Interfacial Regulation of Acid Ceramidase Activity

STIMULATION OF CERAMIDE DEGRADATION BY LYSOSOMAL LIPIDS AND SPHINGOLIPID ACTIVATOR PROTEINS*

Thomas LinkeDagger , Gundo WilkeningDagger , Farsaneh SadeghlarDagger , Heidi MozcallDagger , Katussevani BernardoDagger §, Edward Schuchman, and Konrad SandhoffDagger ||

From the Dagger  Kekulé-Institut für Organische Chemie und Biochemie, D-53121 Bonn, Germany, the  Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, and the § Institut für Medizinische Mikrobiologie und Hygiene, Goldenfelsstrasse 1, D-50935 Köln, Germany

Received for publication, July 31, 2000, and in revised form, November 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosphate, phosphatidylinositol, and dolichol phosphate were incorporated into substrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated that acid ceramidase remained bound to the liposomal surface during catalysis. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators of ceramide hydrolysis in the presence of anionic phospholipids. This finding was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffering from prosaposin deficiency. Strong protein-lipid interactions were observed for both SAP-D and acid ceramidase in surface plasmon resonance experiments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occurred at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are required for efficient hydrolysis of ceramide by acid ceramidase.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glycosphingolipids (GSLs)1 are characteristic components of the extracytosolic leaflet of the plasma membrane of eukaryotic cells. GSLs are composed of a hydrophilic carbohydrate chain linked to a hydrophobic ceramide moiety. They form cell type-specific patterns on the cell surface, which can change with cell growth, differentiation, viral transformation, or oncogenesis (1).

Degradation of GSLs takes place in the acidic compartments of the cell, namely endosomes and lysosomes. According to a recent model for the topology of lysosomal digestion, GSLs reach the lysosome after endocytosis from the plasma membrane in the form of intraendosomal and intralysosomal vesicles (2). In the lysosomes, GSLs are degraded by acidic exohydrolases. The final step of GSL catabolism is the hydrolysis of ceramide into sphingosine and fatty acid by acid ceramidase (AC, N-acylsphingosine amidohydrolase, EC 3.5.1.23 (3)).

An inherited deficiency of AC activity is the principal cause of Farber disease, a rare, autosomal recessive inherited sphingolipid storage disorder. Farber disease is characterized by a massive accumulation of ceramide in lysosomes of various tissues such as liver, spleen, lung, and heart (4). A total of seven different clinical subtypes of Farber disease have been reported, six of which are believed to be primarily caused by mutations in the AC gene. Farber disease type 7 is the result of a complete lack of sphingolipid activator proteins (SAPs) due to a mutation in the initiation codon of the SAP precursor protein prosaposin and is also known as sphingolipid activator protein deficiency. This deficiency not only affects the degradation of ceramide by AC but also the degradation of other GSLs such as glucosylceramide and galactosylceramide by glucocerebrosidase and galactocerebrosidase, respectively (4).

The lysosomal degradation of GSLs with short oligosaccharide head groups requires the coordinate action of both acidic hydrolases and SAPs. SAPs are a group of small, heat-stable, enzymatically inactive glycoproteins. While the GM2 activator protein (GM2-AP) is encoded by its own gene, SAP-A, -B, -C, and -D are derived from a common precursor protein, the SAP precursor or prosaposin, through proteolytic processing (5). All four SAPs display a high degree of homology such as conserved glycosylation sites, matching patches of hydrophobic amino acid residues, and identical location and connectivity of disulfide bridges (6-8).

To measure AC activity in vitro, ceramide has to be solubilized in a complex mixture of synthetic nonionic and anionic detergents (3). The efficient hydrolysis of ceramide by AC in vivo requires the presence of SAPs. The requirement of AC for SAP-D was demonstrated by metabolic labeling studies using fibroblasts from a patient suffering from a prosaposin deficiency. Supplementing the cell culture medium with purified SAP-D specifically reduced the ceramide accumulation observed in these cells (9). However, how SAP-D precisely cooperates with AC to effect this reduction in ceramide storage has not yet been investigated. Furthermore, the mechanism by which water-soluble AC interacts with its membrane-bound substrate ceramide, and how this interaction is modulated by SAPs or by other cofactors, is currently unknown.

A number of studies have shown that some lysosomal hydrolases, such as lactosylceramidase, glucocerebrosidase, and acid sphingomyelinase, are stimulated by different acidic phospholipids including phosphatidylserine, phosphatidic acid, phosphatidylinositol (PI), bis(monoacylglycero)phosphate (BMP), and dolichol phosphate (10-13). Of these lipids only BMP occurs exclusively in the acidic compartments of the cell, and its concentration of the total phospholipids, obtained from purified rat liver lysosomes, is reported to be in the range of 4-17 mol % (14-16). Indeed, Kobayashi and coworkers (17) recently demonstrated that intraendosomal structures are highly enriched in BMP.

