From the Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany
Received for publication, August 31, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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According to a recent hypothesis,
glycosphingolipids originating from the plasma membrane are degraded in
the acidic compartments of the cell as components of intraendosomal and
intralysosomal vesicles and structures. Since most previous in
vitro investigations used micellar ganglioside GM2 as substrate,
we studied the degradation of membrane-bound ganglioside GM2 by
water-soluble The degradation of glycosphingolipids
(GSLs)1 endocytosed from the
plasma membrane takes place in the acidic compartments of the cell.
According to a recently proposed hypothesis of the topology of
lysosomal digestion (1), GSLs reach the lysosomal compartments as
membrane-bound components of intraendosomal and intralysosomal vesicles
and structures. The degradation of GSLs with short oligosaccharide head
groups and ceramide requires two proteins: a water-soluble lysosomal
sphingolipid hydrolase and a sphingolipid activator protein (SAP) (2,
3). Recent studies suggest that SAPs mediate the interaction of
water-soluble sphingolipid hydrolases with their respective
membrane-bound substrates (4).
The enzymatic, lysosomal degradation of the ganglioside GM2 is
catalyzed by Human lysosomal Defects or mutations in the genes of the subunits GM2AP is one of five known SAPs. Saposin (SAP)-A, -B, -C, and -D are
derived from a single precursor protein, prosaposin, through
proteolytic processing (2). GM2AP is first synthesized as a 22-kDa
precursor protein, which is subsequently processed in the lysosomes to
the mature 20-kDa protein (13). Physicochemical measurements with lipid
monolayers demonstrated the membrane-active properties of the GM2AP.
GM2AP can immerse into monolayer membranes with a lateral pressure of
up to 25 millinewtons/m. The lateral pressure of biological
membranes is believed to be significantly higher (14). It is
conceivable, however, that the lateral pressure of the inner membranes
of late endosomes and lysosomes could be less than 25 millinewtons/m due to their high degree of curvature and the
enrichment of the unusual lysophospholipid
bis(monoacylglycero)phosphate (BMP) (15).
Studies on the degradation of glucosylceramide show that
glucocerebrosidase requires negatively charged detergents or
phospholipids to reach its full enzymatic activity in vitro
(16, 17). Negatively charged lipids such as dolichol phosphate (DoP)
and BMP, which were identified as components of the membrane of late
endosomes and lysosomes (15, 18), stimulated the degradation of
membrane-bound glucosylceramide by glucocerebrosidase and SAP-C in a
detergent-free, liposomal system up to 35-fold (4).
So far, in vitro studies on the degradation of GM2 were
mostly performed using micellar ganglioside GM2 as substrate and have never been executed in the presence of anionic phospholipids. To
analyze the influence of anionic lysosomal phospholipids on the
hydrolysis of the membrane bound ganglioside, GM2 was incorporated into
liposomes of varying composition, mimicking intralysosomal vesicles.
The degradation rate of membrane-bound GM2 by HexA and GM2AP was
studied as a function of the concentration of anionic lysosomal
phospholipids in liposomes at acidic pH values, which prevail in the
lysosomal compartment. We also investigated the influence of membrane
curvature on the degradation rate using liposomes of varying size.
Commercial Products--
Phosphatidylcholine (egg yolk) (PC),
phosphatidylinositol (bovine liver) (PI), phosphatidic acid (egg yolk)
(PA), dolichol (porcine liver), dolicholphosphate (porcine liver)
(DoP), and cholesterol (Chol) were purchased from Sigma. Lichroprep
RP18 was obtained from Merck. All other chemicals were of analytical grade or the highest purity available.
Chemical Synthesis--
Ganglioside GM2 was tritiated by the
galactose oxidase/[3H]NaBH4 method of Novak
(1979) (19) to a specific activity of 104 Ci/mol. TLC followed by
autoradiography did not reveal any contamination of the labeled
GM2.
BMP was prepared as described previously (4).
