEN,MOSER,ANDMOSER
HUMAN
LYSOSOMAL
ACID CERAMIDASE
cell lines were incubated with NBD-ceramide suspension under the conditions described abo,ve. After NBD-ceramide was removed, monolayer cells were dissociated with trypsin. Suspended cells were washed and immediately examined by fluorescence microscopy. As shown in Figs. 1C and D, fluorescent NBD-ceramide has a diffuse pattern and is clearly located in the intracellular structures of Farber’s cells at 37°C. No fluorescence was detected on the surface of cells. Incorporation of [3H]Oleylsphingosine into Farber’s and Normal Fibroblasts To investigate quantitatively the incorporation of ceramide into fibroblasts, monolayers of Farber’s and normal fibroblasts were incubated with [3H]oleylsphingosine suspension as described under Experimental Procedures. Ceramide, methyl oleate, and complex sphingolipids were separated by two-dime:nsional thin-layer chromatography and the radioactivity associated with these fractions was determined. As shown in Fig. 2, nearly identical amounts of [3H]oleylsphingosine were accumulated in Farber’s and normal fibroblasts. The rate of accumulation was linear with time up to 12 h before reaching a plateau. After both cells were incubated for 12 h, only about 20% of [3H]ceramide was recovered from culture media. The distribution of [3H]oleic acid in the total lipid fractions of these cells was determined. It showed that more than 85% of the radioactivity was associated with ceramide at each time point. The remaining radioactivity was found mainly (more than 60%) in methyl [3H]oleate probably derived from triglyceride and glycerophospholipids by mild alkaline methanolysis. Although the cells were incubated under these conditions for up to 24 h, fewer than 10% of the cells had divided, as judged by the cell count and protein content.
ROLE IN CERAMIDE
0
449
METABOLISM
6 I2 18 INCUBATION TIME t hr)
i 2r I
FIG. 2. Incorporation of [3H]oleylsphingosine into Farber’s and normal fibroblasts. Incubation conditions and analysis were as described under Experimental Procedures. 0, [SH]oleylsphingosine for Farber’s cells; A, [3H]oleic acid associated with other lipids for normal cells: 0, [3H]oleylsphingosine in cells; A, [3H]oleic acid associated with other lipids.
Turnover of [3H]Oleylsphinghosine Farber’s and Normal Fibroblasts
in
Monolayers of Farber’s and normal cells were incubated with [3H]oleylsphingosine at 37°C for 24 h and then incubated with fresh medium supplemented with 13% fetal calf serum for indicated time intervals as described under Experimental Procedures. No detectable increase of radioactivity was found in the lipid fractions of these media. The radioactivity in medium was found mainly in the aqueous soluble fraction. The distribution of radioactivity in these cells showed that more than 85% of the radioactivity in the lipid fraction was associated with ceramide. Figure 3 shows the remaining [3H]oleylsphingosine in Farber’s and normal fibroblasts at each time interval, At 6 h the turnover of [3H]oleylsphingosine in normal fibroblasts was 61.8% compared to 16.0% in Farber’s cells. Between 6 and 12 h an additional 2.5% of the [3H]oleylsphingosine was hydrolyzed in normal cells compared to 17.9% in Farber’s cells. At 24 h,
FIG. 1. Fluorescence and light micrographs of monolayer cells and suspended Farber’s cells treated with NBD-ceramide. Incubations were carried out at 37°C for 60 min. Suspended cells were prepared by the treatment with trypsin. (A) Fluorescence micrograph of monolayer cell, (B) light micrograph of monolayer; (C) fluorescence micrograph of suspended cells; and (D) light micrograph of suspended cells.
450
CHEN, MOSER, AND MOSER
INCUBATION
TIME (hr)
FIG. 3. Turnover of [3H]oleylsphingosine in Farber’s (0) and normal (@) fibroblasts. Incubation conditions and analysis were as described under Experimental Procedures.
the turnover in normal cells was 67.3%, compared to 36.7% in Farber’s cells. Even though a similar amount of [3H]ceramide was accumulated in both Farber’s and normal fibroblasts, as shown in Fig. 2, the rate of uptake in Farber’s fibroblasts may be different due to the different rate of turnover (Fig. 3). The apparent rate of turnover of [3H]ceramide which was originally taken up from the medium may be estimated by the rate of disappearance during the first 6 h of the chasing period (Fig. 3). Table II presents the estimated rate of turnover in both cells and shows that the apparent rate of degradation in normal cells was more than fourfold higher than that in Farber’s cells. The turnover of ceramide in normal cells was drastically decreased after 6 h, while the turnover in Farber’s cells continued up to 12 h before reaching a plateau. The decreased rate of turnover in Farber’s cells correlated with the deficiency of acid ceramidase in these cells. Since the net accumulation of rH]ceramide, as shown in Fig. 2, represented the difference between uptake and degradation of ceramide in these cells, it was therefore possible to estimate the rate of uptake by correction for the degradation of ceramide. Table II also shows that the exogenous ceramide take up by Farber’s cells (3.27 pg/h/mg of protein) was less than that taken up by the normal cells (5.6 pg/h/mg of protein).
The remaining [3H]ceramide, 63.3% in Farber’s fibroblasts and 32.7% in normal cells, remained more or less constant during the additional 12-h incubation. This result was consistent with the possibility that there are two distant pools of ceramide in these fibroblasts, and the S.&fold accumulation of ceramide in Farber’s cells as shown in Table I may be located in the slow-turnover pool. To investigate the subcellular localization of the slow-turnover fraction of [3H]ceramide in Farber’s fibroblasts, we carried out the following experiments. Localization of Lysosomal Fraction Farber’s and Normal Fibroblasts
in
The subcellular fractionation of Farber’s and normal fibroblasts was carried out by differential centrifugation and isopycnic TABLE
II
ESTIMATED RATES OF CERAMIDE DEGRADATION AND CERAMIDE UPTAKE IN FARBER’S AND NORMAL FIBROBLASTS
Human fibroblast line
Measurement Acid ceramidase activity (nmol/h/mg protein) Concentration of ceramide In culture medium (PM) Endogenous ceramide (pg/mg protein) Accumulated from culture medium (pg/mg protein) Estimated rate Ceramide degradation (wg/h/mg protein) Ceramide accumulation (wg/h/mg protein) Ceramide uptake (pg/h/mg protein)
Normal skin fibroblasts 1.23 177
Farber’s skin fibroblasts 0.039 177
5.27
14.80
27.53
25.78
2.34
0.69
2.76
2.58
5.60
3.27
Note. For each experiment about 590,060 cells were used. The specific activity of exogenous [ 3H]oleylsphingosine (18.13 x lo6 cpm/mg) was used to calculate these rates. For other details see Experimental Procedures.
HUMAN
LYSOSOMAL
ACID CERAMIDASE
centrifugation in a sucrose gradient as described under Experimental Procedures. After centrifugation of the postnuclear fraction, the activities of lysosomal hydrolases, /3-N-acetylglucosaminidase, arylsulfatase A, and p-glucosidase, distributed between soluble and membrane fractions were determined. More than 90% of /3-glucosidase was found in the membrane fraction. The recoveries of arylsulfatase A and P-N-acetylglucosaminidase activities in the membrane fraction were lower, about ‘78 and 66%, respectively. The finding of arylsulfatase A and ,&N-acetylglucosaminidase in the soluble fraction suggested that some lysosomes had been disrupted during the preparation. The ultrastructural study of purified lysosomes of Farber’s fibroblasts, as previously reported, confirmed this conclusion (39). The lysosomal fraction isolated from a sucrose density gradient was identified by measurement of the activities of the lysosomal enzymes, P-Nacetylglucosaminidase, /3-glucosidase, and arylsulfatase A. Figure 4 illustrates that as previously described (40-42) the /3-glucosidase in normal fibroblasts is located in a fraction with a sucrose density of approximately 42%. In contrast in an identical sucrose gradient, the lysosomal/3-glucosidase in Farber’s fibroblasts was detected in a fraction with a sucrose density of about 32%. The lysosomal &acetylglucosaminidase and arylsulfatase A activities were also detected in the 32% sucrose layer in Farber’s fibroblasts. Since, as shown in Table I, there is a 2.8fold accumulation of ceramide in Farber’s cells over that in normal cells, it seems likely that the lowering of the density of the lysosomal fraction in Farber’s cells is due to the accumulation of ceramide (and possibly of other lipids) in this subcellular organelle. To test this possibility, the subcellular localization of accumulated [3H]oleylsphingosine was studied as described below. Localization qf Accumulated [3H]Oleylsplaingosine in Lysosomal Fraction of -Farber’s Fibroblasts
To determine the localization of accumulated [3H]oleylsphingosine in Farber’s fi-
ROLE IN CERAMIDE
METABOLISM
451
FRACTIONS
FIG. 4. Fractionation of postnuclear supernatant preparation of Farber’s and normal fibroblasts on sucrose density gradient. Subcellular fractionations were performed and lysosomal fl-glucosidase activity, protein concentration, and sucrose density were determined as described under Experimental Procedures. (A) Distribution of P-glucosidase determined in fractions of Farber’s fibroblasts (0) and normal fibroblasts (0); (B) protein concentration of fractions prepared from Farber’s fibroblasts (A) and normal fibroblasts (A); and (C) sucrose density of each fraction m.