With the goal of providing further support for a previously presented model concerning the topology of lysosomal digestion, we directed the main focus of our investigation toward the degradation of membrane-bound ceramide by AC in the presence of lysosomal lipids and SAPs. We established a detergent-free, liposomal assay system for AC with the purpose of studying ceramide degradation as an interfacial process. To mimic the acidic environment of lysosomes as closely as possible, not only lysosomal lipids, such as BMP, PI, and dolichol phosphate, but also a number of lipid degradation products such as fatty acids and dolichol were incorporated into ceramide-bearing liposomes.

In addition, we wanted to gain new insights into how SAPs and AC interact with and bind to phospholipid membranes. We therefore complemented our enzymatic experiments with surface plasmon resonance spectroscopy binding studies using immobilized lipid bilayers of varying composition.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Phosphatidylcholine (egg yolk), phosphatidylinositol (bovine liver), cholesterol, dolichol (porcine liver), and dolichol phosphate (porcine liver) were purchased from Sigma. Pioneer L1 sensor chips were purchased from BiaCore (Freiburg, Germany). Bis-(1-myristoyl-sn-glycero-3)-phosphoric acid was purchased from Avanti Polar Lipids (Birmingham, AL). Bis-(1-palmitoyl-sn-glycero-3)-phosphoric acid (BMP) was synthesized as described previously (18). Ceramides were purchased from Matreya Lipids (Pleasant Gap, PA) or synthesized according to Okazaki et al. (19). All other reagents were of the highest purity available.

Purification of AC and SAPs-- SAP-D-free, native acid ceramidase (AC) was purified from human placenta to apparent homogeneity as described previously (20). Briefly, fresh human placenta were freed from the amnion and umbilical cord and homogenized in 0.25% (w/v) Nonidet P-40 in water. Following centrifugation, the supernatant was subjected to a fractionated ammonium sulfate precipitation. Precipitated proteins were collected by centrifugation and dissolved in buffer. AC was purified by sequential chromatography using concanavalin A-Sepharose, octyl-Sepharose, Matrix Gel Red-Sepharose, and DEAE-Fractogel.

SAP-A, -B, -C, and -D were purified to apparent homogeneity by sequential chromatography using DEAE Poros HQ, Superdex 75, and reverse phase-high pressure liquid chromatography from Gaucher spleen according to established protocols (9). The purity and identity of the individual SAPs was tested by SDS-polyacrylamide gel electrophoresis, Western blot analysis, and matrix-assisted laser desorption ionization-mass spectrometry.

Preparation of Liposomes-- Large unilamellar liposomes (LUVs) were prepared as described previously (21). Briefly, appropriate aliquots of phosphatidylcholine (PC), cholesterol (Chol), ceramide, and other lipids were dissolved in organic solvents, mixed, and then dried under a stream of nitrogen and kept under a vacuum for at least 1 h. The dried lipid mixture was rehydrated to a concentration of 2.5 mM in a 2 mM Tris/HCl buffer, pH 7.0. The lipid suspension was freeze-thawed 10 times in liquid nitrogen. The lipid suspension was then passed 21 times through two stacked polycarbonate membranes (pore size diameter: 200, 100, or 50 nm as indicated in the legend to the figures, Nucleopore) in a miniextruder (Liposofast, Avestin).

SUVs were prepared by ultrasonic irradiation of LUVs with a Microtip sonicator (Branson, Danbury, CT) at 0 °C for 40 min (intervals of 15 s of sonification and 30 s of pause).

Neutral substrate-carrying liposomes were composed of 70 mol % PC, 20 mol % Chol, and 10 mol % ceramide. Unless otherwise stated, negatively charged liposomes generally consisted of 45 mol % PC, either 25 mol % BMP or PI, 20 mol % Chol, and 10 mol % ceramide. When the concentration of negatively charged lipids was varied, the appropriate amount of PC was replaced, whereas the mol % of all other components were kept constant.

Liposomal AC Assay-- The assay mixture for the determination of AC activity contained the following in a final volume of 100 µl: 25 µl of NaAc buffer (40 mM, pH 4.0; 600 mM NaCl), 2.5-3 µg of AC (in 10 µl), LUVs, or SUVs as indicated (40 µl, 2.5 mM), and 25 µl of water. Incubations were carried out for 30 min at 37 °C. High pressure liquid chromatographic analysis and quantification of the liberated sphingosine was performed as described previously (3).

