From the Department of Energy and Hydrocarbon
Chemistry, Graduate School of Engineering, Kyoto University, Sakyo-ku,
Kyoto 606-8501, Japan, the § Department of Dementia
Research, National Institute of Longevity Sciences, Gengo 36-3, Morioka, Obu 474-8522, Japan, the
Graduate School of Biostudies,
Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, and ** Glyco-chain
Expression Laboratory, Supra-biomolecular System Research, RIKEN
Frontier Research System, Wako 351-0198, Japan
Received for publication, January 11, 2001, and in revised form, March 12, 2001
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ABSTRACT |
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GM1 ganglioside-bound amyloid The critical step in the development of Alzheimer's disease
(AD)1 is the conversion of
soluble, nontoxic amyloid Yanagisawa et al. (7) discovered GM1
ganglioside-bound A In this study, the effects of GM1 and cholesterol contents in the
membranes on the binding of A Peptides--
Human A Lipids--
GM1 and cholesterol were purchased from Sigma.
Porcine brain L- Lipid Vesicle Preparation--
Large unilamellar vesicles (LUVs)
for fluorescence experiments were prepared and characterized as
described elsewhere (19). Briefly, lipids were mixed in a
chloroform/methanol mixture. The solvent was removed by evaporation in
a rotary evaporator. The residual lipid film, after drying under vacuum
overnight, was hydrated with buffer (10 mM Tris, 150 mM NaCl, 2 mM CaCl2, pH 7.4) and
vortex-mixed to produce multilamellar vesicles. A physiological concentration of Ca2+ was included because this divalent
ion is known to interact with gangliosides (20). For PS LUV
preparation, CaCl2, which induces aggregation and fusion of
vesicles, was omitted, and 1 mM EDTA was added. The
suspension was subjected to five cycles of freezing and thawing and
then extruded through polycarbonate filters (100-nm pore size filter,
31 times) using a Liposofast extruder (Avestin, Ottawa, Canada). The
lipid concentration was determined in triplicate by phosphorus analysis
(21).
Small unilamellar vesicles for CD experiments were prepared by
sonication of multilamellar vesicles under a nitrogen atmosphere for 15 min (three times for 5 min each) using a probe-type sonicator. Metal
debris from the titanium tip of the probe was removed by centrifugation.
Fluorescence--
Fluorescence measurements were carried out on
a Shimadzu RF-5000 or RF-5300 spectrofluorometer with a cuvette holder
thermostatted at 30 °C. After blank subtraction (and volume
correction in titration experiments), the spectra were corrected using
the spectrum correction attachment provided by the manufacturer.
Fluorescence Titration--
DAC-A
For competitive binding experiments, GM1-rich cholesterol-rich LUVs (20 µM) were mixed with a DAC-A Excimer Fluorescence--
LUVs containing 5 or 10 mol % BODIPY-GM1 (Molecular Probes, Inc., Eugene, OR) were placed in a quartz
cuvette. Fluorescence emission spectra were recorded at an excitation
wavelength of 480 nm. Fluorescence anisotropy (r) was
determined at an emission wavelength of 520 nm (monomer peak) using
polarizers placed in both the excitation and emission light paths
(23).
CD--
Native human A Lipid Specificity of A
To confirm that DAC-A
Thus, DAC fluorescence can be utilized to assess the binding affinity
of A Cholesterol-induced A
Binding isotherms were obtained from Fig. 4A as follows.
Rmax values were estimated by linear
extrapolation of R versus DAC-A Detection of GM1 Cluster--
Excimer formation of BODIPY-GM1 was
utilized to detect the GM1 cluster. The fluorophore BODIPY is known to
form an excimer that emits red-shifted fluorescence (~630 nm)
compared with monomer (~520 nm) (27). The excimer formation, which
occurs upon collision of two dye molecules (one is in the excited
state), is facilitated by higher local concentration of the dye as well
as lower membrane rigidity. Fig. 5 shows
the fluorescence emission spectra of BODIPY-GM1-labeled liposomes. The
spectra are normalized to the monomer peaks because the excimer/monomer
fluorescence ratio is directly proportional to local dye concentration
(28). BODIPY-GM1/GM1/cholesterol/SM (10:30:30:30) liposomes
corresponding to the GM1-rich cholesterol-rich system exhibited a large
excimer fluorescence (trace 1). In contrast, BODIPY-GM1/PC (10:90)
liposomes with the identical dye content showed much weaker excimer
fluorescence (trace 2). The anisotropy value of monomer fluorescence
(r) as a measure of membrane rigidity of the latter membrane
(0.046) was significantly smaller than that of the former (0.104).