broblasts, Farber’s cells were incubated with [3H]oleylsphingosine suspension for 24 h and then with fresh medium supplemented with 13% fetal calf serum for an additional 24 h under the conditions given under Experimental Procedures. These cells were then mixed with a larger quantity of nonradioactive Farber’s cells (30 x lo6 cells) and subjected to subcellular fractionation as described under Experimental Procedures. It was found that some intact fibroblasts (about 10%) were recovered in the nuclear pellet. Nevertheless approximately 80% of the [3H]oleylsphingosine was found in the postnuclear
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somal fraction. The lysosomal fraction in these diseased cells is therefore substantially altered and appears to be markedly low in density. To investigate the role of lysosomes in the cellular metabolism of ceramide, a comparative study of ceramide degradation in normal and Farber’s fibroblasts was undertaken. The metabolism of ceramide is linked to the synthesis and degradation of sphingolipids, but we chose experimental conditions under which the exogenous ceraut, , , , ,lU mide taken up by these cells was primarily 2 4 6 8 IO FRACTIONS degraded, and little or none was utilized for the synthesis of sphingolipids (see Fig. 3 FIG. 5. Distribution of [3H]oleylsphingosine and pand Results). This permitted us to focus on glucosidase in subcellular fractions of Farber’s fibrothe breakdown of ceramide. To investigate blasts. Subcellular fractionation and the measurement the uptake of ceramide, we initially used of p-glucosidase were performed as described in the legend to Fig. 4. Distribution of [3H]oleylsphingosine exogenous fluorescent NBD-eeramide. We (A) and lysosomal P-glucosidase (0). found that at 37°C the fluorescent ceramide was localized in cytoplasmic structures supernatant fraction. Virtually all of the ra- of monolayer iibroblasts with a predomidioactive ceramide was recovered in the nantly particulate pattern. The distribumembrane fraction. This membrane frac- tion of fluorescent ceramide in suspension tion was further separated by centrifugacells showed a diffuse pattern with a reltion on a sucrose gradient. Activities of atively lower intensity. It was known that lysosomal hydrolases, P-N-acetylglucosafibroblasts often contained some fluoresminidase, P-glucosidase and arylsulfatase cent materials within lysosomes, but the A, and radioactivity of [3H]oleylsphingononspecific fluorescence, particularly in sine were determined in each fraction. Fig- suspension cells, was not intense enough ure 5 shows that the distribution of for the photographic detection. In our [3H]oleylsphingosine is similar to the distristudy, the fluorescent pattern of both bution of lysosomal p-glucosidase and that monolayer and suspension fibroblasts the peaks of [3H]oleylsphingosine radioac- clearly showed that no fluorescent certivity and of p-glucosidase activity were amide was detected on the surface of the both found at the 32% sucrose layer. There cells. These results were consistent with was very little detectable [3H]oleylsphingothe uptake of phospholipid liposomes via sine and lysosomal p-glucosidase at the fusion, lipid exchange, and endocytosis (ldensity in which the lysosomal fraction of 6). The combination of the uptake of flunormal fibroblasts is located. This finding orescent ceramide and the subcellular disprovides evidence that the accumulated tribution of r3H]-ceramide suggested that ceramide in Farber’s fibroblasts is localized the exogenous ceramide was indeed incorin the lysosomal fraction, and suggests that porated into the inside of the fibroblasts the accumulated ceramide is responsible at and did not merely adhere to the surface least in part for the alteration of the den- of the cells. On the basis of this finding we sity of the lysosomal fraction in Farber’s were able to investigate the cellular degdiseased fibroblasts. radation of ceramide in Farber’s and normal fibroblasts. DISCUSSION To analyze quantitatively the incorporaThis study has demonstrated that the tion of ceramide into both Farber’s and norwe used exogenous ceramide which accumulates in Farber’s fi- mal fibroblasts, broblasts resulting from the deficiency of [3H]oleylsphingosine and found that the net acid ceramidase is localized in the lyso- accumulation of ceramide in both cells was
HUM.AN
LYSOSOMAL
ACID
CERAMIDASE
nearly identical. As shown in Table II, a large amoun.t of [3H]oleylsphingosine, approximately a half of the ceramide content in normal cells (about 2.6 pg/mg of cell protein) was accumulated within an hour when a substantial quantity of exogenous [3H]ceramide was present in the medium (1’77 PM). In normal cells, the apparent rate of degradation (2.84 pg/h/mg of protein) was equally rapid, and therefore a low cellular content of ceramide was maintained under normal growth conditions. On the other hand the deficiency of acid ceramidase in Far’ber’s cells resulted in a decreased rate of degradation and the estimated rate of ceramide degradation (0.69 pg/h/mg of protein) was about one-fourth of that in normal cells. Based on the apparent rate of ceramide degradation in both cells, we found that the estimated rate of ceramide taken up by normal cells (5.60 pg/h/mg of protein) was higher than that by Farber’s cells (3.27 lg/h/mg of protein). If these calculations were carried out by correcting the specific activity of ceramide with the endogenous ceramide level shown in Table I, the obtained values would be slightly altered. But the degradation rate of ceramide in normal fibroblasts (3.38 pg/h/mg of protein) was still more than 3-fold higher than that in Farber’s cells (1.08 pg/h/mg of protein) and the rate of exogenous ceramide taken up by normal cells (6.14 pg/h/mg of protein) was 1.7-fold higher than that by Farber’s cells (3.66 pg/‘h/mg of protein) In view of the previous report by Goldstein et ~2. (43), prior incubation of cultured human fibroblasts with Triton WR 1339 was shown to block the proteolytic degradation of lowdensity lipoprotein (LDL)3 and to prevent the cholesterol of LDL from being taken up by cells. In addition it was also reported that this detergent not only concentrated in lysosomes (12, 32) but also caused hyperlipidemia (44,45). It is conceivable that the impairment of lysosomal hydrolysis may affect the cellular uptake processes. In previous studies it was shown that, in addition to acid ceramidase, an alkaline ceramidase activity was present in human fibroblasts, and this activity was not deficient in Farber’s cells. It is not unexpected
ROLE
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METABOLISM
453