Metabolic Labeling and Isolation of Cellular SLs-- Normal human and SAP precursor-deficient fibroblasts were incubated for 24 h with L-[3-14C]serine (54 µCi/µmol; 1 µCi per ml of medium) in minimum Eagle's medium containing 0.3% fetal bovine serum. The medium was then removed and exchanged for a medium containing both L-serine (185 nmol/ml medium; 0.06% FKS) and purified SAPs (25 µg/ml). After 120 h, cells were washed with phosphate-buffered saline, detached with 0.25% trypsin, and collected by centrifugation. Cell pellets were resuspended in water, and total lipids were extracted with 7 ml of chloroform/methanol/water/pyridine (60/160/6/1, by volume) overnight at 50 °C. Phospholipids were removed by alkaline hydrolysis with 2 ml of methanolic KOH (50 mM) for 2 h at 37 °C. Lipids were desalted with reversed phase chromatography. Aliquots were measured for radioactivity by scintillation counting. Labeled lipids were applied to TLC plates and chromatographed with chloroform/methanol/0.22% aqueous CaCl2 (60/35/5 by volume). GSLs and SLs were identified by their Rf values. Radioactive spots were evaluated with the Fujix BAS 1000 PhosphorImager system.

Surface Plasmon Resonance (BiaCore)-- Surface plasmon resonance (SPR) was measured at 25 °C using real time bimolecular interaction analysis in a BiaCore instrument (BiaCore X). Lipid bilayers were immobilized on the surface of a PioneerTM L1 sensor chip (BiaCore, Freiburg, Germany). LUVs of varying composition were diluted in TES buffer to a final concentration of 0.1 mM (10 mM TES, 300 mM NaCl, 1 mM CaCl2, pH 7.0) and injected into the system until the sensor chip surface was saturated as indicated by a constant signal plateau at ~8000 response units. AC (100 nM) and SAP-D (2.5 µM) were injected in running buffer (50 mM NaAc buffer, pH 4.0, unless otherwise indicated) at a flow rate of 20 µl/min.

Presentations of Data-- All data presented are the means of at least duplicate determinations. Determinations were carried out in duplicate. All individual values were in the range of ±5% up to ±15% of the mean.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ceramide Degradation Is Significantly Enhanced by Lysosomal Phospholipids-- Several studies have shown that the enzymatic activity of various lysosomal proteins, including GSL hydrolases and phospholipases, is stimulated by negatively charged phospholipids such as phosphatidic acid, phosphatidylserine, PI, and BMP (10-13). Of these lipids BMP and PI were found to be enriched in the endosomes of BHK cells and in the lysosomal fractions of rat liver and in cultured skin fibroblasts (14-16, 22).

As a starting point of our investigation of the interfacial activity of AC, we incorporated BMP and PI into ceramide-containing large unilamellar vesicles (LUVs, diameter >100 nm). Fig. 1 shows that increasing concentrations of BMP incorporated into LUVs stimulate the rate of ceramide hydrolysis. PI was also able to enhance the rate of ceramide degradation; however, it was not as effective as BMP, especially at higher concentrations of negatively charged phospholipid (Fig. 1).



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Fig. 1.   BMP and PI stimulate the degradation of membrane-bound ceramide. AC activity was measured in the presence of increasing concentrations of BMP (black-square) and PI () in ceramide-bearing LUVs in the absence of SAPs. All assay mixtures were prepared as described under "Experimental Procedures." The data presented are the means of three determinations. All individual values were in the range of ±5 up to ±10% of the mean.

The Hydrolysis of Membrane-bound Ceramide Is Stimulated by SAP-D and SAP-C-- The hydrolysis of ceramide in vivo requires the presence of SAP-D and possibly SAP-C (9). To verify this observation in vitro we added increasing amounts of SAP-D and SAP-C to our liposomal assay mixture. We also wanted to find out whether the stimulatory activity of SAP-D and SAP-C depended on the concentration of negatively charged lipid. Fig. 2, A and B, shows that SAP-D required the presence of at least 10 mol % BMP or PI before it significantly stimulated ceramide hydrolysis. In contrast, SAP-C was able to enhance ceramide degradation to a minor degree even in the absence of negatively charged phospholipid (Fig. 2, C and D). However, as Fig. 2, A-D, demonstrates, both SAP-D and SAP-C were most effective when LUVs contained 25 mol % of negatively charged phospholipid. Under these conditions, the addition of 2.5 µM SAP-D increased ceramide degradation ~3-fold in both BMP- and PI-containing LUVs. Under the same conditions, the addition of 2.5 µM SAP-C resulted in a 2-fold stimulation for BMP-containing LUVs and a 3-fold stimulation of PI-containing LUVs. These enzymatic assays prove that a combination of BMP and either SAP-D or SAP-C afforded the highest in vitro ceramide degradation rates. The combination of 25 mol % BMP and 2.5 µM SAP-D increased the ceramide degradation rate nearly 30-fold, and the combination of 25 mol % BMP and 2.5 µM SAP-C stimulated ceramide hydrolysis nearly 20-fold when compared with assay mixture devoid of activator proteins and BMP.



