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J. Biol. Chem., Vol. 278, Issue 39, 37169-37174, September 26, 2003
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From the
Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU) and the Departamento de Bioquímica, Universidad del País Vasco, 48080 Bilbao, Spain and ¶Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, March 28, 2003 , and in revised form, June 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Sphingomyelin is a phospholipid localized mainly at the outer leaflet of the plasma membrane, where it was originally conceived to act in a structural manner to provide a semipermeable barrier to the extracellular environment (2). In 1987, however, it was reported that 1,2-diacylglycerols induce sphingomyelinase activation in GH3 pituitary cells (3), and it was suggested for the first time that this response may activate a sphingomyelin-based signaling pathway (4, 5). Subsequently, Okazaki et al. (6, 7) confirmed this notion, demonstrating that receptor binding of vitamin D3 induced sphingomyelinase activation and that the generated ceramide served as a second messenger in a differentiation pathway of HL-60 cells. Over the past decade, ceramide and other sphingolipids have been identified as primary regulators of signal transduction involved in multiple biological functions (8, 9). Recent studies have suggested that much of the sphingomyelin in the plasma membrane exists in preformed microdomains in mammalian cells (10) called rafts and that this is the site of ceramide generation and signal transmission (11).
Ceramide was originally conceived of as a classic second messenger, directly and stoichiometrically activating a set of target proteins (see below) (8, 9). However, recent data suggest that ceramide may act in part by physically altering the structure and properties of membrane rafts. In this context, it has been suggested that ceramide generated in rafts induces their coalescence into larger platforms and that these macrodomains serve as a site for oligomerization of cell surface receptors such as Fas and CD40 (12–14) and the internalization of bacteria such as Pseudomonas aeruginosa and Neisseria gonorrhoeae (15, 16). In this paradigm, acid sphingomyelinase is initially translocated onto the outer leaflet of the plasma membrane where it catalyzes the hydrolysis of raft-associated sphingomyelin.
Ceramide affects a number of membrane properties in model membrane systems, which may impact platform formation in biological membranes; ceramide increases lipid order, gives rise to ceramide-rich separate domains, and destabilizes the lamellar structures, inducing membrane permeabilization and membrane fusion (see Ref. 17 for a review). As a consequence, treating defined model membrane systems containing sphingomyelin with sphingomyelinase has been found to produce, via ceramide formation, vesicle efflux (18, 19), membrane fusion (20, 21), and vesicle budding (22). These events are believed to occur through the transient formation of non-bilayer intermediates (17, 23, 24).
Ceramide structural effects on membranes are interesting in the context of the sphingomyelin pathway (9). Evidence shows that the sphingomyelin pathway is a ubiquitous signaling pathway conserved through evolution and that it signals events as distinct as differentiation, senescence, proliferation, and cell cycle arrest (11, 25, 26). A large body of research has focused on its role in stress responses (11, 27–29). Over the past decade, a number of direct targets for ceramide have been explored as mediators of ceramide signaling. Ceramide reportedly interacts directly with kinase suppressor of Ras (identical to ceramide-activated protein kinase) (30, 31), ceramide-activated protein phosphatase (32), the protein kinase C isoforms 
, 
, and 
(33, 34), c-Raf-1 (35, 36), and other proteins. A recent report by Kashiwagi et al. (33) demonstrated that ceramide binds to the C1b domain of protein kinase C- in COS7 and Chinese hamster ovary-K1 cells, directing its translocation to the intracellular membrane compartments. Ceramide exhibited a high affinity toward the C1b domain, exceeding the binding affinity of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Other potential ceramide targets, such as kinase suppressor of Ras and c-Raf-1, also contain C1b domains, but whether the C1b domain targets these proteins to sites of ceramide generation, such as plasma membrane rafts, is presently unknown. However, these targets are intracellular, and it is presently uncertain whether ceramide generated on the outer leaflet of the plasma membrane can initiate their activation.
