80
Biochimicu 0 1992 Elsevier
BBALIP
Science
Publishers
et Biophysicu Actu.
B.V. All rights reserved
I 124 (1992) X0-87
0005-2760/92/$0.5.00
53840
Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts Eugenio
Monti, August0 Preti, Albert0 Novati, Maria Francesca Maria Luisa Clemente and Sergio Marchesini
Depurtment
of Biomedicul
Sciences unci Biotechnology,
(Revised
Key words:
Fluorescent
sphingolipid;
Fluorescent
Aleo,
Unir~ersity of Bresciu, Bresciu (Itulyl
(Received 14 March 1991) manuscript received 3 June 1991)
sulfatide:
Sulfatide
metabolism;
Lissamine
rhodamine:
(Human
skin fibrohlast)
The sulfatide fluorescent analogue N-l&amine rhodaminyl-(12-aminododecanoyl)cerehroside 3-sulfate was administered in the form of albumin complex to normal human skin tibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.
Introduction Several fluorescent sphingolipid analogues have been synthesized in recent years [1,2], which have been proved to be a useful tool in studying the uptake, the metabolism, the intracellular traffic and the molecular sorting of lipids in cultured cells [3-51, as well as the chemicophysical properties of model membranes [2,6]. Fluorescent derivatives of ceramide and glucocerebroside have provided elegant means for studying the role of Golgi apparatus in the traffic of sphingolipids
[41. A model of sphyngomyelin transport and recycling has been proposed by Koval et al. [7,8] following the uptake and metabolism of an NBD-sphingomyelin analogue by fluorescent microscopy and conventional biochemical procedures. Recently, fluorescent derivatives of cerebroside sulfate have also been synthesized [9,10]. These com-
pounds have been used to examine the influence of the sulfatide molecules on the physical properties of model membranes [6] and as substrates to determine arylsulfatase A activity both in vitro [9] and in vivo [11,12]. In this manuscript we describe the uptake and subsequent metabolism of a fluorescent sulfatide, namely N-lissamine rhodaminyl-(12-aminododecanoyl)cerebroside 3-sulfate (LRh-CS) [lo], in cultured skin fibroblasts. The kinetics of the uptake and the main products of the metabolic processes are characterized. The use of LRh-CS was suggested because this sulfatide resulted from preliminary experiments to be extensively taken up by cells, whereas other sulfatides containing eosine, fluoresceine or NBD were not (unpublished data). The synthesis of ceramide, galactosylceramide, glucosylceramide, free acid and sphingomyelin, all containing the fluorophore lissamine rhodamine are also reported for the first time. Materials
Abbreviations: LRh, l&amine rhodamine; LRh-CS, N-lissamine rhodaminyl-(12.aminododecanoyl) cerebroside 3-sulfate; LRh-SPM, N-lissamine rhodaminyl-(12-aminododecanoyl)sphingosine l-phosphorylcholine; Cer, ceramide, GalCer, galactosylceramide; GluCer, glucosylceramide: LRh-FA, N-lissamine rhodaminyl-12.aminododecanoic acid; TLC, thin-layer chromathography. Correspondence: S. Marchesini, Department of Biomedical Sciences and Biotechnology, University of Brescia, Via Valsabbina 19, 25123 Brescia, Italy.
and Methods
Cells Skin fibroblasts from a 2-year-old late infantile metachromatic leukodistrophy (MLD) patient and a 15 year old normal subject, were grown from forearm skin biopsies in Eagle’s minimum essential medium (Flow Laboratories, Irvine, Scotland) supplemented with 10% FCS (fetal calf serum), glutamine, non-essential amino
81 acids and antibiotics (Flow Laboratories, Irvine, Scotland); maintained at 37°C in an atmosphere of 5% CO, in air. Hanks’ Balanced Salt Solution was supplied from Flow Laboratories (Irvine, U.K.). Trypan Blue Stain (0.4%) was purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). General procedures and chemicals
Sulfatide and cerebroside (galactosylceramidel were extracted from bovine brain following the procedure of Hara et al. [13], omitting the periodic acid oxidation. Glucosylceramide isolated from Gaucher’s spleen and sphingosylphosphorylcholine obtained by acid hydrolysis of bovine brain sphingomyelin [14] were a generous gift of Professor S. Gatt. Ceramide was prepared from bovine brain galactocereboside by periodic acid/acid hydrolysis procedure [ 151. Deacylation of sulfatide, galactosylceramide, glucosylceramide and ceramide were performed using the procedure described by Dubois et al. [161. 12Aminododecanoic acid (aminolauric acid), 9-fluorenylmethylchloroformate (FMOC), N-hydroxysuccinimide, N-N-dicyclohexylcarbodiimide, piperidine and other reagents were purchased from Aldrich Chemie (Steinheim, F.R.G.). Lissamine rhodamine B sulfonyl chloride and fluorescein conjugate of bovine serum albumin were obtained from Molecular Probes (Eugene, OR, U.S.A.). Synthesis of fluorescent sulfatide
LRh-sulfatide was prepared by reacting N-(12aminododecanoyl)cerebroside 3-sulfate with lissamine rhodamine B sulfonylchloride in dimethylformamide using the procedure described by Marchesini et al. [ 101. Synthesis of fluorescent ceramide, galactosyl- and glucosyl ceramide
Fluorescent (LRh) Cer, GalCer, GluCer were synthesized as described for sulfatide, by reacting the N-hydroxysuccinimide ester of FMOC-aminolauric acid with sphingosine, galactosylsphingosine or glucosylsphingosine, respectively, followed by deprotection of the aminogroup and reaction with lissamine rhodamine B sulfonyl chloride. Lissamine rhodamine B sulfonyl chloride, as already observed in the synthesis of LRhCS [lo], resulted in a mixture of two compounds having very similar excitation and emission spectra; therefore, conjugation with the amine acceptor always produced the formation of two compounds. The two could be separated on an anionic resin in acetate form, (Sep-Pak Accell QMA cartridge, Millipore, Bedfort, MA, U.S.A.): the compound not adsorbed on the resin had a R, in chloroform/ ethyl acetate/n-propanol/0.25% KCl/methanol (25 : 25 : 25 : 9 : 16, v/v) a little higher than the corresponding fluorescent compound which was strongly attached to the resin. The latter could be recovered only by eluting the cartridge with acidified
chloroform/ methanol. All the sphingolipids used in this work were those not adsorbed to the resin. The final purification of Cer, GalCer and GluCer was achieved by a first TLC on silica gel 60 plates in chloroform/ ethyl acetate/ndeveloped propanol/0.25% KCl/methanol (25 : 25 : 25 : 9 : 16, v/v), followed by a second one, developed in acetonitrile/30% ammonium hydroxyde/water (45 : 2.5 : 2.5, v/v). Synthesis of fluorescent sphingomyelin