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Fig. 2.   SAP-D and SAP-C enhance the hydrolysis of ceramide in negatively charged liposomes. AC activity was measured with ceramide-bearing LUVs in the presence of increasing concentrations of either SAP-D (A and B) or SAP-C (C and D) in the assay mixture. A, liposomes contained 0 mol % (open circle ), 5 mol % (×), 10 mol % (black-triangle), 25 mol % (black-square), and 50 mol % () BMP. B, liposomes contained 0 mol % (open circle ), 5 mol % (×), 10 mol % (black-triangle), 25 mol % (black-square), and 50 mol % () PI. C, liposomes contained 0 mol % (open circle ), 5 mol % (×), 10 mol % (black-triangle), 25 mol % (black-square), and 50 mol % () BMP. D, liposomes contained 0 mol % (open circle ), 5 mol % (×), 10 mol % (black-triangle), 25 mol % (black-square), and 50 mol % () PI. All assay mixtures were prepared as described under "Experimental Procedures." The data presented are the means of at least duplicate determinations. All individual values were in the range of ±12 up to ±17% of the mean.

Dolichol Phosphate but Not Dolichol Stimulates Ceramide Degradation-- Dolichol is one of the largest naturally occurring lipids in the cell and consists of 18-20 isoprene units. Dolichol and its phosphate ester dolichol phosphate were identified as lipid components of lysosomal membranes (23). Whereas dolichol was not able to influence markedly ceramide degradation even in the presence of SAP-D or SAP-C (Fig. 3A), dolichol phosphate was able to stimulate ceramide degradation ~2-fold at 10 mol %. Under these conditions, only SAP-C, and not SAP-D, afforded an additional 2.5-fold increase in the rate of ceramide hydrolysis (Fig. 3B).



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Fig. 3.   Only the phosphorylated form of dolichol stimulates the hydrolysis of membrane-bound ceramide. Increasing concentrations of dolichol (A) and dolichol phosphate (B) were incorporated into substrate-bearing liposomes. Incubation mixtures contained either no SAP (open circle ), 2.5 µM SAP-D (black-triangle), or 2.5 µM SAP-C (black-square). All assay mixtures were prepared as described under "Experimental Procedures." The data presented are the means of at least duplicate determinations. All individual values were in the range of ±5 up to ±8% of the mean.

A number of other negatively charged lipids, including gangliosides and fatty acids, were also tested for their ability to stimulate the degradation of membrane-bound ceramide by AC. The gangliosides GM1 and GD1a were incorporated into LUVs at a concentration of 10 mol %; however, they had no effect on the rate of ceramide hydrolysis. Incorporated fatty acids including lauric and palmitidic acid inhibited the degradation of membrane-bound ceramide to a minor degree at a concentration of 10 mol % (data not shown).

Bilayer Curvature Significantly Affects the Degradation of Membrane-bound Ceramide-- According to our topology model, GSLs are degraded as membrane-bound components of intralysosomal vesicles and structures. These vesicles were observed in fibroblasts of patients suffering from a prosaposin deficiency. According to electron microscopy studies, the size of these vesicles was estimated to be in the range of 40-100 nm (24). It was therefore speculated that this high degree of bilayer curvature could have an influence on ceramide degradation rates. To test this hypothesis we prepared neutral and anionic liposomes with varying mean diameter. The effect of vesicle size on ceramide hydrolysis was especially apparent in neutral liposomes composed of PC, Chol, and ceramide. The rate of ceramide hydrolysis was nearly 5-fold higher in the absence of SAP-D and ~7-fold higher in the presence of SAP-D in small unilamellar vesicles (SUVs, diameter <50 nm) as compared with large unilamellar vesicles (LUVs, diameter >100 nm, Fig. 4). In negatively charged, PI-containing liposomes, however, ceramide degradation rates were clearly dominated by the presence of anionic lipids. Ceramide degradation rates were only slightly higher in negatively charged SUVs than in LUVs.



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Fig. 4.   Increased membrane curvature enhances ceramide hydrolysis. LUVs and SUVs with varying mean diameter were prepared as described under "Experimental Procedures." Liposomes either contained none or 25 mol % PI. In addition, incubation mixtures contained either none or 2.5 µM SAP-D. The data presented are the means of at least duplicate determinations. All individual values were in the range of ±5 up to ±15% of the mean.

Ceramide Degradation Is a Membrane-bound Process-- To test whether ceramide was perhaps solubilized from the lipid bilayer by either AC or SAPs during degradation, we performed a series of dilution experiments. With these experiments we wanted to distinguish whether ceramide degradation takes places either as a membrane-bound process, in the aqueous phase, or perhaps in both phases simultaneously. The incubation mixtures were diluted up to 7.5-fold with assay buffer, whereas the amount of AC and SAPs were kept constant. Fig. 5A demonstrates that the rate of ceramide hydrolysis in neutral LUVs decreased by ~50% both in the absence and presence of SAP-C and SAP-D. In contrast, degradation rates were not significantly diminished when BMP-containing LUVs were used as substrate-bearing vesicles. The rate of ceramide hydrolysis in the presence of SAP-D or SAP-C, however, was slightly reduced (Fig. 5B). These results indicate that electrostatic interactions provide a mean of efficiently binding AC, SAP-C, and SAP-D to the liposomal surface and that ceramide is not solubilized into the aqueous phase by SAP-C and SAP-D.