therefore that in Farber’s cells ceramide turnover is only partially impaired. We found that the rate of ceramide turnover in Farber’s fibroblasts was approximately %rd to l/4th that of normal fibroblasts, while the in vitro measurement of acid ceramidase activity in these diseased cells was only 1/32nd that of normal cells. These results indicated that both alkaline and acid ceramidases were involved in catalyzing the degradation of ceramide, and that the decreased rate of ceramide degradation in these diseased fibroblasts was still sufficient to maintain normal cellular growth. Nevertheless, the findings of a decreased rate of ceramide turnover and a 2.8-fold accumulation in Farber’s cells support the previous suggestion that the enzymatic defect causing lysosomal storage is the metabolic basis of this disorder (27-29). To obtain direct evidence that the accumulated ceramide in Farber’s fibroblasts is localized in lysosomes and that sphingolipids are degraded in these organelles, we carried out a subcellular fractionation of fibroblasts by differential centrifugation and isopycnic centrifugation on a sucrose gradient. We observed first, that the lysosomal fraction isolated from Farber’s fibroblasts was low in density, and second, that the accumulated [3H]ceramide in these diseased cells was localized in these buoyant lysosomes. Our finding of a low-density lysosomal fraction suggests that the concentric lamellar vacuoles which were observed after overloading with exogenous ceramide as previously reported (33) are indeed lysosomal inclusions. It is also consistent with the notion that the catabolism of sphingolipids in intact cells takes place, at least in part, in lysosomes. It has been reported (12, 32, 44-47) that administration of indigestible materials such as Triton WR 1339, colloidal carbon, and polyvinylpyrrolidone indextrans, duced storage of lipids in lysosomes. The altered density of overloaded lysosomes permits easy isolation of this lysosomal fraction. Certain amphiphilic cationic drugs such as chloroquine might also induce storage of lipids in lysosomes (12, 13, 15, 17). Such a drug-induced lipidosis is characterized by the occurrence of acid phosphatase-
454
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MOSER,
positive cytoplasmic inclusions and it is thought to be attributed to the interference with general lysosomal function by increasing pH (10). Cellular changes, resembling those of lipidoses, were produced in normal fibroblasts by incubating them with excessive amounts of either polar lipids or ceramide (33, 48, 49). These observations strongly suggest that the density of lysosomes is influenced by the environmental conditions. Moreover, in the atheromatous aorta, a broad distribution of lysosomes, differing markedly in density, was observed upon subcellular fractionation (50, 51). This diverse distribution of lysosomes was also shown in human skin fibroblasts (40), rat hepatocytes (50-52), and two different cultured hamster fibroblast lines (53, 54). In addition, recent reports demonstrated that the 1251-asialoceruloplasmin was taken up into rat hepatocytes by endocytosis, and a-L-iduronidase incorporated into cultured human fibroblasts was initially localized in the lysosomal fraction of lower density and later seen in the dense fraction (40, 52). These findings are also consistent with the thesis that the decreased density of lysosomes, resulting from the incorporation of exogenous lipids, is a transitional state in the normal metabolic cycle. On the other hand, the accumulation of lipids in lipidosis fibroblasts, resulting from the deficiency of lysosomal hydrolases, may impair lysosomal digestion processes and, thus, may block the normal cycle of lysosomal metabolism. It is conceivable, though it has not been reported, that only a buoyant lysosomal fraction is found in these lipidosis fibroblasts. In Farber’s fibroblasts, the finding of a lysosomal fraction with markedly low density supports this hypothesis. A systematic study of the enzymatic abnormality and biochemical composition of isolated lysosomal fractions from different lipidosis fibroblasts, as well as the mechanism causing these changes, may greatly increase our understanding not only of the pathology of these disorders, but also of normal lysosomal metabolism. ACKNOWLEDGMENTS We wish to thank Mrs. lent technical assistance.
Patricia Veno for her excelWe also thank Drs. D. K.
AND
MOSER
Struck and R. E. Pagan0 for the gift of Cnitrobens-2oxa-1,3-diazole-%aminododecanoic acid and for the advice and help in investigating the uptake of NBD-ceramide, Drs. G. H. Thomas and S. W. Craig for help in the preparation of fluorescence micrographs, and Dr. Y. Kishimoto for help in the synthesis of ceramides. We gratefully acknowledge Dr. E. F. Neufeld for helpful discussion and criticism and Dr. Pamela Talalay for her expert editor-al help.
REFERENCES
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13. WIBO, M., AND POOLE, B., (1974) J. Cell Biol. 63, 430-440. 14. LULLMANN, H., LULLMANN-RAUCH, R., AND WASSERMANN, 0. (1978) Biochem. P~A~w,co~. 27, 1103-1108. 15. REIJNCOUD, D. J., AND TAGER, J. M. (1977) Biochim. Biophys. Acta 472,419-429. 16. STEIN, Y., EBIN, V., BAR-ON, H., AND STEIN, 0. (1977) Biochim. Bkphys. Acta 486, 286-297. 17. FL~REN, C. H., NORDGREN, H., AND NILSSON, A. (1977) Eur. J. Chem. 80, 331-340. 18. CHAJEK, T., FRIEDMAN, G., STEIN, O., AND STEIN, Y. (1977) B&him. Biophys. Acta 488, 270-279.
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19. G~LDSTEII\I, J. L., DANA, S. E., TAUST, J. R., BEAUDET, A. L., AND BROWN, M. S. (1975) J. Biol. Chem. 250,8487-8495. 20. GOLDSTEIN, J. L., DANA, S. E., AND BROWN, M. S. (1975) Proc. Nat. Acad. Sci. USA 71, 428% 4292. 21. FARBER, S., COHEN, J., AND UZMAN, L. L. (195’7) J. Mt. Sinai Hosp. 24, 816-837. 22. MOSER, H. W. (1978) in The Metabolic Basis of Inherited Diseases (Straubury, J. B., Wynaarden, J. B., and Fredrickson, D., eds.), 4th ed., pp. 705-717, McGraw-Hill, New York. 23. MOSER, H. W., PRENSKY, A. L., WOLFE, H. J., ROSMAN,, N. P. CARR, S., AND FERRERA, G. (1969) Amer. J. Med. 47, 869-890. 24. SAMUELSSON, E., AND ZETTERSTROM, R. (1971) Stand. J Clin. Lab. Invest. 27, 393-405. 25. SUGITA, M. IWAMORI, M. EVANS, J. MCCLUER, R. H., DULANEY, J. T., AND MOSER, H. W. (1974) J. Lipid Res. 15, 223-226. 26. SUGITA, M., DULANEY, J. T., AND MOSER, H. W. (1972) Science 178, 1100-1102. 27. DULANEY, J. T., MILUNSKY, A., SIDBURY, J. B., HOBOLTH, N., AND MOSER, H. W. (1976) J. Ped. 89, 59-61. 28. DULANEY, .J. T., AND MOSER, H. W. (1977) in Practical Enzymology of the Sphingolipidoses (Glew, R. H., and Peters, S. P., eds.), pp. 283296, Liss, New York. 29. SUGITA, M., WILLIAMS, M., DULANEY, J. T., AND MOSER, H. W. (1975) Biochim. Biophys. Acta 398, 125-130. 30. BATTIN, J., VITAL, C. L., AND A~ANZA, X. (1970) Ann. Dermatol. Syphiligr. 97, 241-248. 31. DUSTIN, P., TONDEUR, M., JONNIAUX, G., VAMOS-HIJRWITZ, E., AND PELE, S. (1973) Bull. Aced. Med. Belg. 128, 733-762. 32. VANHOOF, F., AND HERS, H. G. (1973) in Lysosome, and Storage Diseases (Hers, H. G., and VanHoof, F., eds.), pp. 559-563, Academic Press, New York. 33. DUTSTAERT, J., TANDEUR, M., VAMOS-HURWITZ, E., AND DUSTIN P. (1977) Lab. Invest. 36, 474-480. 34. KISHIMOTO, Y. (1975) Chem. Phys. Lipids 15, 33-36.