LRh-SPM was synthesized as follows: sphingosyl phosphorylcholine was reacted for 2 h with a slightly molar excess of N-hydroxysuccinimide ester of FMOC-aminolauric acid [lo] in 5% aqueous NaHCO,-ethanol (9 : 1, v/v>. The N-(12-FMOCaminododecanoyl)sphingosyl phosphorylcholine was recovered from the reaction mixture as described by Ahmad et al. [18]. After deprotection in piperidine [lo], N-(12-aminododecanoyl)sphingosyl phosphorylcholine was reacted with LRh-SO,Cl in dimethyl formamide/ triethylamine (40 : 1, v/v) under stirring for 3 h at room temperature. After evaporation of the solvent, the reaction mixture was dissolved in water and passed through a small reverse-phase column (Sep-Pak Cl8 cartridge, Millipore, Bedfort, MA, U.S.A.): free fluorophore was eluted with water and crude SPM was recovered washing the column with methanol. Two SPMs, differing in the fluorophore moiety could be separated on Sep-Pak Accell QMA cartridge as described above for the other sphingolipids. TLC on silica gel 60 plates developed in chloroform/ methanol/30% ammonium hydroxide/ water (72: 48: 2: 9, v/v> of the non-adsorbed fraction resolved two products, LRh-SPMl (RF = 0.48) and LRhSPM2 CR, = 0.45), both hydrolyzed in vitro by sphingomyelinase (Streptomyces sp, Sigma, St. Louis, MO, U.S.A.). However, the time curves of hydrolysis were not the same for the two SPM’s. LRh-SPMl was hydrolyzed only up to 35% after 30 min, whereas LRh-SPM2 was hydrolyzed up to 92% to a ceramide having the same R, than the ceramide obtained by chemical synthesis. Furthermore, LRh-SPM2 has the same R, in different solvent systems of LRh-SPM produced by cells after incubation with LRh-CS. Thus, we identified LRh-SPM2 as the natural p-erythro form. Synthesis of jkorescent
fatty acid
The conjugation of lissamine rhodamine fluorophore to aminolauric acid was achieved in isopropanol/5% aqueous NaHCO, (4: 1, v/v) reacting a molar excess of LRh-SO&l with aminolauric acid under ovenight stirring. The reaction mixture, after evaporation of isopropanol, was diluted in water and chromatographed on a reverse-phase column (Sep-Pak C18) with increas-
82
ing concentration of methanol in water: crude LRh-FA was recovered from the column with methanol. Anion-exchange chromatography (Sep-Pak Accell QMA) resolved LRh-FA into products: the first one eluted with 0.01 M sodium acetate in chloroform/ methanol (8 : 2, v/v); the second one was released from the column washing with chloroform/ methanol/acetic acid (8: 2: 0.5, v/v>. The two fluorescent fatty acids differed in the fluorophore moiety, as observed for the above sphingolipids. The first one was used as a standard in these experiments. Preparation of sulfatide-albumin complexes Sulfatide-albumin complex was prepared immediately before use as described by Viani et al. [12] for pyrene containing sulfatide. Briefly, for every 10 nmol of the fluorescent sulfatide in dry form, 2.5 ~1 of dimethylsulfoxide were added, and the mixture was heated at 60°C for 10 min. An appropriate amount of fatty acids free human albumin (Sigma Chemical Company, St. Louis, MO, U.S.A.) in 0.9% NaCl was added in order to obtain an albumin/fluorescent sulfatide mole ratio of 1 : 1. The solution was ready for use after a 20 min incubation at 37°C. Administration of fluorescent sulfatide to cells and analysis of cell lipid extracts LRh-sulfatide complexed to human albumin (fatty acids free), was diluted with nutrient medium, devoid of FCS, to the desired concentration and added to 7 cm2 flasks of confluent cells (average protein content: 0.08 mg) in a final volume of 2 ml. In pulse experiments after the desired period of incubation at 37°C the medium was decanted, the cells washed with 0.9% NaCI, harvested by trypsinization and again washed twice with physiological saline. In ‘pulse-chase’ experiments after 6 h-pulse the cells were washed two times with Hanks’ Balanced Salt Solution, the medium containing the fluorescent sulfatide was replaced with fresh medium containing 10% FCS and devoid of substrate. The lipids were extracted from the pellet with 2 ml of chloroform/ methanol (6 : 4, v/v> and the total cell associated fluorescence was quantified. The percentage of sulfatide degradation was detected separating the degradation products on DEAESephadex A-25 (Pharmacia AB, Uppsala, Sweden) as described by Marchesini et al. [9] and reading the fluorescence of the eluates. The identification and quantification of the metabolic products was carried out by thin-layer chromatography (Silica gel 60 precoated plates, E. Merck, Darmstadt, F.R.G.) using chloroform/ ethyl acetate/ n-propanol/0.25% KCI/ methanol (25 : 25 : 25 : 9 : 16, v/v) as solvent system.
Fluorescent were scraped, form/methanol measured.
spots detected under ultraviolet light, extracted from the gel using chloro(1: 1, v/v>, and their fluorescence
Analysis of the fluorescent lipids in the media The media of the chase experiments were extracted with 2 volumes of chloroform/methanol (6: 4, v/v). After vortexing and centrifuging, the upper phase containing the Phenol-Red was discarded; the lower phase was concentrated and used for total fluorescence determination. The identification and quantification of metabolic products was performed as described for cell lipid extracts. Fluorescence determinations All fluorescence determinations of LRh-containing lipids were carried out in a Jasco FP-770 spectrofluorometer using chloroform/ methanol (6 : 4, v/v>, as solvent. An excitation wavelength of 565 nm and an emission of 575 nm were used. The LRh-sulfatide concentration in chloroform/ methanol (6: 4, v/v) was determined using for the fluorescent sulfatide a molar extinction coefficient of 95000 A per mol per liter [lo]. The same fluorescence per mol was assumed for the fluorescent sulfatide and for their fluorescent degradation products. The absolute amount of LRh-lipid species recovered from TLC were determined by reference to a known amount of fluorescent lipid chromatographed and analyzed under the same conditions. For fluoresceinated albumin dissolved in water, an excitation wavelength of 502 nm and an emission of 520 nm were used. Microscopy Fibroblasts were grown on 4-well chamber/slide (Miles Scientific, Miles Laboratories, Naperville, IL, U.S.A.), washed several times with 0.15 M potassium phosphate buffer (pH 7.2), 0.137 M NaCl, and fixed with 5% formaldehyde in the same buffer. The coverslips were mounted on plastic slides with glycerol. The slides were viewed with a Leitz Diaplan microscope equipped with Leitz filter pack N2 (E. Leitz Wetzlar GMBH, Wetzlar, F.R.G.). Photographs were taken using a Wild-MPS 52 camera equipped with a WildMPS46 photoautomat (Wild-Leitz, Heerbrugg, Switzerland), using a Kodak TMY 400 ASA film (Kodak Limited, U.K.). Presentation of the data Each experiment was repeated at least three times, but only representative experiments are presented in figures.