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Fig. 5.   Effect of dilution on the ceramide degradation rates in both neutral and negatively charged liposomes. The degradation of membrane-bound ceramide by AC was measured, and incubation mixtures were diluted up to 7.5-fold with assay buffer. Liposomes either contained no BMP (A) or 25 mol % BMP (B). Ceramide hydrolysis was determined without SAPs (open circle ) or in the presence of 2.5 µM SAP-D (black-square) or 2.5 µM SAP-C (black-triangle). All assay mixtures were prepared as described under "Experimental Procedures." The data presented are the means of at least duplicate determinations. All individual values were in the range of ±9 up to ±15% of the mean.

Substrate Specificity of Acid Ceramidase toward Membrane-bound Ceramides-- Increased degradation rates of membrane-bound GSLs were observed when the ceramide moiety contained short acyl chains (10, 12). We therefore investigated how the acyl chain length of ceramide affected the rate of hydrolysis by AC. Fig. 6A demonstrates that the degradation rates markedly decreased with increasing acyl chain length. The degradation rate of N-stearoylsphingosine was ~15-fold lower compared with N-lauroylsphingosine. The degree of stimulation by SAP-D and SAP-C was also slightly lower for the hydrolysis of N-stearoylsphingosine. Slightly increased degradation rates were observed with N-oleoylsphingosine as substrate compared with N-stearoylsphingosine, both in the absence and presence of SAPs (Fig. 6A). We also tested if ceramide derivatives with truncated sphingoid base structures were degraded by AC in our liposomal assay system. Fig. 6B shows that the truncated ceramide derivatives were hydrolyzed 2-fold faster than ceramides with the normal sphingoid backbone. SAP-D additionally stimulated the degradation of these truncated ceramide derivatives 3-fold, whereas SAP-C enhanced the rate of hydrolysis ~2-fold (Fig. 6B).



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Fig. 6.   AC displays higher interfacial activity toward truncated ceramide derivatives. A, ceramide derivatives with different acyl chain length were incorporated as substrates into BMP-containing liposomes. B, ceramide derivatives with different chain length of the sphingoid backbone were incorporated into BMP-containing vesicles. Assays were carried out in the absence of SAPs or in the presence of either 2.5 µM SAP-D or SAP-C. All assay mixture were prepared as described under "Experimental Procedures". The data presented are the means of at least duplicate determinations. All individual values were in the range of ±5 up to ±12% of the mean.

Ceramide Degradation Is Stimulated by SAP-A, -C, and -D but Not by SAP-B-- Previous experiments showed that ceramide degradation was not only stimulated by SAP-D but also by SAP-C. We further tested whether SAP-A and -B were able to enhance the degradation of membrane-bound ceramide. Fig. 7A shows that SAP-A, -C, and -D but not SAP-B stimulated the degradation of ceramide in the liposomal assay system. To verify the results, we performed a cell culture study in which the purified, individual SAPs were added to the culture medium of radioactively labeled fibroblasts of a patient suffering from prosaposin deficiency (5). The addition and presumed endocytosis of SAP-A, -B, -C, and -D resulted in a reduction of ceramide storage after feeding of SAP-A, -C, -D but not SAP-B (9, 25) (Fig. 7B). SAP-D and -A reduced ceramide storage by ~50% and SAP-C by nearly 30%. These cell culture results were in good agreement with the experimental data obtained from our in vitro liposomal assay system.



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Fig. 7.   SAP-A, -C, and -D stimulate ceramide hydrolysis in vitro and in vivo. A, ceramide hydrolysis was measured in neutral (open bar) and negatively charged BMP-containing LUVs (25 mol %, closed bars) in the presence of 2.5 µM SAP-A, -B, -C, or -D. B, effect of SAP-A, -B, -C, and -D on the turnover of labeled SL in fibroblasts of a patient suffering from prosaposin deficiency (and therefore being devoid of SAP-A, -B, -C, -D) and healthy control fibroblasts. Cells were labeled with L-[3-14C]serine and fed with SAP-A, -B, -C, or -D (25 µg/ml). Then the lipids isolated and visualized as described under "Experimental Procedures." The data presented are the means of at least duplicate determinations. All individual values were in the range of ±7 up to ±13% of the mean.

Binding Studies of SAP-D and AC with Lipid Bilayers-- The previous studies suggested that binding of AC and SAP-D to lipid membranes is required for efficient ceramide hydrolysis. We used surface plasmon resonance spectroscopy (SPR) to characterize the interaction of AC and SAP-D to lipid membranes in greater detail. The first experiments illustrate that the binding of SAP-D to negatively charged bilayers containing BMP was nearly 2-fold higher than to uncharged membranes (Fig. 8, A and B).