Enzyme Preparation--
HexA was purified from human liver to
apparent homogeneity according to Schütte (20). Purity and
identity were ascertained by SDS-polyacrylamide gel
electrophoresis and Western blot. One unit of enzyme activity
(U) is defined as the amount of enzyme that catalyzes the hydrolysis of
1 µmol of
4-methylumbelliferyl-2-acetamido-2-deoxy- Activator Preparation--
Recombinant GM2AP was expressed in
insect cells using the baculovirus expression system and purified as
described previously (21).
SAP-B was isolated from the spleen of a patient with Gaucher disease
(3). The purity and identity of the final preparation was ensured by
SDS-polyacrylamide gel electrophoresis and Western blot analysis.
Vesicle Preparation--
Large unilamellar vesicles (LUVs) were
prepared by the following procedure: PC (50 mM,
toluol/ethanol 2:1), BMP (5 mM, chloroform/methanol 2:1),
PI (2.5 mM, chloroform), Chol (25.6 mM,
chloroform/methanol 2:1), dolichol (2 mM, chloroform), DoP
(0.75 mM, chloroform), PA (1.8 mM,
chloroform/ethanol 1:1), and 3H-labeled GM2 (0.5 mM, toluol/ethanol 1:1) were dissolved in organic solvents.
Appropriate aliquots of the lipid solutions were mixed and dried under
nitrogen. The lipid mixture was hydrated to a total lipid concentration
of 2 mM in Tris/HCl buffer (1 mM, pH 7.4) and
freeze-thawed ten times in liquid nitrogen to ensure solute
equilibration between trapped and bulk solutions.
Unilamellar vesicles were prepared by passage through two polycarbonate
filters (pore size, 100 nm; Avestin) mounted in tandem in a
mini-extruder (Liposo-Fast; Avestin). Samples were subjected to 19 passes as recommended (22).
Small unilamellar vesicles (SUVs) were produced by sonification of LUVs
with a Microtip sonicator (Branson, Danbury, CT) at 0 °C for 40 min
(intervals of 15-s sonification, 30-s pause) under a stream of argon to
minimize oxidation of lipids. Samples were centrifuged at 100,000 × g for 15 min. The liposomes used for the MUG assays were
prepared as described above, but without the addition of GM2.
Liposomes used for the surface plasmon resonance spectroscopy were
prepared as described above, but hydrated to a lipid concentration of
0.5 mM in PBS buffer (10 mM phosphate, 140 mM NaCl, 10 mM KCl, pH 7.4). Liposomes used for
surface plasmon resonance spectroscopy contained unlabeled GM2.
Concentrations of liposomal lipid (LUVs and SUVs) were determined by
measurement of the radioactively labeled GM2. For liposomes that did
not contain radioactive GM2 but were prepared by the same protocol,
identical concentrations were assumed.
Enzyme Assays--
Enzyme assays using MUG as substrate
contained the following components in a final volume of 200 µl:
sodium citrate buffer (pH 4.2, 50 mM), MUG (2 mM), unilamellar liposomes (LUVs, 1 mM, amount of total liposomal lipids) composed
of 20 mol % Chol and 80 mol % PC or 20 mol % Chol, 60 mol % PC, and
20 mol % PA. Enzyme assays were incubated for 15 min at 37 °C.
Incubations were stopped by adding 1 ml of sodium carbonate/glycine
buffer (pH 9.5, 200 mM each). After vigorous shaking the
amount of liberated 4-methylumbelliferone was measured fluorometrically.
The standard incubation mixture using GM2 as substrate contained the
following components in a final volume of 50 µl: bovine serum albumin
(50 µg/ml), sodium citrate buffer (pH 4.2, 50 mM), unilamellar liposomes (LUVs or SUVs, total lipid concentration = 1 mM), GM2AP (0.08-2 µg), and
Liposomes had the following composition: [3H]GM2 (10 mol
%, 1.8 Ci/mol), Chol (20 mol %), PC (50-80 mol %), and BMP (0-25
mol %), dolichol (0-10 mol %), DoP (0-10 mol %), PI (0-30 mol
%), or PA (0-25 mol %) as indicated in the legends to all of figures.