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35. KISHIMOTO, Y., DAVIES, W. E., AND RADIN, N. S. (1965) J. Lipid Res. 6, 525-509. 36. FOLCH, J., LEES, M., AND SLOAN STANLEY, G. H. (1957) J. Biol. Chem. 226, 497-509. 37. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 38. GLEW, R. H., AND PETERS, S. P. (eds.) (1977) Practical Enzymology of the Sphingolipidoses, Liss, New York. 39. CHEN, W. W., DECKER, G. L., AND MOSER, H. W. (1980) Fed. Proc. 39, 2185. 40. ROME, L. H., GARVIN, A. J., ALLIETTA, M. M., AND NEUFELD, E. F. (1979) Cell 17, 143-153. 41. PERDUE, J. F. (1974) in Methods in Engymology (Fleischer, S., and Packer, L., eds.), Vol. 31, pp. 162-168, Academic Press, New York. 42. TROUET, A. (1974) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 31, pp. 326-329, Academic Press, New York. 43. GOLDSTEIN, J. L., BRUNSCHEDE, G. L., AND BROWN, M. S. (1975) J. Biol. Chem. 250,78547862. 44. BYERS, S. O., FRIEDMAN, M., ANDSUGIYAMA, T. (1963)Amer. J. Physiol. 294, 1100-1102. 45. SCANU, A., ORIENTE, P., SZAJEWSKI, J. M., MCCORMACK, L. J., AND PAGE, I. H. (1961) J. Exp. Med. 114, 279-293. 46. WERB, Z., AND DINGLE, J. T. (1976) in Lysosomes in Biology and Pathology (Dingle, J. T., and Dean, R. T., eds.), Vol. 5, pp. 127-156, North-Holland, Amsterdam. 47. ABRAHAM, R., AND RINGWOOD, N. (1977) Exp. Mol. Pathol. 26, 13-27. 48. STERN, J. (1972) Lab. Invest. 26, 509-514. 49. STERN, J. (1973) Lab. Invest. 28, 87-93. 50. PETERS, T. J., AND DE DUVE, C. (1974) Exp. Mol. Pathol. 20, 228-256. 51. TAKANO, T., BLACK, W. J., PETERS, T. J., AND DEDWE, C. (1974) J. Biol. Chem. 249, 67326737. 52. PERTOFT, H., WARMEGARD, B., AND HOOK, M. (1978) Biochem. J. 174, 309-317. 53. WARBURTON, M. J., AND WYNN, C. H. (1976) Eur. J. Biocbm. 65, 341-348. 54. BROTHERUS, J., AND RENKONEN, 0. (1977) J. Lipid Res. 18, 191-202.
Vol.
OF BIOCHEMISTRY
208, No. 2, May,
AND BIOPHYSICS pp. 444-455, 1981
Role of Lysosomal Acid Ceramidase in the Metabolism Ceramide in Human Skin Fibroblastsl WINSTON
W. CHEN,2 John
F.
ANN
B. MOSER,
AND HUGO
of
W. MOSER
Kennedy Institute Johns
and the Department of Neurology, Hopkins University School of Medicine, Baltimore, Maryland 21205 Received December 3, 1980
A Z&fold accumulation of ceramide was demonstrated in cultured skin fibroblasts from a patient with Farber’s disease, an inborn error of metabolism in which acid ceramidase activity is deficient. To investigate the role of acid ceramidase in the metabolism of ceramide in fibroblasts, we have investigated the lysosomal degradation of ceramide that was taken up by fibroblasts from an exogenous lipid suspension. Fluorescent 4-nitrobenz-2-oxa-1,3-diazole-7-aminododecanoyl-sphingosine (NBD-ceramide) from an exogenous ceramide suspension was incorporated into the intracellular structures of fibroblasts at 37°C. Study of the cellular uptake of exogenous [3H]oleylsphingosine showed that the rate of ceramide accumulation was nearly identical in Farber’s disease and normal fibroblasts. The deficiency of acid ceramidase in Farber’s fibroblasts resulted in the decrease of cellular degradation and uptake of ceramide and the increase of retention time of ceramide in these diseased cells. Studies of subcellular fractionation of these fibroblasts showed that the accumulated ceramide was located in the lysosomal fraction. As a result, the density of the lysosomal fraction of Farber’s fibroblasts was found to be less than that of controls. These results suggest the defect of cellular metabolism in this inherited disease is located within the lysosome.
Previous studies have shown that cultured fibroblasts rapidly incorporated exogenous lipids via uptake processes of liposomes and low-density lipoprotein (l-9). Lysosomes have also been implicated in the catabolism of exogenous lipids, since degradation of lipids in lysosomes is impaired by amphiphilic cationic drugs, such as chloroquine, which are known to affect lysosomal processes (10-18). Moreover, direct evidence that the degradation of cholesterol esters takes place in lysosomes was provided by studies on skin fibroblasts cultured from patients with a genetically determined deficiency of acid cholesterol esterase and in which cholesterol esters accumulated (19, 20). However, little is known about the uptake and metabolism of ceramide in intact fibroblasts. The present ‘This work has been supported in part by Grants 2ROl-NS13513-02 and NS 16955-01 from the National Institutes of Health and PEW Foundation. “To whom requests for reprints should be addressed. 0003-9861/81/060444-12$02.00/O Copyright C 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
studies were undertaken to investigate the incorporation of an exogenous ceramide suspension into normal cultured skin fibroblasts, and to follow the turnover of this compound in these cells. We then compared these activities with those in fibroblasts from patients with Farber’s disease, which have been shown in vitro to be severely deficient in acid ceramidase. Farber’s disease (lipogranulomatosis) is a rare disorder with an autosomal recessive inheritance (21, 22). It is characterized by accumulation of ceramide in the spinal cord, brain stem, and dorsal root ganglia (23,24) and excretion of excessive amounts of ceramide in the urine (25). The accumulation of ceramide in kidney and cerebellum has been shown to correlate with the deficiency of acid ceramidase (26). The deficiency of this enzyme has also been demonstrated in cultured skin fibroblasts (27) and leukocytes (28). These tissues also contain an alkaline ceramidase but this does not appear to be deficient in Farber’s disease (29).
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Ultrastructural studies of Farber’s subcutaneous and visceral tissues have shown inclusions that contain lamellar, rectilinear or curvilinear structures (30-32). In cultured Farber’s skin fibroblasts, an accumulation of small curvilinear structures has been demonstrated after ceramide was added to the culture medium (33). Although i.t is clear that the deficiency of acid ceramidase is a primary defect in Farber’s disease, and it has been suggested that this deficiency leads to the accumulation of ceramide in pathological tissues (27-29), how the deficiency of ceramidase affects the accumulated ceramide and its subcellular location has not been reported. In this report, we have investigated the metabolic role of acid ceramidase in cultured skin fibroblasts by permitting the cells to take up ceramide and then studying the degradation of the accumulated ceramide in lysosomes. This report describes a comparison of the accumulation and metabolism of ceramide between normal and Farber’s diseased fibroblasts, and shows that ceramide accumulates within the lysosomal fraction, thus significantly lowering its density. EXPERIMENTAL
PROCEDURES
Materials and chromatographic procedure. 9,16[3H]Oleic acid (sp act, 5.66 Ci/mmol) was purchased from New England Nuclear (Boston, Mass.). Eagle’s minimum essential medium, penicillin, streptomycin, glutamine, and fetal calf serum were purchased from Grand Island Biological Company (Grand Island, N. Y.). Sphingosine, oleic acid, sphingomyelin, cerebroside, ceramide, galactoceramide, lactosylceramide, and GM3 gangliaside were obtained from Supelco Inc. (Bellefonte, Pa.). 4-Nitrobenz-2-oxa-1,3-diazole-7aminododecanoic acid (NBD-dodecanoic acid)3 was generously supplied by Drs. D. K. Struck and R. E. Pagano, Carnegie Institution of Washington, Baltimore, Maryland. Triphenylphosphine and 2,2’-dipyridyldisulfide (Aldithiol 2) were purchased from Eastman Kodak (Rochester, N. Y.). Methylene chloride was obtained from Burdick and Jackson Laboratories (Muskegon, Mich.). Thin-layer chromatography was
3 Abbreviations used: NBD-dodecanoic acid, 4-nitrobenz-2-oxa-1,3-diazole-7-aminododecanoic acid; NBD-ceramide, 4-nitrobenz-2-oxa-l,3-diazole-7-aminododecanoyl-sphingosine; LDL, low-density lipoprotein; BSA, bovine serum albumin.