The present study was intended to address, using biophysical techniques, the question of how sphingomyelin hydrolysis on one side of a bilayer might induce transbilayer lipid reorganization. We have developed two novel systems to examine ceramide-induced transfer across a membrane bilayer, one of which we have applied to large unilamellar vesicles and the other to both vesicles and resealed erythrocyte ghosts. In all three cases, we observe transmembrane lipid movements in parallel with sphingomyelinase activity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Liposome Preparation—SM/PE/Ch (2:1:1 molar ratio) large unilamellar vesicles (LUV) were prepared by the extrusion method (38) using Nuclepore filters of 0.1-µm pore diameter at room temperature, as detailed previously (39). Chemical analysis of the extruded vesicles showed that the extrusion procedure did not modify their composition.
LUV were prepared in 10 mM HEPES, 200 mM NaCl, 10 mM CaCl2, 2 mM MgCl2, pH 6.5. When required, neuraminidase (0.16 unit/ml) was added to the hydration buffer. In this case, non-entrapped neuraminidase was removed by gel filtration through Sephadex G-75.
Asymmetric Incorporation of Glycolipids—To incorporate GM3 ganglioside to the outer monolayer of liposomes prepared as described above, the glycolipids in organic solvent were dried and resuspended in a volume of methanol that was 5% of the vesicle suspension volume. Vesicles were added to this methanolic glycolipid solution so that GM3 was 10 mol % of the total lipids. After vortex mixing, they were left to incubate for 15 min at room temperature (40).
Purification of Anti-neuraminidase IgG—The antibody raised in rabbits against neuraminidase was separated from hemoglobin by using a HiTrap protein G affinity column; 1 ml of the mixture was passed through the column, and the HiTrap column was washed 3 times with a 20 mM phosphate, pH 7, buffer to remove hemoglobin. Anti-neuraminidase IgG was eluted from the affinity column with a 0.1 mM glycine-HCl, pH 2.7, buffer.
Enzyme Assay—All experiments were performed at 37 °C, lipid concentration was 0.3 mM, and enzymes were used at 1.6 units/ml for sphingomyelinase on LUV, 0.16 unit/ml for sphingomyelinase on erythrocytes, and 0.16 unit/ml for neuraminidase.
Sphingomyelinase activity was assayed by determining phosphorous content (41) in the aqueous phase of an extraction mixture (chloroform/methanol/hydrochloric acid, 200/100/1, by volume) after the addition of aliquots from the reaction mixtures at different times. Sialic acid formed after treatment of liposomes containing GM3 with neuraminidase was determined by using a modification of the resorcinol-sialic acid assay (42). When required, neuraminidase activity was tested with an artificial substrate, N-acetylneuraminyl-lactose.
Neuraminidase Inhibition by Anti-neuraminidase IgG—LUV containing entrapped neuraminidase were prepared as above and diluted to 0.3 mM lipid. The vesicle suspension was treated with Triton X-100 (0.1% w/v) to release the enzyme; 100 µl of the anti-neuraminidase IgG (0.5 µg/µl) was then added, and the reaction mixture was left to incubate for 30 min. Then, 0.03 mM N-acetylneuraminyl-lactose was added, and the production of sialic acid was measured after 1 h. A control in the absence of IgG was performed simultaneously.