The standard incubation conditions were 37 °C for 30 min.
Enzyme assays were stopped by the addition of 50 µl of methanol.
Terminated enzyme assays were loaded onto a reverse phase column (RP18, 1 ml) equilibrated with a solution of
chloroform/methanol/0.1 M KCl (3:48:47, v/v/v). The column
was eluted with 2 ml of the same solvent, and the radioactivity in the
effluents was measured in a scintillation counter.
Surface Plasmon Resonance Spectroscopy
(Biacore)--
Biomolecular interaction analysis (surface plasmon
resonance spectroscopy) was carried out at 25 °C with a Bialite
instrument (Biacore).
Sensorchips providing a preimmobilized surface with lipophilic anchors
attached to a dextran matrix (Pioneer L1 chip) were obtained from
Biacore. LUVs (total lipid concentration = 0.5 mM), diluted in PBS buffer, were injected into the system at a flow rate of
5 µl/min in two steps (first injection, 60 µl; second injection, 40 µl). This resulted in a RU shift of 5000-7000 RU. 10 µl sodium
hydroxide (25 mM) were then injected at 100 µl/min to
remove multilamellar structures and to stabilize the base line, resulting in a RU shift of about 20-50 RU. GM2AP (0.2 µM) in running buffer (50 mM sodium citrate
buffer, pH 4.2) was injected into the flow cells at a rate of 20 µl/min.
Presentation of Data--
All data are means of at least
duplicate determinations. All individual values are in the range of
±5% up to ±15% of the mean.
Basic Experiments
To investigate the influence of acidic lipids on the rate of GM2
degradation under lysosome-like conditions, GM2 was incorporated into
liposomes and incubated with HexA and GM2AP. In a series of preliminary
experiments the influence of pH, time, and substrate concentration on
GM2 degradation was investigated. Optimal degradation rates were
obtained at pH 4.2 for ganglioside GM2 and at pH 4.5 for the artificial
substrate MUG (data not shown). It was found that the rate of
degradation was linear for up to 30 min using liposomes containing 10 mol % [3H]GM2 and 20 mol % PA (data not shown).
Experiments with increasing amounts of [3H]GM2 (0-10 mol
%) in liposomes containing 20 mol % PA resulted in an almost linear
dependence of the degradation rate on substrate concentration (data not
shown). Studies using the water-soluble synthetic substrate MUG showed
no influence of GM2AP on the degradation rate. The hydrolysis was also
not affected by the presence of liposomes containing 20 mol % PA or by
the presence of both, GM2AP and the anionic lipid (data not shown).
The Influence of Anionic Lysosomal Lipids on the Degradation of
Liposomal GM2
The Effect of BMP on the Hydrolysis of Membrane-bound GM2--
BMP
is a unique, anionic lysophospholipid found specifically in the
lysosomes (23-26) and in intravesicular structures of late endosomes
(15). It may play a role in destabilizing these structures, thus
facilitating the degradation of their components by lysosomal hydrolases. To determine the influence of BMP on the degradation of
GM2, we tested how increasing concentrations of GM2AP affected the GM2
degradation rate in liposomes containing differing amounts of BMP (Fig.
1). This experiment demonstrated that GM2
hydrolysis requires the presence of GM2AP. Increasing amounts of BMP
stimulated GM2 degradation in the presence of GM2AP, but not in its
absence, whereas maximum stimulation of GM2 degradation was achieved by 0.5-1 µM GM2AP. We also tested how increasing amounts of
BMP stimulate the GM2 degradation. LUVs containing 10 mol % [3H]GM2 were spiked with increasing amounts of BMP and
incubated with 15 milliunits of HexA in the presence of 0.5 µM GM2AP. Under these conditions, the incorporation of 25 mol % BMP into liposomes increased the GM2 degradation rate 180-fold
compared with BMP-free liposomes (Fig.
2A).