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performed on precoated plates (Analtech, Newark, Del.) in one of the following solvent systems: Solvent A, CHC13:CH30H:2 N NHIOH in Hz0 (40:1&l); Solvent B, CHC13:CH30H:Hz0 (65:25:4); and Solvent C, CHC13:CH30H:CH&OOH (94:2:5). Two-dimensional thin-layer chromatography was performed in the same plates in Solvent C and then in Solvent D, CHC13:CH30H:Hz0 (65265). The radiochromatogram was obtained by scanning with a Berthold Model LB 2760 radiochromatogram scanner. Lipids and fatty acids were detected by spraying the plate with 0.001% sodium anilinonaphthalsulfanate or by exposing it to Iy vapor. The radioactivity associated with lipids and fatty acids was detected by scraping segments of silica gel into scintillation vials for counting. Cells. The normal human fibroblasts used in this study were the same strains as those used in our previous studies (27,28). The specific activity of acid ceramidase and the ceramide content in these cells were in the same range as those in 10 other normal fibroblasts derived from skin biopsies of healthy adults and children (Table I). The fibroblasts from the patient with Farber’s disease were derived from a Syear-old girl with typical clinical manifestations, whose skin fibroblasts have been previously shown to be severely deficient in acid ceramidase (27). All cells were grown in monolayers and studied between the 3rd and 12th passages. Cells were maintained in a humidified 5% COZ-95% air atmosphere at 37°C in 60-mm petri dishes containing 5 ml of growth medium which consisted of Eagle’s minimum essential medium supplemented with penicillin (100 units/ml), streptomycin (100 rg/ml), glutamine (34 mM), and 13% fetal calf serum. Confluent monolayers of cells from 60-mm petri dishes were dissociated with 0.2% trypsin/0.02% EDTA solution and seeded at 1 X 105 cells per dish into 60-mm dishes containing 5 ml of growth medium with 13% fetal calf serum. On Day 3 the medium was replaced with 5 ml of fresh growth medium with 13% fetal calf serum. On Day 6, when the cells were not yet confluent, each monolayer was washed with 3 ml of Dulbecco’s phosphate-buffered saline. Studies of the incorporation of [3H]oleylsphingosine and NBD-ceramide were carried out with these cells (see below). Larger quantities of cells were grown in roller bottles (Corning, 143.9 x 108.72 mm). Dissociated cells from confluent monolayers were seeded at 1 X 106 cells per bottle into a roller bottle and incubated with 100 ml of growth medium supplemented with 13% fetal calf serum in a humidified incubator at 37°C under 5% CO,-95% air atmosphere. Cells were harvested by the procedure described above on Day 7 when the cells were not yet confluent (density, 30 x 106 cells per bottle). The determinations of acid ceramidase and of ceramide cellular content and the subcellular localization of lysosmal hydrolases were carried out with these cells (see below).
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of [Hloleylsphingosine and NBDmoved and the cells were treated with 0.02% try[3H]01eylsphingosine and NBD-ceramide spin/O.028 EDTA for 30 s. After removal of the trypwere prepared by a modification of the procedure of sin solution from the culture dishes, the cells were Kishimoto (34). Radioactively labeled 9,1@[3H]oleic washed twice with 2-ml portions of Dulbecco’s phosacid 0.5 mg (1 mCi), 3.5 mg of triphenylphosphine, and phate-buffered saline. The coverslips were removed 2.9 mg of 2,2’-dipyridyldisulfide were dissolved in 80 from the culture dishes and examined in a fluorescence ~1 of methylene chloride and added to 40 ~1 of a methmicroscope (Ortholux 2, Leitz Wetzlar). The light ylene chloride solution containing 1 mg of sphingosine. source from a 200-W mercury lamp was passed through a heat filter and a G, filter for uv excitation, The reaction was carried out for 18 h at room temperaand fluorescence emission was examined through a ture with vigorous stirring. The reaction was stopped by evaporation of the methylene chloride under Ne gas suppression filter K-490. Cellular incorporation and turnover of [3H]oleylat room temperature, and 1 ml of CHCl, : 0.21 N NaOH in CH,OH (2: 1) was added. The oleate ester derivasphingosine in jihroblasts. A [3H]oleylsphingosine tive was removed by mild alkaline methanolysis in 1 suspension was prepared by sonication of 200 pg of ml of CHCl,:0.21 N methanolic NaOH (2: 1) (35). After [3H]oleylsphingosine (about 36 X 106 cpm) in 2 ml of termination of methanolysis by the addition of 0.2 ml Eagle’s minimum essential medium at 0°C for 15 min. of 0.35 N acetic acid, the product [3H]oleylsphingosine Farber’s and normal fibroblasts (about 600,000 cells) was isolated by the Folch procedure (36) and purified in 60-mm Petri dishes were incubated with [3H]oleylby thin-layer chromatography in Solvent A. sphingosine suspension in a humidified incubator at 37°C for 6, 12, and 24 h. The uptake of ceramide in [3H]01eylsphingosine with R, 0.63 was recovered. The these cells was measured at these times. To measure [3H]oleylsphingosine was analyzed by thin-layer chromatography in Solvents B and C. More than 99% of the degradation of ceramide, Farber’s and normal the radioactivity comigrated with both internal and cells were incubated with [3H]oleylsphingosine suspension for 24 h and then the [3H]oleylsphingosine was external authentic oleylsphingosine. Unlabeled oleylsphingosine was prepared by the same procedure. removed. Cells were washed twice with 2 ml each of Fluorescent NBD-ceramide was prepared by using Eagle’s minimum essential medium and incubated 5 mg of NBD-dodecanoic acid under the conditions dewith 5 ml of growth medium supplemented with 13% scribed above, and the methanolysis, purification, and fetal calf serum for an additional 6, 12, and 24 h. At the analysis also were carried out by the same procedure. indicated times, cells were harvested by the following Both [3H]oleylspingosine and NBD-ceramide were procedure. The growth medium was removed, and the cells were washed with 1 ml of 0.05% trypsin/0.02% found to be hydrolyzed to labeled fatty acid by the acid ceramidase in human postmortem kidney according to EDTA solution and dissociated with 1 ml of 0.2% the procedure previously described (29). trypsin/0.02% EDTA for 1 to 2 min. One milliliter of Measurement of cemmide and of acid ceramidase growth medium (0°C) was added to stop the trypsin activity in Farber’s and normal skin jbroblasts. The reaction. Dissociated cells were transferred into a 15 ceramide in fibroblasts (30 x 106 cells) was extracted ml conical tube and centrifuged at 6009 for 10 min. Cell with 2 ml of chloroform :methanol 2 : 1 (v/v) according pellets were suspended in 5 ml of Dulbecco’s phosto the Folch procedure (36). The extracted lipids were phate-buffered saline and centrifuged, and this washsubjected to mild alkaline methanolysis in 1 ml of chlo- ing procedure was repeated twice. roform:0.21 M methanolic NaOH to remove phosphoThe protein content of the cell pellet was deterlipids (35). The isolation and analysis of ceramides mined by the method of Lowry et al. (37). Total lipids were carried out according to the method of Sugita et were extracted by the Folch procedure (36). The al. (25). Determination of the acid ceramidase activity ester-linked fatty acids were hydrolyzed by mild alkain Farber’s and normal fibroblasts was carried out on line methanolysis (35). After addition of a mixture of 30 x 108 cells, according to the method previously de- standards including oleylsphingosine, methyl fatty scribed (27). acid ester, free fatty acid, sphingomyelin, galactocereIncorporation of NBD-cemmide into jbroblasts. broside, lactosylceramide, and GM, ganglioside (10 pg Farber’s and normal fibroblasts were seeded in 60-mm of each), the [3H]oleylsphingosine was isolated by twopetri dishes containing two coverslips and incubated dimensional thin-layer chromatography in Solvents C with 5 ml of growth medium for 2 days to 30 to 40% and D as described above. confluency. NBD-ceramide suspension was prepared Subcellular localization of lysosomal fraction in by sonic treatment of 500 pg ceramide containing 50 human jibroblasts. Large quantities of Farber’s and mg of NBD-ceramide in 5 ml of Dulbecco’s phosphatenormal fibroblasts were harvested from roller bottles buffered saline at 0°C for 15 min. Farber’s and normal as described above. The cells (30 X 10s cells) were susfibroblasts in the petri dishes were incubated with 1 pended in 4 ml of 25 mM Tris-HCl buffer, pH 7.4, conml of NBD-ceramide suspension in a humidified incutaining 0.25 M sucrose, 1 mM CaCl,, and 0.01 mg/ml of bator at 37°C for 30,60, and 90 min. At these indicated BSA” (designated Tris-HCl buffer) and kept at room time intervals, the NBD-ceramide suspension was retemperature for 15 min before homogenization. FibroPreparation cemmide.