Ghost Membrane Preparation—Human erythrocyte ghost membranes were obtained by a modified Steck and Kant method (43, 44). The membranes obtained from 20 ml of erythrocyte concentrate (as provided by the blood bank) were washed three times by centrifugation in 0.9% NaCl, and the pellet was resuspended with cold 1.2 mM acetic acid, 4 mM MgSO4, pH 3.2, buffer, left for 30 min at 4 °C, and centrifuged. In the next step, the pellet was suspended in 3 volumes of 10 mM HEPES, 200 mM NaCl, 10 mM CaCl2, 2 mM MgCl2, pH 6.5, containing 3 µM NBD-PE, and then membranes were stabilized and allowed to reseal by incubating them for 1 h at 37 °C. After washing 3 times by centrifugation in the same buffer, the resulting membrane suspension was layered over 43% sucrose in 25 mM Tris, pH 7.4, and centrifuged for 60 min at 27,500 x g (TST 55.5 rotor, Kontron) (45). The resealed ghosts floating on top of the sucrose solution were harvested and washed 3 times in the 10 mM HEPES, 200 mM NaCl, 10 mM CaCl2, 2 mM MgCl2, pH 6.5, buffer. At this step, the ghosts were labeled with NBD on both sides of the membranes. To eliminate the outer leaflet label, we used 0.6 mM sodium dithionite, which reduced outer NBD and thus abolished its fluorescence. Reduction was monitored in an LS50 spectrofluorometer (PerkinElmer Life Sciences) at room temperature with a continuously stirred cuvette. Excitation and emission wavelengths were 465 and 530 nm, respectively. A cut-off filter (515 nm) was used to prevent contamination from scattered light. Labeled ghosts were separated from excess sodium dithionite by centrifugation. The pellet was resuspended in the buffer described above.
LUV Labeled in the Inside Bilayer with NBD-PE—SM/PE/Ch LUV were prepared by using the method described above, including 0.6 mol % NBD-PE. To eliminate the outer leaflet NBD-PE fluorescence, the same dithionite method described above for ghost membranes was used, except that to separate LUV from excess sodium dithionite, a Sephadex G-75 chromatography column was used.
Fluorimetric Assays—All experiments were performed at 37 °C, lipid concentration was 0.3 mM for LUV assays and 0.09 mM for ghost assays, and sphingomyelinase was used at 1.6 unit/ml and 0.16 unit/ml for LUV and ghost assays, respectively. Aliquots (50 or 100 µl for LUV or ghost membranes, respectively) were removed from the reaction mixture at regular intervals and mixed with 10 µl of Rho-IgG in 10 mM HEPES, 200 mM NaCl, 10 mM CaCl2, 2 mM MgCl2, pH 6.5, buffer to a final volume of 500 µl. Energy transfer was monitored by using an LS50 spectrofluorometer (PerkinElmer Life Sciences) under the same conditions described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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10 mol % of total lipid) located exclusively on the outer leaflet (40). These vesicles contained neuraminidase and when kept at 4 °C, the vesicles were stable for at least 12 h; no significant amount of GM3 was hydrolyzed in this period of time. Addition of sphingomyelinase (1.6 units/ml) to a suspension of these vesicles induced SM hydrolysis, which reached equilibrium after 20 min, when 40% of SM had been hydrolyzed (Fig. 1A). Addition of Triton X-100 after 60 min caused membrane disruption, but SM hydrolysis did not go beyond 50% at 30 min after detergent addition, i.e. 90 min after sphingomyelinase addition. Previous experiments had shown that this Triton X-100 concentration did not inhibit sphingomyelinase or neuraminidase.
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Aliquots of the vesicle suspension were removed at fixed times after the addition of sphingomyelinase and analyzed for the GM3 product of neuraminidase activity, sialic acid. GM3 was hydrolyzed almost in parallel with SM, except that no saturation was observed (Fig. 1B). After the addition of Triton X-100, virtually all GM3 was cleaved by neuraminidase. One interpretation of these data is that, as a consequence of sphingomyelinase activity, GM3 was flipping to the inner leaflet, thus becoming accessible to neuraminidase.