To test the influence of the membrane curvature on the rate of
degradation, SUVs were doped with increasing amounts of BMP and
subjected to HexA and GM2AP. They displayed GM2 degradation rates that
ranged about 40% above those measured for LUVs (data not shown).
In a study by Wu and co-workers (27), it was shown that the
GM2AP-dependent degradation of micellar GM2 is increased by the
additional presence of SAP-B in high concentrations (40-fold excess
over GM2AP). The authors conclude that SAP-B might play a role in the
degradation of GM2 in vivo. To test this hypothesis in our
detergent-free assay system, we prepared LUVs containing 10 mol % [3H]GM2 and either 70 mol % PC and 20 mol % Chol or 45 mol % PC, 20 mol % Chol, and 25 mol % BMP. GM2-containing LUVs were
incubated with HexA in the presence of either SAP-B, GM2AP, or a
mixture of the two activator proteins, using a 2-fold excess of SAP-B over GM2AP. In the absence of BMP, minimal GM2 degradation was detected
in the presence of GM2AP and in the mixture of GM2AP and SAP-B, whereas
no degradation took place in the presence of SAP-B alone, as expected
(Fig. 3). Using BMP-containing liposomes, high degradation rates were detected in the presence of GM2AP. This,
however, was not the case when SAP-B was used as activator protein
(which, again, was expected), and no additional increase in the
degradation rate was observed when SAP-B was added to GM2AP-containing incubation mixtures.
Since we do not observe an effect of SAP-B on the
GM2AP-dependent degradation of GM2 in our liposomal system
using similar amounts of SAP-B and GM2AP, we conclude that the effect
observed in the micellar system by Wu and co-workers (27) does probably not play a role under physiological conditions.
Influence of Other Lysosomal Lipids on the Degradation of GM2 in
Liposomes--
Not only BMP, but also the negatively charged
phospholipid PI, has been found in lysosomes in relatively high
concentrations (15, 23-25). Dolichol is a polyisoprenoide and one of
the largest lipids occurring in mammalian cells. It consists of an
Liposomes containing 10 mol % [3H]GM2 and increasing
amounts of one of these four lipids were incubated with HexA either in the presence or in the absence of GM2AP. Neither dolichol nor GD1a or
dolichol phosphate showed a significant stimulatory effect on GM2
degradation. Degradation rates were in the range of the measuring
accuracy for all concentrations measured (0-2 nmol/h U) (data
not shown). However, a pronounced stimulation was detected in the
presence of PI and GM2AP (Fig. 2B). A concentration of 30 mol % PI in the liposomes stimulated the degradation of membrane-bound GM2 150-fold.
Interaction of GM2AP with Liposomes of Different Compositions
To analyze how GM2AP interacts with lipid bilayers, surface
plasmon resonance measurements were performed. The effect of the presence of GM2 and acidic phospholipids in the lipid bilayers was
studied in detail. In the first set of experiments, liposomes composed
of neutral lipids and rising concentrations of GM2 were immobilized on
a Pioneer L1 chip. The strongest binding of GM2AP to the immobilized
lipid bilayer was observed when the membrane structures did not contain
GM2 (Fig. 4A). Unexpectedly,
the binding signal progressively decreased with rising amounts of
GM2.
-hexosaminidase A in the presence of the GM2 activator
protein in a detergent-free, liposomal assay system. Our results show
that anionic lipids such as the lysosomal components
bis(monoacylglycero)phosphate or phosphatidylinositol stimulate the
degradation of GM2 by
-hexosaminidase A up to 180-fold in the
presence of GM2 activator protein. In contrast, the degradation rate of
GM2 incorporated into liposomes composed of neutral lysosomal lipids
such as dolichol, cholesterol, or phosphatidylcholine was significantly
lower than in negatively charged liposomes. This demonstrates that
both, the GM2 activator protein and anionic lysosomal phospholipids,
are needed to achieve a significant degradation of membrane-bound GM2
under physiological conditions. The interaction of GM2 activator
protein with immobilized membranes was studied with surface plasmon
resonance spectroscopy at an acidic pH value as it occurs in the
lysosomes. Increasing the concentration of bis(monoacylglycero)phosphate in immobilized liposomes led to a
significant drop of the resonance signal in the presence of GM2
activator protein. This suggests that in the presence of
bis(monoacylglycero)phosphate, which has been shown to occur in inner
membranes of the acidic compartment, GM2 activator protein is able to
solubilize lipids from the surface of immobilized membrane structures.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hexosaminidase A (HexA) and requires the GM2 activator
protein (GM2AP) as cofactor (5).