HUMAN
LYSOSOMAL TABLE
ACID
CERAMIDASE
ROLE
IN CERAMIDE
METABOLISM
447
RESULTS
I
CONCENTRATION OF CERAMIDE CONTAINING NONHYDROXY FATTY ACID AND SPECIFIC ACTIVITY OF ACID CERAMIDASE IN HUMAN SKIN FIBROBLASTS
Fibroblast line
Specific activity of acid ceramidase (nmol/h/mg protein)
Concentration of ceramide (wdmg protein)
Normal skin fibroblasts Farber’s skin fibroblasts
1.23 0.039
5.27 14.8
Note. Ceramide and acid ceramidase activity were measured as described under Experimental Procedures.
Acid Ceramidase Activity and Ceramide Content in Cultured Farber’s and Normal Skin Fibroblasts Table I shows that there was a 2.8-fold accumulation of ceramide in Farber’s fibroblasts compared with normal fibroblasts. As anticipated, acid ceramidase activity was deficient in Farber’s fibroblasts, while the activity of another lysosomal enzyme, P-N-acetylglucosaminidase, was normal. Cellular Incorporation of NBD-Ceramide into Farber’s and Normal Fibroblasts
To investigate the effect of acid ceramidase on ceramide metabolism in intact cells, we studied the incorporation of exogfluorescent NBD-ceramide into blasts were homogenized by 30 strokes at room tem- enous perature in a 5-ml glass homogenizer equipped with a Farber’s and normal fibroblast cells. MonoTeflon-coated pestle. The cell homogenate was then layers of Farber’s and normal fibroblasts immediately cooled to 0°C and centrifuged at 6009 for were incubated with NBD-ceramide sus10 min at 4°C. The pellet of unbroken cells was reho- pension at 37°C as described under Experimogenized by the same procedure and centrifuged at mental Procedures and examined by fluo600g for 10 min at 4°C. The two supernatant fractions rescence microscope. After incubation for were combined and centrifuged at 100,OOOgfor 1 hr. 30 min at 37°C a large quantity of fluoresThe membrane fraction which was recovered from the cent NBD-ceramide was observed inside pellet was suspended in 0.5 ml of 25 mM Tris-HCl Farber’s and normal cells. The intensity buffer, pH 7.4, containing 22% sucrose, 1 mM CaCly, and 0.01 mg/ml BSA and transferred on top of a 4-ml and pattern of fluorescence were similar in normal and Farber’s cells, although the inlinear sucrose gradient, from 22 to 50% sucrose, in Tris-HCl buffer. This gradient was centrifuged at tensity of fluorescence was found to be in100,OOOg for 12 h. Ten fractions of 0.45 ml were col- creased with time of incubation. Figures lected from the gradient with a Densi Flow II 1A and B, which were obtained with (Buchler). The protein content of each fraction was Farber’s cells, show that the fluorescent measured by the method of Lowry et al. (37). LysoNBD-ceramide is located mainly in the insomal b-glucosidase, /3-N-acetylglucosaminidase, and tracellular structures of cells in the vicinity arylsulfatase A were measured according to the of nuclei. A similar fluorescent NBD-ceramethods previously described (38). mide pattern was observed in normal cells Subcellular localization of [3H]oleylsphingosine in (data not shown, see Fig. 1). Farber’s Jibroblasts. To determine the localization of In a duplicate experiment, to determine accumulated [3H]oleylsphingosine in Farber’s fibrothat remaining NBD molecules were assoblasts, these cells were incubated with [3H]oleylsphingosine suspension for 24 h at 37°C and then with fresh ciated with ceramide, Farber’s and normal medium supplemented with 13% fetal calf serum for cells were incubated with NBD-ceramide an additional 24 h as described under “Incorporation suspension for 90 min at 3’7°C. Total lipids and Turnover of [“HH]Oleylsphingosine in Fibroblasts.” were extracted by Folch’s extraction proThese cells, which have accumulated [3H]oleylsphincedure (36) and analyzed by two-dimengosine, were mixed with a large quantity of nonrasional thin-layer chromatography. It was dioactive Farber’s cells (30 x 106 cells) and subjected found that the NBD fluorescence was assoto subcellular fractionation (see above). Lysosomal hyciated exclusively with the NBD-ceramide drolases, P-glucosidase, P-N-acetylglucosaminidase, fraction. and arylsulfatase A, were determined in each fraction To determine the localization of the (38). Aliquots (0.1 ml) of each fraction were used to measure radioactivity. NBD-ceramide in cell suspensions, both
448
CHEN,MOSER,ANDMOSER
HUMAN
LYSOSOMAL
ACID CERAMIDASE
cell lines were incubated with NBD-ceramide suspension under the conditions described abo,ve. After NBD-ceramide was removed, monolayer cells were dissociated with trypsin. Suspended cells were washed and immediately examined by fluorescence microscopy. As shown in Figs. 1C and D, fluorescent NBD-ceramide has a diffuse pattern and is clearly located in the intracellular structures of Farber’s cells at 37°C. No fluorescence was detected on the surface of cells. Incorporation of [3H]Oleylsphingosine into Farber’s and Normal Fibroblasts To investigate quantitatively the incorporation of ceramide into fibroblasts, monolayers of Farber’s and normal fibroblasts were incubated with [3H]oleylsphingosine suspension as described under Experimental Procedures. Ceramide, methyl oleate, and complex sphingolipids were separated by two-dime:nsional thin-layer chromatography and the radioactivity associated with these fractions was determined. As shown in Fig. 2, nearly identical amounts of [3H]oleylsphingosine were accumulated in Farber’s and normal fibroblasts. The rate of accumulation was linear with time up to 12 h before reaching a plateau. After both cells were incubated for 12 h, only about 20% of [3H]ceramide was recovered from culture media. The distribution of [3H]oleic acid in the total lipid fractions of these cells was determined. It showed that more than 85% of the radioactivity was associated with ceramide at each time point. The remaining radioactivity was found mainly (more than 60%) in methyl [3H]oleate probably derived from triglyceride and glycerophospholipids by mild alkaline methanolysis. Although the cells were incubated under these conditions for up to 24 h, fewer than 10% of the cells had divided, as judged by the cell count and protein content.
ROLE IN CERAMIDE
0
449
METABOLISM
6 I2 18 INCUBATION TIME t hr)
i 2r I
FIG. 2. Incorporation of [3H]oleylsphingosine into Farber’s and normal fibroblasts. Incubation conditions and analysis were as described under Experimental Procedures. 0, [SH]oleylsphingosine for Farber’s cells; A, [3H]oleic acid associated with other lipids for normal cells: 0, [3H]oleylsphingosine in cells; A, [3H]oleic acid associated with other lipids.
Turnover of [3H]Oleylsphinghosine Farber’s and Normal Fibroblasts
in
Monolayers of Farber’s and normal cells were incubated with [3H]oleylsphingosine at 37°C for 24 h and then incubated with fresh medium supplemented with 13% fetal calf serum for indicated time intervals as described under Experimental Procedures. No detectable increase of radioactivity was found in the lipid fractions of these media. The radioactivity in medium was found mainly in the aqueous soluble fraction. The distribution of radioactivity in these cells showed that more than 85% of the radioactivity in the lipid fraction was associated with ceramide. Figure 3 shows the remaining [3H]oleylsphingosine in Farber’s and normal fibroblasts at each time interval, At 6 h the turnover of [3H]oleylsphingosine in normal fibroblasts was 61.8% compared to 16.0% in Farber’s cells. Between 6 and 12 h an additional 2.5% of the [3H]oleylsphingosine was hydrolyzed in normal cells compared to 17.9% in Farber’s cells. At 24 h,
FIG. 1. Fluorescence and light micrographs of monolayer cells and suspended Farber’s cells treated with NBD-ceramide. Incubations were carried out at 37°C for 60 min. Suspended cells were prepared by the treatment with trypsin. (A) Fluorescence micrograph of monolayer cell, (B) light micrograph of monolayer; (C) fluorescence micrograph of suspended cells; and (D) light micrograph of suspended cells.