However, it was necessary to rule out the possibility of neuraminidase coming out from the vesicles as a result of sphingomyelinase activity. Ceramides increase membrane permeability (18, 19, 45), and an efflux of molecules up to 40 kDa has been observed in sphingomyelin-treated vesicles (19). Neuraminidase has a molecular mass of 70 kDa. To clarify this aspect, neuraminidase activity outside the sphingomyelinase-treated vesicles was assayed with the water-soluble substrate N-acetylneuraminyl-lactose. For this purpose, aliquots of the vesicle suspension were filtered through YM-100 Centricon filters, which allow the easy passage of neuraminidase. Neuraminidase activity in the filtrates is shown in Fig. 2A. Even in the absence of sphingomyelinase activity (time zero), 18% of the total enzyme activity was recovered in the filtrates. This is probably because of vesicle breakdown due to shear stress during the filtration procedure. Vesicles containing GM3 but not sphingomyelinase or internal neuraminidase were treated with neuraminidase at the concentration found in the filtrates. Neuraminidase was added to the vesicle suspension from the outside to ensure enzyme-substrate interaction. In this case (as seen in Fig. 2B), the time course of GM3 hydrolysis is totally different from the one depending on sphingomyelinase activity. Thus, the results in Fig. 1B cannot be explained on the basis of neuraminidase efflux from the vesicles.
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An additional control was performed in which any extravascular neuraminidase would have been neutralized by a specific antibody. With this aim, a polyclonal antineuraminidase antibody was raised in rabbits. We verified that the purified antineuraminidase IgG at 50 µg/ml completely abolished neuraminidase activity at 0.16 unit/ml. The same concentration of IgG had no effect on sphingomyelinase activity. When the experiment described in Fig. 1 was repeated with 50 µg/ml antineuraminidase antibody in the medium (Fig. 2C), no significant differences were found between the fractions of hydrolyzed GM3 in the presence and absence of antibody (Student's t test, p > 0.05 for all pairs of data points). We concluded that GM3 hydrolysis was catalyzed by neuraminidase inside the vesicles; thus, sphingomyelinase activity had caused GM3 to flip to the inside lipid monolayer.
Fluorescence Energy Transfer in Lipid Vesicles—A completely different assay was developed to test the transmembrane movement of lipids in a system in which sphingomyelinase activity was likely to cause membrane permeabilization to solutes of Mr 
40,000. This novel procedure was based on fluorescence resonance energy transfer between NBD-PE and Rho-IgG. Energy transfer between NBD and rhodamine is well known and often used in membrane studies. Our modification consisted of using rhodamine bound to an irrelevant IgG, whose large Mr (150,000) ensured that, in principle, it could not penetrate the vesicles.
LUV were prepared as usual, except that 0.6 mol % NBD-PE was included in the lipid mixture. After preparation, the vesicle suspension was treated with the essentially impermeable reducing agent sodium dithionite, so that NBD in the outer monolayers was reduced and its fluorescence was abolished. Excess dithionite was removed immediately by gel filtration. LUV labeled with NBD only in their inner lipid leaflets were then treated with sphingomyelinase. Enzyme activity was the same as described in Fig. 1A. Aliquots were removed from the reaction mixture at regular intervals and mixed with Rho-IgG in buffer. As the sphingomyelinase reaction proceeded, NBD-PE flopped toward the outer monolayer, and energy transfer to Rho-IgG could take place. Consequently, when the fluorescence of NBD-PE was excited, its emission intensity decreased with time, and the emission intensity of Rho-IgG increased accordingly. The corresponding data are shown in Fig. 3. Control experiments, also shown in the figure, indicated that in the absence of sphingomyelinase, NBD-PE fluorescence remained invariant with time. Thus, fluorescence energy transfer measurements confirm that sphingomyelinase activity induces the transmembrane movement of lipids.