83 Results Uptake and degradation of jluorescent sulfatide by cultured skin fibroblasts in pulse experiments
The uptake of the fluorescent sulfatide by cultured skin fibroblasts was linear with the amount added to the medium up to 7.5 nmol/ml. (Fig. 1A). The fluorescence of the cell extracts followed with time (Fig. 1B) using 2.5 nmol of LRh-CS/ml of media, showed after a rapid phase, an incorporation at a lower and a constant rate. MLD cells had the same kinetic behaviour, however, at least with the cell-line used in these experiments, they incorporated LRh-CS at a slightly higher extent than normal fibroblasts. The degradation at 24 h-pulse reached 18% in normal fibroblasts, whereas it was not detectable in MLD cells (Table I). A thin-layer chromatogram of cell lipid extracts from normal and MLD cells is shown in Fig. 2. When the LRh-CS/albumin complex was dissolved in the medium containing 10% FCS and added to the cells, the amount of LRh-CS taken up in 24 h-pulse was only one fourth of the amount taken up in a parallel experiment without FCS (Table I). The total catabolites were remarkably lower (0.68 nmol/mg protein) in the cell extract of the cells incubated in the presence of FCS than in the extract of the cells incubated in the absence of FCS (2.45 nmol/mg protein) (Table I). A thin-layer-chromatogram of a cell extract at 24 h-pulse, in presence or without FCS, is shown in Fig. 5A. In order to understand whether the intact LRh-CS/ albumin complex or only the fluorescent sulfatide is taken up by cells, the following experiment was performed: 5 nmol of LRh-CS complexed to fluores-
2.5
5
FLUORESCENT
SULFATIDE
7.5
1
ceinated albumin were incubated for 24 h in a medium devoid of FCS. The cell pellet was resuspended in water and fluorescence determination was carried out. No fluorescein was detected. Uptake and metabolic fate of fluorescent pulse-chase experiments
sulfatide in
A large part of the fluorescence taken up during a 6 h-pulse was already released into the medium during the first 4 h of the chase (Fig. 3). With an 18 h-chase 55% of the fluorescence in the cell lipid extract was
2
(PM )
3
2
Fig. 2. TLC of fluorescent lipids extracted from cells incubated with 2.5 PM LRh CS for 24 h. Solvent system: chloroform/ethyl acetate/n-propanol/0.25% KCl/methanol (25 : 25: 25 : 9: 16, v/v). The chromatogram was photographed under ultraviolet light. Lane 1, standard fluorescent lipids. From top to bottom: ceramide, free fatty acid, glucosylceramide, galactosylceramide, sulfatide, sphingomyelin; Lane 2, fluorescent lipids from a MLD patient; Lane 3, fluorescent lipids from normal subject.
8 INCUBATION
16
24
TIME (hours)
Fig. 1. LRh-CS uptake by human skin fibroblasts in pulse experiments. (A) Cells incubated at 37°C in medium devoid of FCS with various concentrations of fluorescent sulfatide for 24 h; (B) cells incubated at 37°C in medium devoid of FCS in presence of 2.5 PM fluorescent sulfatide up to 24 h. In both cases the cell-associated lipids were extracted, the amount of fluorescent lipids determined and normalized to total cell protein.
2 1
4
PULSE
,
I
I
6
2
4
t
I
I
12
18
CHASE
INCUBATION
TIME
(hours)
Fig. 3. LRh-CS uptake by human skin fibroblasts in pulse-chase experiment. Cells were pulsed at 37°C in a medium devoid of FCS up to 6 h in presence of 2.5 PM fluorescent sulfatide and chased for various times up to 18 h in a medium containing 10% FCS. At each time, the cell associated lipids were extracted, the amount of fluorescent lipids was determined and normalized to total cell protein.
associated to metabolic products. MLD cells assayed under the same conditions showed only 0.9% of sulfatide degradation (Table I). When the pulse was done in the presence of fetal calf serum, the total fluorescence associated to cells after an 18 h-chase was one fourth of the value obtained in the absence of fetal calf serum (Table I). The degradation products, expressed as a percentage of the total fluorescence, reached more or less the same level. A typical thin-layer chromatogram of a cell extract at 18 h-chase, in the presence or absence of FCS in the pulse medium is shown in Fig. 5B. When fetal calf serum was omitted during both pulse and chase, a three-fold increase in cell associated fluorescence (2.87 nmol/mg protein) was observed. The identification of the different fluorescent lipids present in the cell-extract has been done on the basis
TABLE
2
4
12 INCUBATION
i
TIME
18 (hours)
Fig. 4. Metabolism of LRh-CS. Cells were pulsed with 2.5 PM fluorescent sulfatide for 6 h in a medium devoid of FCS, washed and incubated for the indicated times in presence of FCS. The cell-associated lipids were extracted, separated by TLC and the fluorescent metabolites were measured and expressed as a percentage of total cell associated LRh-lipid. (0) LRh-CS; (0) LRh-ceramide; (A) LRh-galactosylceramide; and (A) LRh-sphingomyelin.
of their RF'sand by chemical or enzymic hydrolysis of the individual spots. In particular, the spot corresponding to standard fluorescent ceramide on thin-layer chromatography was identified as ceramide because it remained unaffected by acid hydrolysis (2.5 M formic acid at 100°C for 18 h); the compound running like standard galactosylceramide was hydrolyzed in formic acid to ceramide and was therefore identified as galactosylceramide; the spot running like standard sphingomyelin was converted by sphingomyelinase to ceramide and therefore identified as sphingomyelin. Fig. 4 shows the percentage distribution of the fluorescence among the metabolic products in cell extract at 6 h-pulse, following 2-, 4-, 12- and 18 h-chase: sulfatide, ceramide, galactosylceramide and sphingomyelin are the only compounds present in well detectable quantities.
I
Uptake and degradation of fluorescent sulfatide under different experimental conditions Cultured skin fibroblasts were incubated with 2.5 PM LRh-CS in form of albumin 24 h-pulse and 6 h-pulse/l8 h-chase. The chase was performed in the presence Fibroblast strain
FCS during pulse
Normal MLD Normal
_ _
nd.,
not detectable.
+
complex in a medium containing 10% FCS or in its absence for of FCS. Values are the meanskS.D. of three measurements. 6 h-pulse/l8
24 h-pulse
h-chase
uptake (nmol/ mg protein)
degradation products (nmol/ mg protein)
%
uptake (nmol/ mg protein)
degradation products (nmol/ mg protein)
%
13.60 + 2.10 16.00 + 2.60 3.40 f 0.60
2.45 f 0.37 n.d. 0.68kO.17
18 _
0.85 + 0.13 0.89*0.16 0.21* 0.05
0.47 * 0.09 0.008 + 0.002 0.11 kO.02
55 0.9 52
20
85
1
2
3
I
2
3
Fig. 5. TLC of fluorescent lipids extracted from human skin fibroblasts incubated with 2.5 FM LRh-CS under different conditions. (A) Cells incubated with fluorescent sulfatide for 24 h. Lane 1. standard fluorescent lipids (as in Fig. 2); Lane 2, incubation in presence of 10% FCS; Lane 3, incubation in absence of FCS. (B) Cells incubated with fluorescent sulfatide in 6 h-pulse/IS h-chase experiments (chase time was always performed in medium plus FCS). Lane 1, standard fluorescent lipids (as in Fig. 2); Lane 2, pulse in presence of 10% FCS; Lane 3, pulse in absence of FCS.
cells or produced, at least in part, by the action of enzymes present in the medium (11, or released into the medium from dead cells (21, the following experiments were performed: (1) 5 nmol of LRh-CS in the form of albumin complex were added to 2 ml of fresh medium containing 10% FCS (medium A) and to the same volume of medium previously incubated for 18 h in the presence of confluent skin fibroblasts (medium B). After 18 h the media were collected and analyzed for catabolites present after removal of sulfatide on DEAE-Sephadex A-25: no significative difference in fluorescence content could be appreciated between medium A and B. Thus, even a partial contribution of the eventually excreted enzymes in the degradation of LRh-CS was excluded; and (2) fibroblasts grown in 96 wells plate were incubated in the presence or absence of fluorescent sulfatide, under typical conditions of pulse-chase. The cell viability was cheked using Trypan-Blue and counting in Biirker’s chamber. The comparison between treated and control cells did not reveal any significant statistical difference.