450
CHEN, MOSER, AND MOSER
INCUBATION
TIME (hr)
FIG. 3. Turnover of [3H]oleylsphingosine in Farber’s (0) and normal (@) fibroblasts. Incubation conditions and analysis were as described under Experimental Procedures.
the turnover in normal cells was 67.3%, compared to 36.7% in Farber’s cells. Even though a similar amount of [3H]ceramide was accumulated in both Farber’s and normal fibroblasts, as shown in Fig. 2, the rate of uptake in Farber’s fibroblasts may be different due to the different rate of turnover (Fig. 3). The apparent rate of turnover of [3H]ceramide which was originally taken up from the medium may be estimated by the rate of disappearance during the first 6 h of the chasing period (Fig. 3). Table II presents the estimated rate of turnover in both cells and shows that the apparent rate of degradation in normal cells was more than fourfold higher than that in Farber’s cells. The turnover of ceramide in normal cells was drastically decreased after 6 h, while the turnover in Farber’s cells continued up to 12 h before reaching a plateau. The decreased rate of turnover in Farber’s cells correlated with the deficiency of acid ceramidase in these cells. Since the net accumulation of rH]ceramide, as shown in Fig. 2, represented the difference between uptake and degradation of ceramide in these cells, it was therefore possible to estimate the rate of uptake by correction for the degradation of ceramide. Table II also shows that the exogenous ceramide take up by Farber’s cells (3.27 pg/h/mg of protein) was less than that taken up by the normal cells (5.6 pg/h/mg of protein).
The remaining [3H]ceramide, 63.3% in Farber’s fibroblasts and 32.7% in normal cells, remained more or less constant during the additional 12-h incubation. This result was consistent with the possibility that there are two distant pools of ceramide in these fibroblasts, and the S.&fold accumulation of ceramide in Farber’s cells as shown in Table I may be located in the slow-turnover pool. To investigate the subcellular localization of the slow-turnover fraction of [3H]ceramide in Farber’s fibroblasts, we carried out the following experiments. Localization of Lysosomal Fraction Farber’s and Normal Fibroblasts
in
The subcellular fractionation of Farber’s and normal fibroblasts was carried out by differential centrifugation and isopycnic TABLE
II
ESTIMATED RATES OF CERAMIDE DEGRADATION AND CERAMIDE UPTAKE IN FARBER’S AND NORMAL FIBROBLASTS
Human fibroblast line
Measurement Acid ceramidase activity (nmol/h/mg protein) Concentration of ceramide In culture medium (PM) Endogenous ceramide (pg/mg protein) Accumulated from culture medium (pg/mg protein) Estimated rate Ceramide degradation (wg/h/mg protein) Ceramide accumulation (wg/h/mg protein) Ceramide uptake (pg/h/mg protein)
Normal skin fibroblasts 1.23 177
Farber’s skin fibroblasts 0.039 177
5.27
14.80
27.53
25.78
2.34
0.69
2.76
2.58
5.60
3.27
Note. For each experiment about 590,060 cells were used. The specific activity of exogenous [ 3H]oleylsphingosine (18.13 x lo6 cpm/mg) was used to calculate these rates. For other details see Experimental Procedures.
HUMAN
LYSOSOMAL
ACID CERAMIDASE
centrifugation in a sucrose gradient as described under Experimental Procedures. After centrifugation of the postnuclear fraction, the activities of lysosomal hydrolases, /3-N-acetylglucosaminidase, arylsulfatase A, and p-glucosidase, distributed between soluble and membrane fractions were determined. More than 90% of /3-glucosidase was found in the membrane fraction. The recoveries of arylsulfatase A and P-N-acetylglucosaminidase activities in the membrane fraction were lower, about ‘78 and 66%, respectively. The finding of arylsulfatase A and ,&N-acetylglucosaminidase in the soluble fraction suggested that some lysosomes had been disrupted during the preparation. The ultrastructural study of purified lysosomes of Farber’s fibroblasts, as previously reported, confirmed this conclusion (39). The lysosomal fraction isolated from a sucrose density gradient was identified by measurement of the activities of the lysosomal enzymes, P-Nacetylglucosaminidase, /3-glucosidase, and arylsulfatase A. Figure 4 illustrates that as previously described (40-42) the /3-glucosidase in normal fibroblasts is located in a fraction with a sucrose density of approximately 42%. In contrast in an identical sucrose gradient, the lysosomal/3-glucosidase in Farber’s fibroblasts was detected in a fraction with a sucrose density of about 32%. The lysosomal &acetylglucosaminidase and arylsulfatase A activities were also detected in the 32% sucrose layer in Farber’s fibroblasts. Since, as shown in Table I, there is a 2.8fold accumulation of ceramide in Farber’s cells over that in normal cells, it seems likely that the lowering of the density of the lysosomal fraction in Farber’s cells is due to the accumulation of ceramide (and possibly of other lipids) in this subcellular organelle. To test this possibility, the subcellular localization of accumulated [3H]oleylsphingosine was studied as described below. Localization qf Accumulated [3H]Oleylsplaingosine in Lysosomal Fraction of -Farber’s Fibroblasts
To determine the localization of accumulated [3H]oleylsphingosine in Farber’s fi-
ROLE IN CERAMIDE
METABOLISM
451
FRACTIONS
FIG. 4. Fractionation of postnuclear supernatant preparation of Farber’s and normal fibroblasts on sucrose density gradient. Subcellular fractionations were performed and lysosomal fl-glucosidase activity, protein concentration, and sucrose density were determined as described under Experimental Procedures. (A) Distribution of P-glucosidase determined in fractions of Farber’s fibroblasts (0) and normal fibroblasts (0); (B) protein concentration of fractions prepared from Farber’s fibroblasts (A) and normal fibroblasts (A); and (C) sucrose density of each fraction m.