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Fig. 8.   BMP increases binding of SAP-D to immobilized lipid bilayers. Increasing concentrations of SAP-D were injected into the flow cell at a flow rate of 20 µl/min in 50 mM NaAc buffer, pH 4.0. Association and dissociation were observed for 5 min, respectively. Bilayers in A were composed of 55 mol % PC, 20 mol % Chol, and 25 mol % BMP. Bilayers in B consisted of 80 mol % PC and 20 mol % Chol. Binding experiments were repeated three times. One representative binding curve is shown.

Fig. 9 demonstrates that AC also bound significantly better to anionic, BMP-containing membranes than to neutral lipid bilayers. The incorporation of ceramide had no noticeable effect on the binding curves of either SAP-D or AC (data not shown). We tested in addition whether SAP-D enhanced the binding of AC to lipid bilayers. For this purpose, SAP-D was first injected into a flow cell and bound to the immobilized negatively charged lipid bilayer, followed by an injection of AC into the same flow cell. However, no increased binding of AC was observed when compared with an identical experiment in the absence of SAP-D (data not shown).



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Fig. 9.   AC binds better to negatively charged membranes. AC in 50 mM NaAc, pH 4.0, was injected at a flow rate of 20 µl/min into the flow cells. Association and dissociation were observed for 5 min, respectively. Lipid bilayers consisted of either 55 mol % PC, 20 mol % Chol, and 25 mol % BMP (solid line) or of 80 mol % PC and 20 mol % Chol (dashed line). Binding experiments were repeated three times. One representative binding curve is shown.

The strength of binding of both SAP-D and AC not only depended on the presence of anionic lipids in the membrane bilayer but also on the pH value. Fig. 10 clearly shows that the interaction of either protein decreased when the pH value of the running buffer approached neutral pH. At pH 6 the binding of SAP-D to the immobilized membrane was 6-fold lower than at pH 4. The difference in membrane interaction as a function of the pH was even more pronounced for AC. At pH 6 the binding of AC to negatively charged membranes was 15-fold lower than at pH 4.0. 



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Fig. 10.   The pH value significantly influences the binding of SAP-D and AC to negatively charged lipid bilayers. A, binding of 2.5 µM SAP-D to immobilized bilayers consisting of 55 mol % PC, 20 mol % Chol, and 25 mol % BMP at pH 4.0, 5.0, and 6.0. B, interaction of AC with immobilized lipid membranes consisting of 55 mol % PC, 20 mol % Chol, and 25 mol % BMP at pH 4.0, 5.0, and 6.0. Each binding experiment was repeated three times. One representative binding curve is shown.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Anionic Phospholipids and Lysosomal Degradation-- According to our proposed model for the topology of lysosomal digestion, GSLs and SLs are degraded in the acidic compartments of the cell as components of intraendosomal and intralysosomal vesicles and structures (2). The major portion of cellular ceramide is catabolized by lysosomal AC. A previous study has shown that SAP-D is an indispensable cofactor for the lysosomal degradation of ceramide in vivo (9).

Ceramides are highly hydrophobic molecules and primarily serve as membrane anchors of GSLs. The solubilization of ceramide for AC activity measurement in vitro requires a mixture of synthetic nonionic and anionic detergents (3). However, detergents do not occur in vivo and form micelles in aqueous solution. To mimic the intraendosomal and intralysosomal environment as closely as possible, we established a detergent-free assay system at pH 4. Ceramide was incorporated into liposomes containing different lysosomal lipids. The objective of the present investigation was to study ceramide hydrolysis by AC as an interfacial process. Analysis of the enzymatic degradation of phospholipids by phospholipase A2 showed that the rate of interfacial catalysis is highly dependent on the components, composition, and structure of the membrane interface (26). Therefore, we varied not only the components and lipid composition but also the size of our substrate-carrying liposomes.

It is demonstrated in Fig. 1 that lysosomal anionic lipids like BMP and PI stimulate the hydrolysis of ceramide. Both lipids are negatively charged under the chosen assay conditions (27, 28). The pI value of human AC was calculated to be 7.2, and AC is therefore likely to be positively charged in a lysosomal environment. This suggests that the translocation of AC to the membrane is primarily promoted by electrostatic interactions and is probably not lipid-specific. A similar binding behavior was also observed for other lysosomal proteins such as glucocerebrosidase and sphingomyelinase (11-13).