Analysis of the fluorescence in the media The medium obtained after harvesting the cells at 18 h-chase was extracted and analyzed for the presence of catabolites: 90.5% of total fluorescence was associated to sulfatide and only 9.5% to catabolites. However, as reported in Fig. 6, as many catabolites were present in medium as in the cells. The distribution of fluorescence among catabolites did not parallel that of the cell lipid extract: much more sphingomyelin (17%) and galactosylceramide (16%) and less ceramide (67%) were always present in the medium. In order to ascertain whether the catabolites detected in the 18 h-chase medium were released from
MEDIUM
0
CERAMIDE
q CEREBROSIDE •m
SULFATIDE
TOTAL
SPHINGOMYELIN
CATABOUTES
Fig. 6. Comparison between fluorescent lipids content in cells and medium. The sulfatide content was obtained as difference between the fluorescence of the total lipid extract and that of total metabolic products separated on DEAE-Sephadex A-25. Quantitative determination of individual metabolic products was performed after their separation on TLC.
Fig. 7. Fluorescence micrograph of human skin fibroblasts incubated with 2.5 WM fluorescent sulfatide, in 6 h-pulse/l8 h-chase experiment. (Original magnification X 3150).
Fluorescence microscopy of cells after 6 h-pulse/l8 h-chase (Fig. 7) indicates a marked perinuclear fluorescence and a weak labeling of plasma membrane. MLD cells, under the same conditions, showed a very similar fluorescence pattern. Discussion
The time-dependent uptake up to 24 h at a sulfatide concentration of 2.5 nmol/ml medium followed a bifasic progress (Fig. lB>, suggesting a relatively rapid incorporation into the plasma membrane, followed by a slower internalization. A comparison between our data on the uptake and those reported by other authors using ‘“C-stearic acid labeled sulfatide [19-211 appears difficult, due to the different experimental conditions. In this work, we chose to administer the fluorescent sulfatide to cells as an albumin complex in a medium deprived of FCS, since in a preliminary experiment (Table I) we had confirmed the inhibition of the sulfatide uptake by FCS [121. Inui et al. [20] reported that FCS plays an important role in the translocation of CS to Iysosomes and, in its absence ‘“C-CS remained unhydrolyzed at plasma membrane. This is not true for LRh-CS: in the absence of fetal calf serum the uptake of the fluorescent sulfatide was strongly enhanched and no reduction of its degradation level was observed (Table I). The contradiction is apparent. In the system used by the above quoted authors, the sulfatide dispersed in the medium devoid of FCS, was probably taken up by cells in the form of aggregate and not transferred to lysosomes. In our system, the human albumin complexed to fluorescent sulfatide is not taken up by cells, as demonstrated by the double-label experiment using ~uoresceinated albumin and therefore it is likely monomeric transfer of LRh-CS to plasma membrane occurs. When FCS is present in the medium it seems plausible that a competition for sulfatide exists between the albumin in the albumin/ sulfatide complex and serum proteins, resulting in a lower transfer of sutfatide to the cell surface. In pulse-chase experiments (Fig. 31, the major part of the fluorescence incorporated at 6 h-pulse, was released into the medium during the chase. However, when the chase was performed in the absence of FCS, much more fluorescence was retained by cells, thus indicating a direct involvement of FCS in pulling out fluorescent lipids from cells. The metabolic fate of LRh-CS in the conditions of maximal degradation, that is in pulse-chase, was quite different from that reported by other authors for 14CCS [21]. The major metabolites derived from LRh-CS were ceramide followed by galactosylceramide and sphingomyelin. The striking difference was the high level of ceramide. Few weak spots detectable on TLC in the
region between SPM and sulfatide, a region in which glycerophospholipids are expected to run in the solvent system used, have not been identified because of lack of standards. It appears that ceramidase degrades LRh-ceramide at a very low rate and the incorporation of the fluorescent fatty acid into glycerophospholipids is impaired. The finding, beside ceramide, of a well detectable quantity of sphingomyeiin suggests a specific transfer of ceramide from lysosomes to GOlgi apparatus, known to bc a site of sphingomyelin formation [4,22-241. The presence in the 18 h-chase medium of a large quantity of LRh-CS, and as many metabolites as in cell extract, is remarkable (Fig. 6). The different percentage distribution of catabolites between cell extract and medium, together with the finding that cells incubated in the presence of LRh-CS are as viable as non-treated cells, eliminates the possibility that the presence of metabolites in the medium is the result of cell lysis. The possibility of a metabolism of sulfatide hy the action of enzymes released into the medium by cells. was also excluded by the experiment in which LRh-CS was exposed in a medium preincubated in the presence of fibroblasts. As to the high sulfatide content observed in the medium after 18 h of chase, the eventuality that it could result from the release of non-specifically bound sulfatide during the pulse could be discarded on the basis of the washes carried out between pulse and chase, and more convincing, of the observation that at 18 h-chase the total fluorescence (cell-associated plus medium-associated) roughly corresponded to the cellassociated fluorescence of 6 h-pulse, detected after cells harvesting by trypsinization (data not shown). In conclusion, fluorescent metabolic products dcrived from internalized sulfatide arc released into the medium together with undegraded sulfatide and this process appears to be enhanced by the presence of FCS. Fluorescence microscopy of the cells at 18 h-chase did not reveal a different distribution of the fluorescence between normal and MLD fibroblasts. This seems in agreement with the finding that unhydrolyzed sulfatide accumulates in MLD fibroblasts not only in lysosomes, but also in plasma membranes and Golgi apparatus [20]. Under our experimental conditions, the fluorescent suffatide was practically undegraded by MLD cells (Table I), thus LRh-CS could successfully substitute the radiolabelled sulfatide in a sulfatideloading test for discrimination between MLD and arylsulfatase A pseudodeficiency. Although the metabolic fate of lissamine-rhodamine sulfatide does not parallel that of “C-sulfatide, and therefore the data obtained can not simply be transferred to the metabolism of natural sulfatide, the slowing of the catabolism observed with lissamine-rhod-
87 amine sulfatide could be of practical use for studies of lipid traffic, provided that short pulses are used. The availability of other sphingolipid analogues, all containing the same fluorophore, i.e., lissaminerhodamine, will allow us to undertake studies aimed to correlate the intracellular distribution of the fluorescence with the subcellular localization of the sphingolipid and its catabolites.
10 Marchesini,
References
16
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11 12 13 14 15
17 18 19 20 21 22 23 24
S., Preti, A., Aleo, M.F., Casella, A., Dagan, A. and Gatt, S. (1990) Chem. Phys. Lipids 53, 165-175. Bach, G., Dagan, A., Herz, B. and Gatt, S. (1987) Clin. Genet. 31, 211-217. Viani, P., Marchesini, S., Cestaro, B. and Gatt, S. (1989) Biochim. Biophys. Acta 1002, 20-27. Hara, A. and Radin, N.S. (1979) Anal. Biochem. 100, 364-370. Cohen, R., Barenholz, Y., Gatt, S. and Dagan, A. (1984) Chem. Phys. Lipids 35. 371-384. Carter, H.G., Rothfus, J.A. and Gigg, R. (1961) J. Lipid Res. 2, 228-234. Dubois, G., Zalc, F., Le Saux, F. and Baumann, N. (1980) Anal. Biochem. 102, 313-317. Sedmak, J.J. and Grossberg, SE. (1977) Anal. Biochem. 79, 544-552. Ahmad, T.Y., Sparrow, J.T. and Morrisett, J.D. (1985) J. Lipid Res. 26, 1160-1165. Kudoh, T. and Wenger, D.A. (1982) J. Clin. Invest, 70, 89-97. Inui, K., Furukawa, M., Okada, S. and Yabuuchi, H. (1988) J. Clin. Invest. 81, 310-317. Trinchera, M., Wiesmann, U., Pitto, M., Acquotti, D. and Ghidoni, R. (19881 Biochem. J. 252, 375-379. Lipsky, N. and Pagano, R.E. (1983) Proc. Natl. Acad. Sci. USA 80. 2608-2612. Futerman, A.H., Steiger, B., Hubbard, A.L. and Pagano, R.E. (1990) J. Biol. Chem. 265, 8650-8657. Jeckel, D., Karrenbauer, A., Birk, R., Schmidt, R.R. and Wieland, F. (1990) FEBS Lett. 261, 1555157.