broblasts, Farber’s cells were incubated with [3H]oleylsphingosine suspension for 24 h and then with fresh medium supplemented with 13% fetal calf serum for an additional 24 h under the conditions given under Experimental Procedures. These cells were then mixed with a larger quantity of nonradioactive Farber’s cells (30 x lo6 cells) and subjected to subcellular fractionation as described under Experimental Procedures. It was found that some intact fibroblasts (about 10%) were recovered in the nuclear pellet. Nevertheless approximately 80% of the [3H]oleylsphingosine was found in the postnuclear
452
CHEN, MOSER, AND MOSER
somal fraction. The lysosomal fraction in these diseased cells is therefore substantially altered and appears to be markedly low in density. To investigate the role of lysosomes in the cellular metabolism of ceramide, a comparative study of ceramide degradation in normal and Farber’s fibroblasts was undertaken. The metabolism of ceramide is linked to the synthesis and degradation of sphingolipids, but we chose experimental conditions under which the exogenous ceraut, , , , ,lU mide taken up by these cells was primarily 2 4 6 8 IO FRACTIONS degraded, and little or none was utilized for the synthesis of sphingolipids (see Fig. 3 FIG. 5. Distribution of [3H]oleylsphingosine and pand Results). This permitted us to focus on glucosidase in subcellular fractions of Farber’s fibrothe breakdown of ceramide. To investigate blasts. Subcellular fractionation and the measurement the uptake of ceramide, we initially used of p-glucosidase were performed as described in the legend to Fig. 4. Distribution of [3H]oleylsphingosine exogenous fluorescent NBD-eeramide. We (A) and lysosomal P-glucosidase (0). found that at 37°C the fluorescent ceramide was localized in cytoplasmic structures supernatant fraction. Virtually all of the ra- of monolayer iibroblasts with a predomidioactive ceramide was recovered in the nantly particulate pattern. The distribumembrane fraction. This membrane frac- tion of fluorescent ceramide in suspension tion was further separated by centrifugacells showed a diffuse pattern with a reltion on a sucrose gradient. Activities of atively lower intensity. It was known that lysosomal hydrolases, P-N-acetylglucosafibroblasts often contained some fluoresminidase, P-glucosidase and arylsulfatase cent materials within lysosomes, but the A, and radioactivity of [3H]oleylsphingononspecific fluorescence, particularly in sine were determined in each fraction. Fig- suspension cells, was not intense enough ure 5 shows that the distribution of for the photographic detection. In our [3H]oleylsphingosine is similar to the distristudy, the fluorescent pattern of both bution of lysosomal p-glucosidase and that monolayer and suspension fibroblasts the peaks of [3H]oleylsphingosine radioac- clearly showed that no fluorescent certivity and of p-glucosidase activity were amide was detected on the surface of the both found at the 32% sucrose layer. There cells. These results were consistent with was very little detectable [3H]oleylsphingothe uptake of phospholipid liposomes via sine and lysosomal p-glucosidase at the fusion, lipid exchange, and endocytosis (ldensity in which the lysosomal fraction of 6). The combination of the uptake of flunormal fibroblasts is located. This finding orescent ceramide and the subcellular disprovides evidence that the accumulated tribution of r3H]-ceramide suggested that ceramide in Farber’s fibroblasts is localized the exogenous ceramide was indeed incorin the lysosomal fraction, and suggests that porated into the inside of the fibroblasts the accumulated ceramide is responsible at and did not merely adhere to the surface least in part for the alteration of the den- of the cells. On the basis of this finding we sity of the lysosomal fraction in Farber’s were able to investigate the cellular degdiseased fibroblasts. radation of ceramide in Farber’s and normal fibroblasts. DISCUSSION To analyze quantitatively the incorporaThis study has demonstrated that the tion of ceramide into both Farber’s and norwe used exogenous ceramide which accumulates in Farber’s fi- mal fibroblasts, broblasts resulting from the deficiency of [3H]oleylsphingosine and found that the net acid ceramidase is localized in the lyso- accumulation of ceramide in both cells was
HUM.AN
LYSOSOMAL
ACID
CERAMIDASE
nearly identical. As shown in Table II, a large amoun.t of [3H]oleylsphingosine, approximately a half of the ceramide content in normal cells (about 2.6 pg/mg of cell protein) was accumulated within an hour when a substantial quantity of exogenous [3H]ceramide was present in the medium (1’77 PM). In normal cells, the apparent rate of degradation (2.84 pg/h/mg of protein) was equally rapid, and therefore a low cellular content of ceramide was maintained under normal growth conditions. On the other hand the deficiency of acid ceramidase in Far’ber’s cells resulted in a decreased rate of degradation and the estimated rate of ceramide degradation (0.69 pg/h/mg of protein) was about one-fourth of that in normal cells. Based on the apparent rate of ceramide degradation in both cells, we found that the estimated rate of ceramide taken up by normal cells (5.60 pg/h/mg of protein) was higher than that by Farber’s cells (3.27 lg/h/mg of protein). If these calculations were carried out by correcting the specific activity of ceramide with the endogenous ceramide level shown in Table I, the obtained values would be slightly altered. But the degradation rate of ceramide in normal fibroblasts (3.38 pg/h/mg of protein) was still more than 3-fold higher than that in Farber’s cells (1.08 pg/h/mg of protein) and the rate of exogenous ceramide taken up by normal cells (6.14 pg/h/mg of protein) was 1.7-fold higher than that by Farber’s cells (3.66 pg/‘h/mg of protein) In view of the previous report by Goldstein et ~2. (43), prior incubation of cultured human fibroblasts with Triton WR 1339 was shown to block the proteolytic degradation of lowdensity lipoprotein (LDL)3 and to prevent the cholesterol of LDL from being taken up by cells. In addition it was also reported that this detergent not only concentrated in lysosomes (12, 32) but also caused hyperlipidemia (44,45). It is conceivable that the impairment of lysosomal hydrolysis may affect the cellular uptake processes. In previous studies it was shown that, in addition to acid ceramidase, an alkaline ceramidase activity was present in human fibroblasts, and this activity was not deficient in Farber’s cells. It is not unexpected
ROLE
IN CERAMIDE
METABOLISM
453
therefore that in Farber’s cells ceramide turnover is only partially impaired. We found that the rate of ceramide turnover in Farber’s fibroblasts was approximately %rd to l/4th that of normal fibroblasts, while the in vitro measurement of acid ceramidase activity in these diseased cells was only 1/32nd that of normal cells. These results indicated that both alkaline and acid ceramidases were involved in catalyzing the degradation of ceramide, and that the decreased rate of ceramide degradation in these diseased fibroblasts was still sufficient to maintain normal cellular growth. Nevertheless, the findings of a decreased rate of ceramide turnover and a 2.8-fold accumulation in Farber’s cells support the previous suggestion that the enzymatic defect causing lysosomal storage is the metabolic basis of this disorder (27-29). To obtain direct evidence that the accumulated ceramide in Farber’s fibroblasts is localized in lysosomes and that sphingolipids are degraded in these organelles, we carried out a subcellular fractionation of fibroblasts by differential centrifugation and isopycnic centrifugation on a sucrose gradient. We observed first, that the lysosomal fraction isolated from Farber’s fibroblasts was low in density, and second, that the accumulated [3H]ceramide in these diseased cells was localized in these buoyant lysosomes. Our finding of a low-density lysosomal fraction suggests that the concentric lamellar vacuoles which were observed after overloading with exogenous ceramide as previously reported (33) are indeed lysosomal inclusions. It is also consistent with the notion that the catabolism of sphingolipids in intact cells takes place, at least in part, in lysosomes. It has been reported (12, 32, 44-47) that administration of indigestible materials such as Triton WR 1339, colloidal carbon, and polyvinylpyrrolidone indextrans, duced storage of lipids in lysosomes. The altered density of overloaded lysosomes permits easy isolation of this lysosomal fraction. Certain amphiphilic cationic drugs such as chloroquine might also induce storage of lipids in lysosomes (12, 13, 15, 17). Such a drug-induced lipidosis is characterized by the occurrence of acid phosphatase-
454
CHEN.
MOSER,
positive cytoplasmic inclusions and it is thought to be attributed to the interference with general lysosomal function by increasing pH (10). Cellular changes, resembling those of lipidoses, were produced in normal fibroblasts by incubating them with excessive amounts of either polar lipids or ceramide (33, 48, 49). These observations strongly suggest that the density of lysosomes is influenced by the environmental conditions. Moreover, in the atheromatous aorta, a broad distribution of lysosomes, differing markedly in density, was observed upon subcellular fractionation (50, 51). This diverse distribution of lysosomes was also shown in human skin fibroblasts (40), rat hepatocytes (50-52), and two different cultured hamster fibroblast lines (53, 54). In addition, recent reports demonstrated that the 1251-asialoceruloplasmin was taken up into rat hepatocytes by endocytosis, and a-L-iduronidase incorporated into cultured human fibroblasts was initially localized in the lysosomal fraction of lower density and later seen in the dense fraction (40, 52). These findings are also consistent with the thesis that the decreased density of lysosomes, resulting from the incorporation of exogenous lipids, is a transitional state in the normal metabolic cycle. On the other hand, the accumulation of lipids in lipidosis fibroblasts, resulting from the deficiency of lysosomal hydrolases, may impair lysosomal digestion processes and, thus, may block the normal cycle of lysosomal metabolism. It is conceivable, though it has not been reported, that only a buoyant lysosomal fraction is found in these lipidosis fibroblasts. In Farber’s fibroblasts, the finding of a lysosomal fraction with markedly low density supports this hypothesis. A systematic study of the enzymatic abnormality and biochemical composition of isolated lysosomal fractions from different lipidosis fibroblasts, as well as the mechanism causing these changes, may greatly increase our understanding not only of the pathology of these disorders, but also of normal lysosomal metabolism. ACKNOWLEDGMENTS We wish to thank Mrs. lent technical assistance.
Patricia Veno for her excelWe also thank Drs. D. K.
AND
MOSER
Struck and R. E. Pagan0 for the gift of Cnitrobens-2oxa-1,3-diazole-%aminododecanoic acid and for the advice and help in investigating the uptake of NBD-ceramide, Drs. G. H. Thomas and S. W. Craig for help in the preparation of fluorescence micrographs, and Dr. Y. Kishimoto for help in the synthesis of ceramides. We gratefully acknowledge Dr. E. F. Neufeld for helpful discussion and criticism and Dr. Pamela Talalay for her expert editor-al help.
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ACID
CERAMIDASE
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