Therefore, BODIPY-GM1 forms an excimer much more easily in the former
DIG-like environment despite its higher rigidity (higher anisotropy),
strongly indicating that GM1 is present in a locally concentrated
state, i.e. in a cluster.
A reduction in total GM1 concentration from 40 to 20% markedly
decreased excimer fluorescence (BODIPY-GM1/GM1/cholesterol/SM = 5:15:40:40, corresponding to the GM1-poor cholesterol-rich system, trace 3), although the intensity was much greater than that of the
control BODIPY-GM1/PC (5:95) system (trace 5). The r value of the former (0.119) was again greater than that of the latter (0.041). It should be noted that the BODIPY-GM1/GM1 ratio remained constant at 1:3 in both GM1 40 and 20% systems. Therefore, if all GM1
molecules had been involved in the cluster formation, the same excimer
fluorescence would have been observed. The weaker excimer fluorescence
in the GM1-poor system indicated that the extent of clustering was
smaller. BODIPY-GM1/GM1/cholesterol/SM (5:15:16:64) membranes
corresponding to the GM1-poor cholesterol-poor system
(r = 0.115, trace 4) exhibited further weaker excimer
fluorescence compared with trace 3. Thus, the extent of clustering was
in the order GM1-rich cholesterol-rich Secondary Structure--
The conformations of A Dye Labeling--
A Lipid Specificity--
DAC-A Binding Isotherms--
Binding of peptides to lipid bilayers has
often been analyzed by a simple partition model that does not include
the concept of binding sites, because in most cases the driving force
of peptide binding is not specific molecular recognition but simple
electrostatic and hydrophobic interactions (34-36). However, A
If individual GM1 molecules constitute binding sites for A
Cooperativity was observed for peptide binding to the GM1-rich
membranes with larger capacities. This may be related to conformational transition from Pathological Implications--
Recently, a great deal of attention
has been focused on the pathological implications of altered
cholesterol metabolism, which is likely to occur with aging or the
expression of apolipoprotein E, in the development of AD. There is
accumulating evidence that the metabolism of amyloid precursor protein,
including A
The results of the present study indicated, for the first time, that
increases in the content of cholesterol in the membrane induce the
formation of GM1/A
Finally, if A-protein
(GM1/A
), found in brains exhibiting early pathological
changes of Alzheimer's disease (AD) including diffuse plaques, has
been suggested to be involved in the initiation of amyloid fibril
formation in vivo by acting as a seed. To elucidate the
molecular mechanism underlying GM1/A
formation, the effects of lipid
composition on the binding of A
to GM1-containing lipid bilayers
were examined in detail using fluorescent dye-labeled human
A
-(1-40). Increases in not only GM1 but also cholesterol
contents in the lipid bilayers facilitated the binding of A
to the
membranes by altering the binding capacity but not the binding
affinity. An increase in membrane-bound A
concentration triggered
its conformational transition from helix-rich to
-sheet-rich
structures. Excimer formation of fluorescent dye-labeled GM1 suggested
that A
recognizes a GM1 "cluster" in membranes, the formation of
which is facilitated by cholesterol. The results of the present study
strongly suggested that increases in intramembrane cholesterol content,
which are likely to occur during aging, appear to be a risk factor for
amyloid fibril formation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-protein (A
) to aggregated, toxic A
rich in
-sheet structures (1). A
has been shown to form amyloid
fibrils, but this requires concentrations of A
(>10
4
to 10
5 M) (2-4) much higher than the
physiological concentration (10
9 M).
Therefore, it has been hypothesized that aggregation of soluble A
in vivo involves seeded polymerization (5, 6).