Biochimicu 0 1992 Elsevier
BBALIP
Science
Publishers
et Biophysicu Actu.
B.V. All rights reserved
I 124 (1992) X0-87
0005-2760/92/$0.5.00
53840
Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts Eugenio
Monti, August0 Preti, Albert0 Novati, Maria Francesca Maria Luisa Clemente and Sergio Marchesini
Depurtment
of Biomedicul
Sciences unci Biotechnology,
(Revised
Key words:
Fluorescent
sphingolipid;
Fluorescent
Aleo,
Unir~ersity of Bresciu, Bresciu (Itulyl
(Received 14 March 1991) manuscript received 3 June 1991)
sulfatide:
Sulfatide
metabolism;
Lissamine
rhodamine:
(Human
skin fibrohlast)
The sulfatide fluorescent analogue N-l&amine rhodaminyl-(12-aminododecanoyl)cerehroside 3-sulfate was administered in the form of albumin complex to normal human skin tibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.
Introduction Several fluorescent sphingolipid analogues have been synthesized in recent years [1,2], which have been proved to be a useful tool in studying the uptake, the metabolism, the intracellular traffic and the molecular sorting of lipids in cultured cells [3-51, as well as the chemicophysical properties of model membranes [2,6]. Fluorescent derivatives of ceramide and glucocerebroside have provided elegant means for studying the role of Golgi apparatus in the traffic of sphingolipids
[41. A model of sphyngomyelin transport and recycling has been proposed by Koval et al. [7,8] following the uptake and metabolism of an NBD-sphingomyelin analogue by fluorescent microscopy and conventional biochemical procedures. Recently, fluorescent derivatives of cerebroside sulfate have also been synthesized [9,10]. These com-
pounds have been used to examine the influence of the sulfatide molecules on the physical properties of model membranes [6] and as substrates to determine arylsulfatase A activity both in vitro [9] and in vivo [11,12]. In this manuscript we describe the uptake and subsequent metabolism of a fluorescent sulfatide, namely N-lissamine rhodaminyl-(12-aminododecanoyl)cerebroside 3-sulfate (LRh-CS) [lo], in cultured skin fibroblasts. The kinetics of the uptake and the main products of the metabolic processes are characterized. The use of LRh-CS was suggested because this sulfatide resulted from preliminary experiments to be extensively taken up by cells, whereas other sulfatides containing eosine, fluoresceine or NBD were not (unpublished data). The synthesis of ceramide, galactosylceramide, glucosylceramide, free acid and sphingomyelin, all containing the fluorophore lissamine rhodamine are also reported for the first time. Materials
Abbreviations: LRh, l&amine rhodamine; LRh-CS, N-lissamine rhodaminyl-(12.aminododecanoyl) cerebroside 3-sulfate; LRh-SPM, N-lissamine rhodaminyl-(12-aminododecanoyl)sphingosine l-phosphorylcholine; Cer, ceramide, GalCer, galactosylceramide; GluCer, glucosylceramide: LRh-FA, N-lissamine rhodaminyl-12.aminododecanoic acid; TLC, thin-layer chromathography. Correspondence: S. Marchesini, Department of Biomedical Sciences and Biotechnology, University of Brescia, Via Valsabbina 19, 25123 Brescia, Italy.
and Methods
Cells Skin fibroblasts from a 2-year-old late infantile metachromatic leukodistrophy (MLD) patient and a 15 year old normal subject, were grown from forearm skin biopsies in Eagle’s minimum essential medium (Flow Laboratories, Irvine, Scotland) supplemented with 10% FCS (fetal calf serum), glutamine, non-essential amino
81 acids and antibiotics (Flow Laboratories, Irvine, Scotland); maintained at 37°C in an atmosphere of 5% CO, in air. Hanks’ Balanced Salt Solution was supplied from Flow Laboratories (Irvine, U.K.). Trypan Blue Stain (0.4%) was purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). General procedures and chemicals
Sulfatide and cerebroside (galactosylceramidel were extracted from bovine brain following the procedure of Hara et al. [13], omitting the periodic acid oxidation. Glucosylceramide isolated from Gaucher’s spleen and sphingosylphosphorylcholine obtained by acid hydrolysis of bovine brain sphingomyelin [14] were a generous gift of Professor S. Gatt. Ceramide was prepared from bovine brain galactocereboside by periodic acid/acid hydrolysis procedure [ 151. Deacylation of sulfatide, galactosylceramide, glucosylceramide and ceramide were performed using the procedure described by Dubois et al. [161. 12Aminododecanoic acid (aminolauric acid), 9-fluorenylmethylchloroformate (FMOC), N-hydroxysuccinimide, N-N-dicyclohexylcarbodiimide, piperidine and other reagents were purchased from Aldrich Chemie (Steinheim, F.R.G.). Lissamine rhodamine B sulfonyl chloride and fluorescein conjugate of bovine serum albumin were obtained from Molecular Probes (Eugene, OR, U.S.A.). Synthesis of fluorescent sulfatide
LRh-sulfatide was prepared by reacting N-(12aminododecanoyl)cerebroside 3-sulfate with lissamine rhodamine B sulfonylchloride in dimethylformamide using the procedure described by Marchesini et al. [ 101. Synthesis of fluorescent ceramide, galactosyl- and glucosyl ceramide
Fluorescent (LRh) Cer, GalCer, GluCer were synthesized as described for sulfatide, by reacting the N-hydroxysuccinimide ester of FMOC-aminolauric acid with sphingosine, galactosylsphingosine or glucosylsphingosine, respectively, followed by deprotection of the aminogroup and reaction with lissamine rhodamine B sulfonyl chloride. Lissamine rhodamine B sulfonyl chloride, as already observed in the synthesis of LRhCS [lo], resulted in a mixture of two compounds having very similar excitation and emission spectra; therefore, conjugation with the amine acceptor always produced the formation of two compounds. The two could be separated on an anionic resin in acetate form, (Sep-Pak Accell QMA cartridge, Millipore, Bedfort, MA, U.S.A.): the compound not adsorbed on the resin had a R, in chloroform/ ethyl acetate/n-propanol/0.25% KCl/methanol (25 : 25 : 25 : 9 : 16, v/v) a little higher than the corresponding fluorescent compound which was strongly attached to the resin. The latter could be recovered only by eluting the cartridge with acidified
chloroform/ methanol. All the sphingolipids used in this work were those not adsorbed to the resin. The final purification of Cer, GalCer and GluCer was achieved by a first TLC on silica gel 60 plates in chloroform/ ethyl acetate/ndeveloped propanol/0.25% KCl/methanol (25 : 25 : 25 : 9 : 16, v/v), followed by a second one, developed in acetonitrile/30% ammonium hydroxyde/water (45 : 2.5 : 2.5, v/v). Synthesis of fluorescent sphingomyelin