-hexosaminidases (EC 3.2.1.52) cleave off terminal
-glycosidically-bound N-acetylglucosamine and
N-acetylgalactosamine residues from a number of
glycoconjugates, including glycoproteins, oligosaccharides, and GSLs
such as GM2, its asialo derivative GA2, and globoside (6). A total of
three different
-hexosaminidase isoenzymes have been identified.
These are composed of different combinations of two noncovalently
linked, structurally related subunits,
and
(7-10). The
-homodimer is called the
-hexosaminidase B, the
-homodimer is
called the
-hexosaminidase S, and the
,
-heterodimer is called
the
-hexosaminidase A. Despite the fact that the active sites of the
and the
subunits are functionally very similar, the three
isoenzymes have different substrate specificities (11). Only HexA is
able to hydrolyze GM2 in the presence of the GM2AP at significant rates
(12).
and
or the
gene encoding the GM2AP lead to the various forms of GM2 gangliosidosis. These storage diseases manifest themselves by a massive
accumulation of GM2 and related glycolipids in neuronal lysosomes,
leading to severe neurodegeneration. Depending on the defect and its
severity, other tissues may also be affected by minor lipid and
oligosaccharide accumulation (6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside (MUG)/min at 37 °C.
-hexosaminidase A (15 milliunits).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BMP and GM2AP stimulate the hydrolysis of
LUV-bound ganglioside GM2. The degradation of ganglioside GM2
inserted into LUVs doped with 0 mol % BMP ( ), 5 mol % BMP (
),
10 mol % BMP (
), 20 mol % BMP (
), or 25 mol % BMP (
) was
measured in the presence of increasing concentrations of GM2AP (0-2.2
µM).
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Fig. 2.
Lysosomal lipids in LUVs stimulate the
enzymatic hydrolysis of ganglioside GM2 in the presence of GM2AP.
All assays were conducted with ganglioside GM2-carrying LUVs as
substrates in the absence ( ) and presence (
) of GM2AP (0.5 µM) as described under "Experimental Procedures,"
using LUVs with various proportions of synthetic BMP (0-25 mol %)
(A) or PI (0-30 mol %) (B).
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Fig. 3.
SAP-B does not stimulate the hydrolysis of
membrane-bound ganglioside GM2 by
-hexosaminidase A, degradation was
measured with ganglioside GM2 carrying LUVs containing 80 mol % PC and
20 mol % Chol (PC) or 55 mol % PC, 20 mol % Chol, and 25 mol % BMP (BMP) in the presence of 2 µg of SAP-B, 1 µg
of GM2-AP, or both activator proteins.
-saturated isoprene unit and occurs either as a free alcohol or in
its phosphorylated form as DoP. Both, dolichol and DoP, were found to
occur in lysosomes in high concentrations compared with other
organelles (18). GD1a is a negatively charged glycosphingolipid, the
degradation of which takes place in the lysosomes. To investigate
whether the stimulation of GM2 degradation by BMP is an unspecific
effect of negatively charged lipids or of lipids that destabilize
membranes such as dolichol (18), the effect of these lipids on the
degradation of liposomal GM2 was determined.
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Fig. 4.
Interaction of GM2 activator protein with
immobilized vesicles. LUVs were immobilized on a Pioneer L.1
sensorchip. GM2AP (0.2 µM) was injected at a flow rate of
20 µl/min in 50 mM sodium citrate buffer (pH 4.2) for
180 s. In the next step, protein-free buffer was injected for
180 s. The measurement started with the injection of GM2AP.