(GM1/A
) in brains exhibiting early
pathological changes of AD and suggested that GM1/A
may serve as a
seed for toxic, amyloid fibril formation. Indeed, immunochemical (8, 9)
and spectroscopic (10-13) studies demonstrated that GM1/A
has a
conformation different from that of soluble A
and accelerates the
rate of amyloid fibril formation of soluble A
in vitro
(12, 14). Interestingly, however, GM1/A
is never found in the normal
brain despite the fact that neuronal membranes are abundant in GM1 and
physiological metabolism of amyloid precursor protein results in
extracellular secretion of A
. Thus, identification of factors that
initiate formation of GM1/A
may be crucial for determination of the
pathogenesis of AD and for development of preventive and curative
treatment strategies. We have recently found that alterations in lipid
composition of the host membrane can be such a factor (13); generation
of GM1/A
is facilitated by the combination of cholesterol and
sphingomyelin (SM) in membranes in proportions similar to the so-called
detergent-insoluble glycolipid-rich domain (DIG) (15), suggesting that
DIG is deeply involved in GM1/A
formation. This hypothesis is in
agreement with the observation that A
is present in DIG in
vivo (16, 17).
to DIG-like lipid bilayers were
examined in detail using fluorescent dye-labeled human A
-(1-40). We
report here that enrichment of cholesterol of the host membranes facilitated the generation of GM1/A
via formation of a GM1
"cluster" that acts as a binding site of A
. A plausible
mechanism of onset of AD will be discussed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-(1-40) labeled with the
7-diethylaminocoumarin-3-carbonyl group at its N terminus (DAC-A
,
Fig. 1) was custom synthesized by the
Peptide Institute (Minou, Japan). The peptide was characterized by
matrix-assisted laser desorption ionization mass spectroscopy (calculated, 4574.07; found, 4574.0) as well as amino acid analysis under two different hydrolysis conditions. The dye-labeled peptide was
always handled in light-protected, capped tubes under a nitrogen atmosphere to avoid photodegradation. Unlabeled human A
-(1-40) was
also purchased from the Peptide Institute. The latter peptide was first
dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (Wako, Osaka, Japan) to
avoid self-aggregation. After removal of the solvent by nitrogen
purging, the peptide was redissolved in pure water (Nanopure) at 30 µM and then mixed with an equal volume of double
concentrated buffer (20 mM Tris, 300 mM NaCl, 4 mM CaCl2, pH 7.4). The labeled peptide was
found to be less stable in this organic solvent and therefore was
directly dissolved in pure water. Physiological A
is present in a
soluble form. To mimic this situation, we removed aggregates, if any,
by ultracentrifugation in 500-µl polyallomer tubes at 100,000 × g at 4 °C for 1 h. Indeed, the supernatant used
adopted unordered structures (see Fig. 6) and did not react with
thioflavin T (data not shown), which has been widely used for the
detection of amyloid aggregation (18). The aggregational state of the
peptide was further characterized by SDS-polyacrylamide gel
electrophoresis using precast 15% polyacrylamide gel SPU-15S (Atto,
Tokyo). Only a band corresponding to monomer was detected by silver
staining (data not shown). The peptide concentration of the supernatant
was determined in triplicate by Micro BCA protein assay (Pierce).
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Fig. 1.
Structure of
DAC-A .
-phosphatidyl-L-serine (PS)
and egg yolk-L-
-phosphatidylcholine (PC) were obtained
from Avanti Polar Lipids (Alabaster, AL). Bovine brain SM was obtained
from Matreya (Pleasant Gap, PA). The molar lipid compositions of the
DIG-like membranes used were GM1/cholesterol/SM (40:30:30),
GM1/cholesterol/SM (40:12:48), GM1/cholesterol/SM (20:40:40), and
GM1/cholesterol/SM (20:16:64), which are referred to as GM1-rich
cholesterol-rich, GM1-rich cholesterol-poor, GM1-poor cholesterol-rich,
and GM1-poor cholesterol-poor, respectively. The GM1-rich and GM1-poor
systems had GM1 contents of 40 and 20%, respectively. The
cholesterol/SM ratios of the cholesterol-rich and cholesterol-poor
systems were 1 and 0.25, respectively.
solution (1 µM, 2 ml) was titrated with aliquots of a concentrated
LUV suspension in a quartz cuvette with gentle stirring. Fluorescence
emission spectra were recorded at an excitation wavelength of 430 nm.
The titration interval was 3 min, which was confirmed to be sufficient
for the establishment of binding equilibrium.
solution (0.16 µM). Under this condition, the binding sites were almost
saturated with DAC-A
. The mixture (2 ml) was titrated with aliquots
of an unlabeled A
-(1-40) solution (280 µM, monomeric
confirmed by SDS-polyacrylamide gel electrophoresis), which was
prepared by dissolving the peptide in 0.02% ammonia on ice followed by
ultracentrifugation (100,000 × g, 3 h, 4 °C)
(22). Fluorescence intensity at 470 nm (excitation at 430 nm) was
monitored during titration. Five minutes were required to establish equilibrium.