LRh-SPM was synthesized as follows: sphingosyl phosphorylcholine was reacted for 2 h with a slightly molar excess of N-hydroxysuccinimide ester of FMOC-aminolauric acid [lo] in 5% aqueous NaHCO,-ethanol (9 : 1, v/v>. The N-(12-FMOCaminododecanoyl)sphingosyl phosphorylcholine was recovered from the reaction mixture as described by Ahmad et al. [18]. After deprotection in piperidine [lo], N-(12-aminododecanoyl)sphingosyl phosphorylcholine was reacted with LRh-SO,Cl in dimethyl formamide/ triethylamine (40 : 1, v/v) under stirring for 3 h at room temperature. After evaporation of the solvent, the reaction mixture was dissolved in water and passed through a small reverse-phase column (Sep-Pak Cl8 cartridge, Millipore, Bedfort, MA, U.S.A.): free fluorophore was eluted with water and crude SPM was recovered washing the column with methanol. Two SPMs, differing in the fluorophore moiety could be separated on Sep-Pak Accell QMA cartridge as described above for the other sphingolipids. TLC on silica gel 60 plates developed in chloroform/ methanol/30% ammonium hydroxide/ water (72: 48: 2: 9, v/v> of the non-adsorbed fraction resolved two products, LRh-SPMl (RF = 0.48) and LRhSPM2 CR, = 0.45), both hydrolyzed in vitro by sphingomyelinase (Streptomyces sp, Sigma, St. Louis, MO, U.S.A.). However, the time curves of hydrolysis were not the same for the two SPM’s. LRh-SPMl was hydrolyzed only up to 35% after 30 min, whereas LRh-SPM2 was hydrolyzed up to 92% to a ceramide having the same R, than the ceramide obtained by chemical synthesis. Furthermore, LRh-SPM2 has the same R, in different solvent systems of LRh-SPM produced by cells after incubation with LRh-CS. Thus, we identified LRh-SPM2 as the natural p-erythro form. Synthesis of jkorescent
fatty acid
The conjugation of lissamine rhodamine fluorophore to aminolauric acid was achieved in isopropanol/5% aqueous NaHCO, (4: 1, v/v) reacting a molar excess of LRh-SO&l with aminolauric acid under ovenight stirring. The reaction mixture, after evaporation of isopropanol, was diluted in water and chromatographed on a reverse-phase column (Sep-Pak C18) with increas-
82
ing concentration of methanol in water: crude LRh-FA was recovered from the column with methanol. Anion-exchange chromatography (Sep-Pak Accell QMA) resolved LRh-FA into products: the first one eluted with 0.01 M sodium acetate in chloroform/ methanol (8 : 2, v/v); the second one was released from the column washing with chloroform/ methanol/acetic acid (8: 2: 0.5, v/v>. The two fluorescent fatty acids differed in the fluorophore moiety, as observed for the above sphingolipids. The first one was used as a standard in these experiments. Preparation of sulfatide-albumin complexes Sulfatide-albumin complex was prepared immediately before use as described by Viani et al. [12] for pyrene containing sulfatide. Briefly, for every 10 nmol of the fluorescent sulfatide in dry form, 2.5 ~1 of dimethylsulfoxide were added, and the mixture was heated at 60°C for 10 min. An appropriate amount of fatty acids free human albumin (Sigma Chemical Company, St. Louis, MO, U.S.A.) in 0.9% NaCl was added in order to obtain an albumin/fluorescent sulfatide mole ratio of 1 : 1. The solution was ready for use after a 20 min incubation at 37°C. Administration of fluorescent sulfatide to cells and analysis of cell lipid extracts LRh-sulfatide complexed to human albumin (fatty acids free), was diluted with nutrient medium, devoid of FCS, to the desired concentration and added to 7 cm2 flasks of confluent cells (average protein content: 0.08 mg) in a final volume of 2 ml. In pulse experiments after the desired period of incubation at 37°C the medium was decanted, the cells washed with 0.9% NaCI, harvested by trypsinization and again washed twice with physiological saline. In ‘pulse-chase’ experiments after 6 h-pulse the cells were washed two times with Hanks’ Balanced Salt Solution, the medium containing the fluorescent sulfatide was replaced with fresh medium containing 10% FCS and devoid of substrate. The lipids were extracted from the pellet with 2 ml of chloroform/ methanol (6 : 4, v/v> and the total cell associated fluorescence was quantified. The percentage of sulfatide degradation was detected separating the degradation products on DEAESephadex A-25 (Pharmacia AB, Uppsala, Sweden) as described by Marchesini et al. [9] and reading the fluorescence of the eluates. The identification and quantification of the metabolic products was carried out by thin-layer chromatography (Silica gel 60 precoated plates, E. Merck, Darmstadt, F.R.G.) using chloroform/ ethyl acetate/ n-propanol/0.25% KCI/ methanol (25 : 25 : 25 : 9 : 16, v/v) as solvent system.
Fluorescent were scraped, form/methanol measured.
spots detected under ultraviolet light, extracted from the gel using chloro(1: 1, v/v>, and their fluorescence
Analysis of the fluorescent lipids in the media The media of the chase experiments were extracted with 2 volumes of chloroform/methanol (6: 4, v/v). After vortexing and centrifuging, the upper phase containing the Phenol-Red was discarded; the lower phase was concentrated and used for total fluorescence determination. The identification and quantification of metabolic products was performed as described for cell lipid extracts. Fluorescence determinations All fluorescence determinations of LRh-containing lipids were carried out in a Jasco FP-770 spectrofluorometer using chloroform/ methanol (6 : 4, v/v>, as solvent. An excitation wavelength of 565 nm and an emission of 575 nm were used. The LRh-sulfatide concentration in chloroform/ methanol (6: 4, v/v) was determined using for the fluorescent sulfatide a molar extinction coefficient of 95000 A per mol per liter [lo]. The same fluorescence per mol was assumed for the fluorescent sulfatide and for their fluorescent degradation products. The absolute amount of LRh-lipid species recovered from TLC were determined by reference to a known amount of fluorescent lipid chromatographed and analyzed under the same conditions. For fluoresceinated albumin dissolved in water, an excitation wavelength of 502 nm and an emission of 520 nm were used. Microscopy Fibroblasts were grown on 4-well chamber/slide (Miles Scientific, Miles Laboratories, Naperville, IL, U.S.A.), washed several times with 0.15 M potassium phosphate buffer (pH 7.2), 0.137 M NaCl, and fixed with 5% formaldehyde in the same buffer. The coverslips were mounted on plastic slides with glycerol. The slides were viewed with a Leitz Diaplan microscope equipped with Leitz filter pack N2 (E. Leitz Wetzlar GMBH, Wetzlar, F.R.G.). Photographs were taken using a Wild-MPS 52 camera equipped with a WildMPS46 photoautomat (Wild-Leitz, Heerbrugg, Switzerland), using a Kodak TMY 400 ASA film (Kodak Limited, U.K.). Presentation of the data Each experiment was repeated at least three times, but only representative experiments are presented in figures.