A, the binding curves were obtained with LUVs bearing Chol
(20 mol %)/PC (various mol %) and 0 mol % GM2 (trace a),
5 mol % GM2 (trace b), 10 mol % GM2 (trace c),
or 20 mol % GM2 (trace d). B, the binding curves
were obtained with LUVs bearing Chol (20 mol %)/BMP (20 mol %)/PC
(various mol %) and 0 mol % GM2 (trace a), 5 mol % GM2
(trace b), 10 mol % GM2 (trace c), or 20 mol % GM2 (trace d). C, the binding curves were
obtained with LUVs bearing Chol (20 mol %)/PC (50 mol %)/BMP (20 mol
%) and GM2 (10 mol %). GM2AP was injected with a concentration of 0.2 µM (trace a), 0.4 µM
(trace b), 1 µM (trace c), or
2 µM (trace d).
In the second set of experiments, 20 mol % of BMP were
incorporated into the bilayers together with rising amounts of GM2 (Fig. 4B). Binding curves followed a similar pattern as
observed for lipid bilayers without BMP, although the binding signals
were somewhat reduced. The most notable effect of the presence of BMP was a dramatic drop of the binding signal shortly after GM2AP injection. This drop was most pronounced for membrane structures containing high amounts of GM2. In all bilayers containing GM2 and BMP,
the signal dropped below the initial base-line value after about
360 s, reaching a minimum of 800 RU in the presence of 20 mol % GM2 (Fig. 4B).
To exclude the possibility that the effect observed for GM2AP is nonspecific, we studied the binding of bovine serum albumin and SAP-B to lipid bilayers containing BMP and GM2. Bovine serum albumin tethers very strongly (11,300 RU), whereas SAP-B binds weaker than GM2AP (250 RU). The binding curves follow a similar pattern as observed for lipid bilayers composed of neutral lipids and GM2 and show no dramatic drop of the binding signal as observed in the presence of BMP and GM2AP (data not shown).
Since the binding of GM2AP to gangliosides had been reported not to be
completely specific for GM2 (5, 28), we decided to analyze whether a
similar effect as that observed for GM2 was also observed for other
glycolipids. To do this, we investigated the binding of GM2AP to lipid
bilayers containing 10 mol % GM1, glucosylceramide, or GM3, all spiked
with 10 mol % BMP, by surface plasmon resonance. The binding curve for
GM3 showed a similar pattern as that observed for lipid bilayers doped
with 10 mol % BMP only (Fig. 5). The
same was true for glucosylceramide, although the total binding signal
was reduced (data not shown). The binding curve of GM2AP to GM1,
however, is comparable with the one observed for GM2, including the
strong drop in the signal upon injection of the activator protein (Fig.
5). These results indicate that GM2AP differentiates between different
glycolipids but is not completely specific for GM2, which is in
accordance with earlier results (5, 28).
|
The observed drop in the plasmon resonance signal suggests that GM2AP
can either extract lipids from immobilized lipid bilayers containing
BMP and GM2 or GM1 or lead to a destabilization of the bilayers
with concomitant loss of lipids.
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DISCUSSION |
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GSLs are amphiphilic components of the plasma membrane of eucaryotic cells. After endocytosis, GSLs reach the lysosomal compartments of the cells where they are catabolized by a series of lysosomal glycosidases. According to our recent model of endocytosis and lysosomal digestion (1, 2), GSLs are degraded as components of intraendosomal and intralysosomal membrane structures, rather than as components of the limiting lysosomal membrane. Such intralysosomal vesicles with a diameter in the range of 50-100 nm were detected in the biopsy tissue of a patient with prosaposin deficiency (29). Several factors such as membrane curvature, membrane pressure, and lipid composition may make such vesicles more accessible for the lysosomal hydrolases than the limiting membrane. To test this hypothesis, we studied the enzymatic hydrolysis of GM2, which requires lysosomal HexA and GM2AP as a cofactor (30-32) in a detergent-free assay system mimicking the intralysosomal conditions. The studies were performed at pH 4.2, which is optimal for the enzymatic reaction and typical for the lysosomal content (33). The substrate was presented to the HexA as a component of liposomal membranes, either of LUVs with an average diameter of 100 nm or of SUVs with an average diameter of 40 nm. Liposomes were composed of lipids that had been detected in the acidic compartment of the cell or had been specifically localized to its inner membranes as BMP. In contrast to the presentation of the substrate as micelles, this is believed to be the most appropriate model for the lysosomal digestion of membrane-bound GSL substrates.