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Fluorescence Energy Transfer in Erythrocyte Ghosts—A similar procedure was applied to erythrocyte ghosts to explore the transbilayer movement of lipids induced by sphingomyelinase in a cell membrane. Unsealed ghosts were treated with NBD-PE, which became incorporated into both membrane leaflets. Ghosts were then resealed and treated with dithionite to reduce outer leaflet NBD. Excess dithionite was removed by repeated centrifugation and washing. Sphingomyelinase activity reached equilibrium in erythrocyte membranes much faster than in liposomes (compare Figs. 1A and 4A). The experiments in Fig. 4 were obtained by using 0.16 unit/ml sphingomyelinase, i.e. 
of the amount used with liposomes. The reasons for this high rate of sphingomyelin degradation in red blood cells are currently the object of a separate investigation in this laboratory. Because of the fast ceramide production, fluorescence resonance energy transfer was detected in ghosts after a very short time (Fig. 4B). The correlation between rates of ceramide production and energy transfer (compare Figs. 3 and 4B) supplies additional evidence that SM hydrolysis is driving the transmembrane lipid movement.
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An additional control experiment was performed to ensure that sphingomyelinase did not stimulate transfer of Rho-IgG across the erythrocyte ghost membrane (Table I). For that purpose, resealed ghosts were prepared containing Rho-IgG and treated with sphingomyelinase for 10 min. The ghosts were then passed through a HiTrap protein G affinity column to retain any Rho-IgG that had leaked out. The fluorescence intensity of the column-eluted ghosts was the same as that of an equivalent ghost preparation untreated with sphingomyelinase, indicating that no loss of Rho-IgG had occurred as a result of enzyme treatment. When ghosts previously treated with Triton X-100 were passed through the HiTrap column, the fluorescence level was 
10% of the former cases, showing that, in the presence of the detergent, loss of Rho-IgG had occurred. Later, the HiTrap column containing any leaked Rho-IgG was eluted with a pH 2.7 buffer. In this case, the eluates from the control and sphingomyelinase-treated ghosts showed virtually no fluorescence, whereas the eluate from the detergent-treated ghosts showed a clear fluorescence signal (Table I).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Under our experimental conditions, which were similar to those used by Ruiz-Argüello et al. (18), a dynamic equilibrium is probably established between lamellar and non-lamellar phase elements within an overall lamellar structure. Of course, only a stable bilayer allows the maintenance of asymmetry. As shown by 31P NMR in Ruiz-Argüello et al. (18), the ceramide-induced formation of isotropic components in a lamellar environment can explain very well the loss of asymmetry. A relationship between flip-flop movement and the formation of non-lamellar lipid structures was already suggested by Cullis and de Kruijff (49). The cryotransmission electron microscopy observations by Basañez et al. (20) are pertinent in this context. These authors examined large unilamellar vesicles containing SM:PE:Ch (2:1:1 molar ratio) treated with sphingomyelinase, i.e. the same system used in the present study. Vesicle aggregation and increase in size were clearly seen, but no images that could be related to pores or non-lamellar intermediates could be seen. This may be because those intermediates were either too small or, probably, too short-lived. However, the data by Basañez et al. (20) confirm that, under our conditions, the lipid organization is essentially lamellar, even after sphingomyelinase treatment. In summary, we suggest that sphingomyelinase generates ceramide and that ceramide induces the transient formation of non-lamellar structures, the latter leading to the (partial) loss of asymmetry in the transbilayer lipid distribution.
Kinetic Aspects—At least two kinetic aspects from the above results deserve some comment. One is the observation, particularly clear in Figs. 1 and 4, that transbilayer movement can go on even after ceramide production has reached equilibrium. With the neuraminidase method (Fig. 1), the amount of ceramide increases between 20 and 60 min by 10% of total sphingolipid, whereas hydrolyzed GM3 increases by 30%. In the case of ghosts (Fig. 4), only 6% of phospholipid is further hydrolyzed between 10 and 120 s, but energy transfer (measured as the increase in rhodamine fluorescence) increases by 42%. This reveals the different nature of the two processes under consideration (sphingomyelin hydrolysis and lipid transbilayer motion). In enzyme catalysis, equilibrium is dictated by the law of mass action, and once it is reached, no net change in product or substrate concentration will be observed. With transbilayer lipid transfer, however, once the minimum proportion of ceramide that allows non-lamellar phase transition is formed, the process will continue until complete symmetry is reached (independently of the changes in ceramide production rate).