83 Results Uptake and degradation of jluorescent sulfatide by cultured skin fibroblasts in pulse experiments
The uptake of the fluorescent sulfatide by cultured skin fibroblasts was linear with the amount added to the medium up to 7.5 nmol/ml. (Fig. 1A). The fluorescence of the cell extracts followed with time (Fig. 1B) using 2.5 nmol of LRh-CS/ml of media, showed after a rapid phase, an incorporation at a lower and a constant rate. MLD cells had the same kinetic behaviour, however, at least with the cell-line used in these experiments, they incorporated LRh-CS at a slightly higher extent than normal fibroblasts. The degradation at 24 h-pulse reached 18% in normal fibroblasts, whereas it was not detectable in MLD cells (Table I). A thin-layer chromatogram of cell lipid extracts from normal and MLD cells is shown in Fig. 2. When the LRh-CS/albumin complex was dissolved in the medium containing 10% FCS and added to the cells, the amount of LRh-CS taken up in 24 h-pulse was only one fourth of the amount taken up in a parallel experiment without FCS (Table I). The total catabolites were remarkably lower (0.68 nmol/mg protein) in the cell extract of the cells incubated in the presence of FCS than in the extract of the cells incubated in the absence of FCS (2.45 nmol/mg protein) (Table I). A thin-layer-chromatogram of a cell extract at 24 h-pulse, in presence or without FCS, is shown in Fig. 5A. In order to understand whether the intact LRh-CS/ albumin complex or only the fluorescent sulfatide is taken up by cells, the following experiment was performed: 5 nmol of LRh-CS complexed to fluores-
2.5
5
FLUORESCENT
SULFATIDE
7.5
1
ceinated albumin were incubated for 24 h in a medium devoid of FCS. The cell pellet was resuspended in water and fluorescence determination was carried out. No fluorescein was detected. Uptake and metabolic fate of fluorescent pulse-chase experiments
sulfatide in
A large part of the fluorescence taken up during a 6 h-pulse was already released into the medium during the first 4 h of the chase (Fig. 3). With an 18 h-chase 55% of the fluorescence in the cell lipid extract was
2
(PM )
3
2
Fig. 2. TLC of fluorescent lipids extracted from cells incubated with 2.5 PM LRh CS for 24 h. Solvent system: chloroform/ethyl acetate/n-propanol/0.25% KCl/methanol (25 : 25: 25 : 9: 16, v/v). The chromatogram was photographed under ultraviolet light. Lane 1, standard fluorescent lipids. From top to bottom: ceramide, free fatty acid, glucosylceramide, galactosylceramide, sulfatide, sphingomyelin; Lane 2, fluorescent lipids from a MLD patient; Lane 3, fluorescent lipids from normal subject.
8 INCUBATION
16
24
TIME (hours)
Fig. 1. LRh-CS uptake by human skin fibroblasts in pulse experiments. (A) Cells incubated at 37°C in medium devoid of FCS with various concentrations of fluorescent sulfatide for 24 h; (B) cells incubated at 37°C in medium devoid of FCS in presence of 2.5 PM fluorescent sulfatide up to 24 h. In both cases the cell-associated lipids were extracted, the amount of fluorescent lipids determined and normalized to total cell protein.
2 1
4
PULSE
,
I
I
6
2
4
t
I
I
12
18
CHASE
INCUBATION
TIME
(hours)
Fig. 3. LRh-CS uptake by human skin fibroblasts in pulse-chase experiment. Cells were pulsed at 37°C in a medium devoid of FCS up to 6 h in presence of 2.5 PM fluorescent sulfatide and chased for various times up to 18 h in a medium containing 10% FCS. At each time, the cell associated lipids were extracted, the amount of fluorescent lipids was determined and normalized to total cell protein.
associated to metabolic products. MLD cells assayed under the same conditions showed only 0.9% of sulfatide degradation (Table I). When the pulse was done in the presence of fetal calf serum, the total fluorescence associated to cells after an 18 h-chase was one fourth of the value obtained in the absence of fetal calf serum (Table I). The degradation products, expressed as a percentage of the total fluorescence, reached more or less the same level. A typical thin-layer chromatogram of a cell extract at 18 h-chase, in the presence or absence of FCS in the pulse medium is shown in Fig. 5B. When fetal calf serum was omitted during both pulse and chase, a three-fold increase in cell associated fluorescence (2.87 nmol/mg protein) was observed. The identification of the different fluorescent lipids present in the cell-extract has been done on the basis
TABLE
2
4
12 INCUBATION
i
TIME
18 (hours)
Fig. 4. Metabolism of LRh-CS. Cells were pulsed with 2.5 PM fluorescent sulfatide for 6 h in a medium devoid of FCS, washed and incubated for the indicated times in presence of FCS. The cell-associated lipids were extracted, separated by TLC and the fluorescent metabolites were measured and expressed as a percentage of total cell associated LRh-lipid. (0) LRh-CS; (0) LRh-ceramide; (A) LRh-galactosylceramide; and (A) LRh-sphingomyelin.
of their RF'sand by chemical or enzymic hydrolysis of the individual spots. In particular, the spot corresponding to standard fluorescent ceramide on thin-layer chromatography was identified as ceramide because it remained unaffected by acid hydrolysis (2.5 M formic acid at 100°C for 18 h); the compound running like standard galactosylceramide was hydrolyzed in formic acid to ceramide and was therefore identified as galactosylceramide; the spot running like standard sphingomyelin was converted by sphingomyelinase to ceramide and therefore identified as sphingomyelin. Fig. 4 shows the percentage distribution of the fluorescence among the metabolic products in cell extract at 6 h-pulse, following 2-, 4-, 12- and 18 h-chase: sulfatide, ceramide, galactosylceramide and sphingomyelin are the only compounds present in well detectable quantities.
I
Uptake and degradation of fluorescent sulfatide under different experimental conditions Cultured skin fibroblasts were incubated with 2.5 PM LRh-CS in form of albumin 24 h-pulse and 6 h-pulse/l8 h-chase. The chase was performed in the presence Fibroblast strain
FCS during pulse
Normal MLD Normal
_ _
nd.,
not detectable.
+
complex in a medium containing 10% FCS or in its absence for of FCS. Values are the meanskS.D. of three measurements. 6 h-pulse/l8
24 h-pulse
h-chase
uptake (nmol/ mg protein)
degradation products (nmol/ mg protein)
%
uptake (nmol/ mg protein)
degradation products (nmol/ mg protein)
%
13.60 + 2.10 16.00 + 2.60 3.40 f 0.60
2.45 f 0.37 n.d. 0.68kO.17
18 _
0.85 + 0.13 0.89*0.16 0.21* 0.05
0.47 * 0.09 0.008 + 0.002 0.11 kO.02
55 0.9 52
20
85
1
2
3
I
2
3
Fig. 5. TLC of fluorescent lipids extracted from human skin fibroblasts incubated with 2.5 FM LRh-CS under different conditions. (A) Cells incubated with fluorescent sulfatide for 24 h. Lane 1. standard fluorescent lipids (as in Fig. 2); Lane 2, incubation in presence of 10% FCS; Lane 3, incubation in absence of FCS. (B) Cells incubated with fluorescent sulfatide in 6 h-pulse/IS h-chase experiments (chase time was always performed in medium plus FCS). Lane 1, standard fluorescent lipids (as in Fig. 2); Lane 2, pulse in presence of 10% FCS; Lane 3, pulse in absence of FCS.
cells or produced, at least in part, by the action of enzymes present in the medium (11, or released into the medium from dead cells (21, the following experiments were performed: (1) 5 nmol of LRh-CS in the form of albumin complex were added to 2 ml of fresh medium containing 10% FCS (medium A) and to the same volume of medium previously incubated for 18 h in the presence of confluent skin fibroblasts (medium B). After 18 h the media were collected and analyzed for catabolites present after removal of sulfatide on DEAE-Sephadex A-25: no significative difference in fluorescence content could be appreciated between medium A and B. Thus, even a partial contribution of the eventually excreted enzymes in the degradation of LRh-CS was excluded; and (2) fibroblasts grown in 96 wells plate were incubated in the presence or absence of fluorescent sulfatide, under typical conditions of pulse-chase. The cell viability was cheked using Trypan-Blue and counting in Biirker’s chamber. The comparison between treated and control cells did not reveal any significant statistical difference.