The incorporation of acidic lipids occurring in lysosomes such as BMP or PI into the liposomes led to a strong stimulation of the degradation of GM2 in the presence of GM2AP (up to 180-fold at 25 mol % BMP). A significant enzymatic degradation of GM2 was only observed in the presence of both, an anionic lipid and the GM2AP.
Although the concentrations of acidic lipids used in this study are quite high, they can still be considered to be in a realistic physiological range. The determination of lipid concentrations in cellular organelles is difficult, and results vary considerably with the cells and the method of organelle isolation used. Nevertheless, several investigations found increased amounts of BMP and PI in lysosomes. The relative amounts of BMP in lysosomes that were reported vary between 7 mol % (23) and 28 mol % (24), but most reports state values of more than 10 mol % of total lysosomal lipid. The corresponding values determined for PI are in the order of 5-10 mol % (25). The determination of lipid concentrations on intraendosomal and intralysosomal membrane structures was not possible to date, so that a further enrichment of the lipids in these structures has to be considered. For BMP it has been demonstrated immunologically that it is localized specifically on intraendosomal membranes (15). Anionic lipids like PA, sulfatide, etc., which also stimulated GM2 hydrolysis in vitro, have not been detected in lysosomes and are therefore of no physiological significance.
Other lipids that occur in the lysosomes such as dolichol, phosphatidylcholine, or dolichol phosphate did not have a similar stimulatory effect on the degradation of GM2 as it was observed for BMP and PI. Acidic gangliosides such as GD1a, which are degraded in the lysosomes and have been shown to disintegrate phospholipid bilayers at concentrations above 20 mol % (34), also showed no significant stimulatory effect at concentrations between 2.5 and 20 mol %. This demonstrates that the effect is not due to an unspecific destabilization of the membranes by anionic lipids. The fact that dolichol or its phosphate do not stimulate the reaction, although they are known to strongly destabilize membranes (18), implicates that a more complex mechanism than the simple perturbation of the membrane is the basis for the stimulatory effect of BMP and PI.
A direct interaction of the acidic lipids with the degrading enzyme, as it has been proposed for the degradation of glucosylceramide by glucocerebrosidase and SAP-C (4), has to be ruled out though, since the degradation of the water-soluble synthetic substrate MUG is not affected by the presence of either GM2AP or liposomes containing acidic lipids.
The specificity of the stimulatory effect of GM2AP on the reaction is high. An additional stimulation of the reaction by SAP-B, which was reported by Wu and co-workers for micellar GM2 (27) was not reproduced in our liposomal system using similar concentrations of both activators. We therefore conclude that this effect is not physiologically significant.
To find out more about the mechanism by which BMP stimulates the lysosomal degradation of GM2, the binding of GM2AP to immobilized lipid bilayers was studied by surface plasmon resonance spectroscopy. Our Biacore studies demonstrate that at pH 4.2 GM2AP binds to neutral lipid bilayers composed of PC and Chol as well as to anionic bilayers, which in addition contain GM2 and/or BMP. At pH 7, the binding of GM2AP to all liposomes tested was much weaker. However, the presence of BMP in the membrane structures did not lead to an increase of GM2AP binding (at pH 4.2) but to a significant drop of the resonance signal even below the base line (Fig. 4B). This effect was not observed in the absence of BMP and indicates a solubilization of lipids from the membranes in the presence of both, BMP and GM2AP, which increases with increasing ganglioside GM2 concentrations. This solubilization is also dependent on concentrations of GM2AP (Fig. 4C). Other proteins (bovine serum albumin, SAP-B) also bind to the membrane, but do not lead to a significant drop of the resonance signal, which indicates that the effect observed is specific for GM2AP and is not caused by an unspecific replacement of lipid structures by protein. These observations suggest an interaction of GM2AP with BMP, which leads to destabilization of the membrane.
