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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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 ( ) 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.
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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 % ( ), 5 mol % (×), 10 mol % ( ), 25 mol % ( ), and 50 mol % ( ) BMP. B, liposomes
contained 0 mol % ( ), 5 mol % (×), 10 mol % ( ), 25 mol % ( ), and 50 mol % ( ) PI. C, liposomes contained 0 mol
% ( ), 5 mol % (×), 10 mol % ( ), 25 mol % ( ), and 50 mol
% ( ) BMP. D, liposomes contained 0 mol % ( ), 5 mol
% (×), 10 mol % ( ), 25 mol % ( ), 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.
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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 ( ), 2.5 µM
SAP-D ( ), or 2.5 µM SAP-C ( ). 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.
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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.
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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 ( ) or in the presence of 2.5 µM SAP-D ( ) or
2.5 µM SAP-C ( ). 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.
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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.
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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.
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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.
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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.
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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.
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 |
DISCUSSION |
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
-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.