Analysis of the fluorescence in the media The medium obtained after harvesting the cells at 18 h-chase was extracted and analyzed for the presence of catabolites: 90.5% of total fluorescence was associated to sulfatide and only 9.5% to catabolites. However, as reported in Fig. 6, as many catabolites were present in medium as in the cells. The distribution of fluorescence among catabolites did not parallel that of the cell lipid extract: much more sphingomyelin (17%) and galactosylceramide (16%) and less ceramide (67%) were always present in the medium. In order to ascertain whether the catabolites detected in the 18 h-chase medium were released from
MEDIUM
0
CERAMIDE
q CEREBROSIDE •m
SULFATIDE
TOTAL
SPHINGOMYELIN
CATABOUTES
Fig. 6. Comparison between fluorescent lipids content in cells and medium. The sulfatide content was obtained as difference between the fluorescence of the total lipid extract and that of total metabolic products separated on DEAE-Sephadex A-25. Quantitative determination of individual metabolic products was performed after their separation on TLC.
Fig. 7. Fluorescence micrograph of human skin fibroblasts incubated with 2.5 WM fluorescent sulfatide, in 6 h-pulse/l8 h-chase experiment. (Original magnification X 3150).
Fluorescence microscopy of cells after 6 h-pulse/l8 h-chase (Fig. 7) indicates a marked perinuclear fluorescence and a weak labeling of plasma membrane. MLD cells, under the same conditions, showed a very similar fluorescence pattern. Discussion
The time-dependent uptake up to 24 h at a sulfatide concentration of 2.5 nmol/ml medium followed a bifasic progress (Fig. lB>, suggesting a relatively rapid incorporation into the plasma membrane, followed by a slower internalization. A comparison between our data on the uptake and those reported by other authors using ‘“C-stearic acid labeled sulfatide [19-211 appears difficult, due to the different experimental conditions. In this work, we chose to administer the fluorescent sulfatide to cells as an albumin complex in a medium deprived of FCS, since in a preliminary experiment (Table I) we had confirmed the inhibition of the sulfatide uptake by FCS [121. Inui et al. [20] reported that FCS plays an important role in the translocation of CS to Iysosomes and, in its absence ‘“C-CS remained unhydrolyzed at plasma membrane. This is not true for LRh-CS: in the absence of fetal calf serum the uptake of the fluorescent sulfatide was strongly enhanched and no reduction of its degradation level was observed (Table I). The contradiction is apparent. In the system used by the above quoted authors, the sulfatide dispersed in the medium devoid of FCS, was probably taken up by cells in the form of aggregate and not transferred to lysosomes. In our system, the human albumin complexed to fluorescent sulfatide is not taken up by cells, as demonstrated by the double-label experiment using ~uoresceinated albumin and therefore it is likely monomeric transfer of LRh-CS to plasma membrane occurs. When FCS is present in the medium it seems plausible that a competition for sulfatide exists between the albumin in the albumin/ sulfatide complex and serum proteins, resulting in a lower transfer of sutfatide to the cell surface. In pulse-chase experiments (Fig. 31, the major part of the fluorescence incorporated at 6 h-pulse, was released into the medium during the chase. However, when the chase was performed in the absence of FCS, much more fluorescence was retained by cells, thus indicating a direct involvement of FCS in pulling out fluorescent lipids from cells. The metabolic fate of LRh-CS in the conditions of maximal degradation, that is in pulse-chase, was quite different from that reported by other authors for 14CCS [21]. The major metabolites derived from LRh-CS were ceramide followed by galactosylceramide and sphingomyelin. The striking difference was the high level of ceramide. Few weak spots detectable on TLC in the
region between SPM and sulfatide, a region in which glycerophospholipids are expected to run in the solvent system used, have not been identified because of lack of standards. It appears that ceramidase degrades LRh-ceramide at a very low rate and the incorporation of the fluorescent fatty acid into glycerophospholipids is impaired. The finding, beside ceramide, of a well detectable quantity of sphingomyeiin suggests a specific transfer of ceramide from lysosomes to GOlgi apparatus, known to bc a site of sphingomyelin formation [4,22-241. The presence in the 18 h-chase medium of a large quantity of LRh-CS, and as many metabolites as in cell extract, is remarkable (Fig. 6). The different percentage distribution of catabolites between cell extract and medium, together with the finding that cells incubated in the presence of LRh-CS are as viable as non-treated cells, eliminates the possibility that the presence of metabolites in the medium is the result of cell lysis. The possibility of a metabolism of sulfatide hy the action of enzymes released into the medium by cells. was also excluded by the experiment in which LRh-CS was exposed in a medium preincubated in the presence of fibroblasts. As to the high sulfatide content observed in the medium after 18 h of chase, the eventuality that it could result from the release of non-specifically bound sulfatide during the pulse could be discarded on the basis of the washes carried out between pulse and chase, and more convincing, of the observation that at 18 h-chase the total fluorescence (cell-associated plus medium-associated) roughly corresponded to the cellassociated fluorescence of 6 h-pulse, detected after cells harvesting by trypsinization (data not shown). In conclusion, fluorescent metabolic products dcrived from internalized sulfatide arc released into the medium together with undegraded sulfatide and this process appears to be enhanced by the presence of FCS. Fluorescence microscopy of the cells at 18 h-chase did not reveal a different distribution of the fluorescence between normal and MLD fibroblasts. This seems in agreement with the finding that unhydrolyzed sulfatide accumulates in MLD fibroblasts not only in lysosomes, but also in plasma membranes and Golgi apparatus [20]. Under our experimental conditions, the fluorescent suffatide was practically undegraded by MLD cells (Table I), thus LRh-CS could successfully substitute the radiolabelled sulfatide in a sulfatideloading test for discrimination between MLD and arylsulfatase A pseudodeficiency. Although the metabolic fate of lissamine-rhodamine sulfatide does not parallel that of “C-sulfatide, and therefore the data obtained can not simply be transferred to the metabolism of natural sulfatide, the slowing of the catabolism observed with lissamine-rhod-
87 amine sulfatide could be of practical use for studies of lipid traffic, provided that short pulses are used. The availability of other sphingolipid analogues, all containing the same fluorophore, i.e., lissaminerhodamine, will allow us to undertake studies aimed to correlate the intracellular distribution of the fluorescence with the subcellular localization of the sphingolipid and its catabolites.
10 Marchesini,
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