Cell culture studies indicate that SAP-D and also SAP-C increased the activity of AC toward its membrane-bound substrate ceramide (Fig. 7). It is clearly shown in Fig. 2 that (a) SAP-D and -C were able to stimulate the degradation of membrane-bound ceramide in vitro, and that (b) SAP-D and -C required a threshold concentration of either BMP or PI in the lipid bilayer before a significant stimulation of ceramide hydrolysis occurred. The strong dependence of SAPs on negatively charged lipids again hints at electrostatics as the prime mechanism of protein-membrane interactions. A similar dependence has also been observed for other membrane-associated enzymes including cytosolic phospholipase A2, protein kinase C, and phosphocholine cytidylyl transferase (29). In contrast to AC, SAP-D and SAP-C (pI ~4.2-4.5) are only weakly positively charged under the chosen assay conditions, pH 4.0.

Fig. 2 also demonstrates for the first time that ceramide degradation in vitro was stimulated by at least two different SAPs, namely SAP-D and SAP-C. At a concentration of 10 mol % of anionic phospholipid in substrate-bearing liposomes, SAP-D was the strongest stimulator of AC activity. With uncharged liposomes, however, SAP-C was able to enhance ceramide degradation to a greater extent than SAP-D. Both activator proteins therefore seem to be capable of supplementing each other under a variety of lysosomal conditions. This compensatory effect of SAP-C also helps to explain why a disease phenotype caused by an isolated SAP-D deficiency has, in contrast to SAP-B and SAP-C, not been observed yet (30-32).

The importance of electrostatic interactions also became evident when either dolichol or dolichol phosphate was incorporated into substrate-bearing liposomes. Both lipids were found to increase membrane fluidity and to perturb the structure of phospholipid bilayer (23). These lipids should therefore promote hydrophobic interactions and partial membrane penetration by AC and SAPs. Only the negatively charged dolichol phosphate, however, was able to increase the rate of ceramide hydrolysis (Fig. 3A), whereas dolichol itself did not significantly affect this activity (Fig. 3B). The stimulation of ceramide degradation by dolichol phosphate was considerably weaker in comparison to BMP or PI (Fig. 3A). The observation that negatively charged, sialic acid-containing glycosphingolipids, such as the gangliosides GD1a and GM1, had no effect on ceramide degradation suggests that the negative surface charge must be located close to the hydrophilic/hydrophobic membrane interface to play a role in ceramide hydrolysis.

Gel filtration experiments demonstrated that AC forms high molecular weight aggregates in the absence of detergents (33). This led to the assumption that some nonpolar amino acids must be located on the surface of AC and that hydrophobic interactions could also play a role in ceramide degradation. This hypothesis is supported by the observation that ceramide degradation in the presence of activators also took place in the absence of anionic phospholipids. To facilitate the formation of nonpolar interactions we prepared liposomes with increased membrane curvature. It has been shown that the increasing curvature of the liposomes causes following: (a) redistribution of lipids from the inner to the outer layer due to geometric constraints, (b) decrease in the lateral surface pressure, (c) increase head group spacing, and (d) increase in the number of packing defects in the lipid bilayer (34, 35). It can be seen from Fig. 4 that increased membrane curvature in neutral liposomes leads to higher ceramide degradation rates both in the absence and presence of SAP-D. This suggests that both AC and SAP-D penetrate into the hydrophobic/hydrophilic membrane interface during the course of enzymatic catalysis. SPR experiments further support the concept that both unspecific ionic and nonpolar interactions mediate membrane binding of AC and SAP-D. Both SAP-D and AC bound better to immobilized, negatively charged membranes at acidic pH values than to bilayers composed of neutral lipids (see Figs. 8 and 9). This observation also explains the decreased binding of proteins near neutral pH values. It is, however, obvious that the differences in binding could not be correlated with the degree of stimulation of AC activity observed in the presence of 25 mol % BMP. Anionic lipids might therefore not only facilitate membrane binding but could also induce a conformational change in AC and/or in the lipid phase, which may affect the rate of interfacial catalysis. A change in the secondary structure of a protein after binding to negatively charged membranes has been recently reported for glucocerebrosidase (36).

The SPR experiments also indicate that only a minor amount of AC was released from the immobilized lipid bilayer during the dissociation period. Further experimental evidence for the strong AC-lipid interaction resulted from dilution experiments with BMP-containing liposomes (Fig. 5B). The fact that AC activity was not significantly affected by a dilution of the liposomal assay mixture suggests that ceramide degradation occurred primarily as a membrane-bound process and that SAPs did not solubilize ceramide into the aqueous solution, a mechanism that has been proposed for the degradation of GM2 by beta -hexosaminidase A in the presence of the GM2 activator protein (5). When combining the SPR and dilution experiments, it may be concluded that, similar to many mammalian phospholipases A2, AC remains bound to the bilayer surface during ceramide hydrolysis (29).