To investigate the specificity of the observed effect for the ganglioside GM2, we used bilayers containing GM1, GM3, or glucosylceramide in the plasmon resonance measurements. Lipid bilayers containing GM3 or glucosylceramide showed a similar GM2AP binding curve as membranes devoid of ganglioside, whereas the GM2AP binding behavior is comparable for bilayers containing either GM1 or GM2, including the strong drop of the signal upon injection of GM2AP. This demonstrates that GM2AP shows some specificity but is not completely specific for GM2, which is in agreement with earlier glycolipid transfer studies using GM2AP (5, 28). An interesting point is that studies on the enzymatic degradation of GM1 led to the conclusion that the GM2AP, similar to SAP-B, stimulates this degradation step in the presence of BMP (35). Whether, therefore, the strong drop in the plasmon resonance signal, which was observed for GM1 and GM2, is due to the same effect that causes the activator function of GM2AP is still speculative, but an interesting point to consider.
The BMP-induced stimulation of glycolipid degradation is a phenomenon
that is not limited to GM2 and the GM2AP. Similar results were obtained
for the degradation of glucosylceramide by glucocerebrosidase and SAP-C
(4) and of GM1 by -galactosidase and SAP-B or GM2AP (35). These
results together with the data presented here strongly support our
hypothesis that the lysosomal degradation of glycosphingolipids takes
place on intralysosomal membrane structures rather than in the limiting
lysosomal membrane. BMP and presumably PI as components of these
structures may contribute to a selective destabilization of such
membrane structures, whereas the BMP-free limiting lysosomal membrane
(15) is not affected. The specific lipid composition of different
membrane structures may therefore serve to differentiate between them,
protecting the limiting lysosomal membrane from digestion on one hand
and marking the components of intralysosomal membranes for degradation
on the other.
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ACKNOWLEDGEMENTS |
---|
We express our thanks to Dr. Thomas Linke for critical discussions and careful reading of the manuscript and to Dr. Günter Schwarzmann for supplying ganglioside GD1a.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 284 and by the Fonds der Chemischen Industrie.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.
Dedicated to Prof. Saul Roseman on the occasion of his 80th birthday.
To whom correspondence should be addressed: Kekulé-Institut
für Organische Chemie und Biochemie, Universität Bonn,
Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany. Tel.: 49-228-73-5834;
Fax: 49-228-73-7778; E-mail: sandhoff@uni-bonn.de.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M007970200
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ABBREVIATIONS |
---|
The abbreviations used are:
GSL, glycosphingolipid;
BMP, bis(monoacylglycero)phosphate;
Chol, cholesterol;
DoP, dolichol phosphate;
GD1a, ganglioside GD1a,
NeuAc.2
3Gal
1
3GalNAc
1
4Gal(3
2
NeuAc)
1
4Glc
1
1Cer;
GM2, ganglioside GM2, GalNAc
1
4Gal(3
2
NeuAc)
1
4Glc
1
1Cer;
GM2AP, GM2 activator protein;
HexA,
-hexosaminidase A,
2-acetamido-2-desoxy-
-D-hexoside-acetamidodesoxyhexohydrolase
A;
LUV, large unilamellar vesicles;
MUG, 4-methylumbelliferyl-2-acetamido-2-deoxy-
-D-glucopyranoside;
SAP, sphingolipid activator protein;
SAP-B, saposin B;
SUV, small
unilamellar vesicle;
PC, phosphatidylcholine (egg yolk);
PI, phosphatidylinositol (bovine brain);
PA, phosphatidic acid (egg yolk);
RU, relative unit(s).
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