Also noticeable is the very fast lipid scrambling in sphingomyelinase-treated erythrocyte ghosts, which is secondary to the equally fast SM hydrolysis (Fig. 4). Apart from confirming the cause-effect relationship between ceramide production and transbilayer movement, the fast kinetics helps to differentiate our observations from those attributed to a putative "scramblase" (1, 50, 51). These authors find that flip-flop of phospholipids ensues when treating red blood cells with calcium and an ionophore. Because we used Ca2+ in the sphingomyelinase assay medium and the enzyme activity permeabilizes the erythrocyte membranes (18), it could be argued that we were, in fact, activating the "scramblase." However, the kinetics of Ca2+/ionophore-induced transbilayer movements has halftimes on the order of 10 min (50, 51) versus less than 10 s in our case (Fig. 4). This large kinetic difference virtually excludes any influence of "scramblase" in our results.
Physiological Implications—Lipid and protein motions are known to be closely interrelated in cell membranes (52). The observation that ceramide formation by sphingomyelinase leads to transbilayer movements of various lipid species may be related to a number of physiological cellular events and properties not well understood at present. An important one is the apparently anomalous distribution of SM predominantly in the outer monolayer of the cell plasma membrane, whereas ceramide receptor proteins are intracellular. Such distribution may appear reasonable in the resting condition, but how are ceramide and receptor both put together after stimulation of the ceramide signaling pathway? Our results are consistent with the fact that the secretory form of the acid sphingomyelinase must be translocated into raft structures on the outer leaflet of the plasma membrane to hydrolyze raft SM and initiate signaling within seconds to minutes, which results in the activation of intracellular signaling cascades. At least in some instances, this may be spatially integrated through ceramide-driven platform formation (12–14). The resulting ceramide, originally generated in the outer leaflet, is soon distributed among both monolayers, thus being able to bind intracellular proteins docking the plasma membrane inner monolayer. Moreover, as shown in Figs. 1, 3, and 4, ceramide motion will be accompanied by the transbilayer transfer of other lipids that will in turn become accessible to cytosolic enzymes. Importantly, SM may be internalized as a result of ceramide formation, thus becoming a substrate for intracellular sphingomyelinases.
Moreover, the present study defines a mechanism for transmembrane signaling distinct from that reported by Tepper et al. (53). In their study, the apoptotic stimulus itself resulted in the loss of membrane asymmetry because of a membrane scramblase, allowing some SM molecules access to the inner leaflet and initiating ceramide formation in a time frame of hours to days after stimulation. However, as noted above, the physiological process of cell signaling is triggered in a period of seconds to minutes, a time scale more compatible with the kinetics of sphingomyelinase-induced transbilayer motion demonstrated in this paper than with the scramblase-dependent mechanism.
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FOOTNOTES |
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Recipient of a predoctoral fellowship from the Basque Government.
|| To whom correspondence should be addressed. E-mail: gbpgourf{at}lg.ehu.es.
1 The abbreviations used are: SM, egg sphingomyelin; PE, phosphatidylethanolamine; Rho-IgG, tetramethylrhodamine goat anti-mouse IgG; GM3, monosialoganglioside; NBD, N-(7-nitrobenzene-2-oxa-1,3-diazol-4-yl); Ch, cholesterol; LUV, large unilamellar vesicles.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, hydrolyzed GM3 ganglioside when LUV not containing neuraminidase were treated with the same amount of enzyme that was released in the experiment in A. Average values of two closely similar experiments; 
, data of 
, controls in the absence of sphingomyelinase. NBD-PE was initially located on the inner leaflet only; Rho-IgG was outside the vesicles during the whole experiment. Rho-IgG fluorescence was excited through NBD-PE by fluorescence resonance energy transfer. Average values ± S.E. (n = 3). a.u., absorbance units.