Substrate Specificity of AC in Terms of Interfacial Catalysis-- In contrast to the degradation of GSLs, in which exposed sugar units are cleaved off by exohydrolases, the amide bond of ceramide is embedded in the hydrophilic/hydrophobic region of the lipid bilayer and is not initially accessible for enzymatic degradation. As a consequence, water-soluble AC must either partially penetrate into the membrane to reach its substrate or the substrate must be lifted out of the membrane toward the active center of the enzyme to be hydrolyzed. Lipids with short hydrophobic tails can be lifted out of membranes more easily than their long chain counterparts. Therefore, it was tested whether this factor also played a role for ceramide hydrolysis by incorporating ceramide derivatives with different acyl length and sphingosine backbones into liposomes. Fig. 6A demonstrates that the rate of degradation decreased with increasing chain length of the acyl moiety of ceramide. This finding indicates that long chain ceramides required more time to reach the active center of AC than the short chain derivatives.

Ceramide Degradation Is Stimulated by Three Activator Proteins-- Until now there have been only two reports where the effect of SAPs on the in vitro activity of AC was investigated. In the first paper (33) the effect of a crude SAP-C preparation on AC activity was studied in a micellar system but failed to notice any stimulatory effect on AC activity. It was shown in the second study (38) that AC was specifically stimulated by SAP-D in a detergent-based assay system using a crude AC preparation from human placenta. Our results prove that not only SAP-D but also SAP-C and -A were able to stimulate the degradation of ceramide in vitro and in cell culture experiments (Figs. 2, 3, and 7). This result helps to explain why, in contrast to SAP-C and -B, an isolated SAP-D deficiency has not yet been discovered as a clinical phenotype. Only if all the four SAPs are missing was ceramide accumulation found (25).

Of the four SAPs, only SAP-B did not stimulate ceramide degradation, despite the fact that it has a high degree of homology to SAP-A, -C, and -D, even though it has been previously described as a physiological detergent (39).

Based on our observations, we suggest the following model for the hydrolysis of membrane-bound ceramide. AC first moves to lipid membranes due to nonspecific electrostatic and hydrophobic interactions. Electrostatic interactions then provide the driving force for the correct orientation of AC on the membrane for ceramide hydrolysis. Partial membrane penetration is facilitated by nonpolar interactions between hydrophobic patches on the surface of AC and membrane lipids. AC remains bound to the liposomal surface during and after catalysis of ceramide degradation. Ceramide hydrolysis takes place when the substrate reaches the active center of AC through diffusion.

Based on our findings we propose that SAP-A, -C, and -D, in accordance with several studies in which the lipid binding properties of SAPs have been described (8, 40), indirectly enhance ceramide hydrolysis by creating packing perturbations in the lipid bilayer. These defects promote the partial penetration of AC into the hydrophilic/hydrophobic interface of the membrane to reach its hydrophobic substrate. This model is also supported by the experimental result that under certain conditions SAPs are able to induce leakage of trapped dyes in LUVs (40). Furthermore, most saposin-like proteins can interact strongly with membranes. These interactions can be as diverse as pore formation by the pore-forming peptides of Entamoeba histolytica (42), the membrane lytic activity of NK-lysin (43), or the enhancement of surface activity of surfactant phospholipids by surfactant protein B (41).

Whether SAP-A, -C, and -D interact with and perhaps allosterically activate membrane-bound AC has not yet been investigated in detail. The allosteric activation of AC by SAP-D has been proposed previously (37). It still remains an unsolved question how SAPs achieve substrate specificity despite their similar properties. As previously mentioned, SAP-B is the only SAP that is not able to stimulate the degradation of ceramide in vivo and in vitro. SAP-A, -C, and -D were all capable to promote ceramide degradation both in vitro and in cell culture. In contrast, glucosylceramide degradation is specifically stimulated by SAP-C in vivo and by SAP-C and SAP-A in vitro.

Further insight into the mechanism of membrane interaction and substrate specificity of SAPs will not only require additional biophysical studies, including differential scanning calorimetry and film balance experiments, but also a structural determination of the SAPs. Our study represents the first report of how the activity of AC is influenced by the presence of lysosomal anionic phospholipids and SAPs.


    FOOTNOTES

* This work was supported by a scholarship (to T. L.) from the Graduiertenkolleg "Pathogenese von Krankheiten des Nervensystem," by Deutsche Forschungsgemeinschaft Grants SFB 400 and SFB 284, and the Ministerium für Schule, Wissenschaft und Forschung des Landes Nordrhein-Westfalen.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Kekulé-Institut für Organische Chemie und Biochemie, Gerhard Domagk Strasse 1, D-53121 Bonn, Germany. Tel.: 49-228-735834; Fax: 49-228-737778; E-mail: sandhoff@uni-bonn.de.

Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M006846200


    ABBREVIATIONS

The abbreviations used are: GSLs, glycosphingolipids; SLs, sphingolipids; AC, acid ceramidase; SAPs, sphingolipid activator proteins; BMP, bis(monoacylglycero)phosphate; PI, phosphatidylinositol; Chol, cholesterol; SUVs, small unilamellar vesicles; LUVs, large unilamellar vesicles; SPR, surface plasmon resonance spectroscopy; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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