(Eq. 1)
Fluorescence intensity was denoted by I, and the
suffixes indicate polarization (in degrees) of the excitation-emission
beams. Fluorescence intensity of the corresponding blank sample without BODIPY-GM1 was negligible.
(Eq. 2)
-(1-40) (15 µM) in
buffer (10 mM Tris, 150 mM NaCl, 2 mM CaCl2) was used for CD measurements. CD
spectra were measured on a Jasco J-720 apparatus interfaced to an NEC
PC9801 microcomputer, using a 1-mm path length quartz cell to minimize the absorbance due to buffer components. The instrumental outputs were
calibrated with nonhygroscopic ammonium
d-camphor-10-sulfonate (24). Eight scans were averaged for
each sample. The averaged blank spectra (vesicle suspension or buffer)
were subtracted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Binding--
Binding of DAC-A
to LUVs
of various lipid compositions was estimated on the basis of DAC
fluorescence. The fluorophore was practically nonfluorescent in aqueous
environments (Fig. 2A,
trace 1). The addition of GM1-rich
cholesterol-rich LUVs at a lipid/peptide ratio (L/P) of 80 induced a
large increase in fluorescence intensity accompanied by a blue shift in
the emission maximum from 483 to 470 nm, indicating that the peptide
was bound to the membrane with the N-terminal DAC moiety embedded in a
hydrophobic environment. Fluorescence spectra of DAC-A
were also
measured in various dioxane/water mixtures. The maximal wavelength of
470 nm in the presence of the membrane corresponded to that in a
dioxane/water (3/1, v/v) mixture with a dielectric constant of ~20,
suggesting that the DAC moiety was located at the interfacial region of
the membrane (25).
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Fig. 2.
Detection of membrane binding of
DAC-A . A, fluorescence
emission spectra of 1 µM DAC-A
(excitation at 430 nm)
were recorded in 10 mM Tris, 150 mM NaCl, 2 mM CaCl2 buffer, pH 7.4 (trace
1) and in the presence of 80 µM
GM1/cholesterol/SM (40:30:30) LUVs (trace 2) at
30 °C. S.D. values for 2-4 preparations are shown by
error bars at the peaks. B,
LUVs (20 µM) composed of GM1/cholesterol/SM (40:30:30)
were mixed with a DAC-A
solution in the buffer (0.16 µM). Under this condition, the binding sites were almost
saturated with DAC-A
. The mixture (2 ml) was titrated with aliquots
of a unlabeled A
-(1-40) solution. Fluorescence intensity at 470 nm
(excitation at 430 nm) was plotted as a function of DAC-A
/unlabeled
A
ratio (closed circles). The open
circle shows the result of the converse experiment in which
the vesicles were pretreated with excess unlabeled peptide and then
DAC-A
was added. Error bars for duplicated measurements are within
the symbols.
behaves similarly to native A
, competitive
binding experiments were carried out. The binding sites of GM1-rich
cholesterol-rich LUVs were almost saturated with DAC-A
. Unlabeled
A
was then added, and a decrease in fluorescence was monitored as a
function of unlabeled A
-to-DAC-A
ratio (Fig. 2B,
closed circles). About 40-fold unlabeled peptide
was needed to replace 50% of the labeled peptide. Conversely, the
pretreatment of the vesicles with excess unlabeled peptide decreased
DAC-A
binding (Fig. 2B, open
circle). These data suggest that both peptides competitively
and reversibly bind to the common binding site with DAC-A
having a
40-fold larger affinity.
for various membranes. As a quantitative measure, relative
fluorescence enhancement (R) is defined as follows.
Fluorescence intensities at 466 nm (peak of raw, uncorrected
spectra) in the presence and absence of LUVs are denoted by F and F0, respectively. Fig.
3 plots the R value as a
function of L/P for various lipids. DAC-A
(Eq. 3)
exhibited strong binding
only to GM1-containing membranes (open circles).
The R value was almost saturated; i.e. the
peptide was completely bound at an L/P of >200. Negatively charged PS
(open diamonds), zwitterionic PC (open squares), or PC/cholesterol (2:1) (closed
squares) bilayers did not bind the amyloid even at the
highest L/P ratio examined (2560).
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Fig. 3.
Lipid specificity of DAC-A
binding. DAC-A
(1 µM) buffer solutions were
titrated with LUVs of various lipid compositions at 30 °C, and
fluorescence spectra were measured as in Fig. 2. Relative fluorescence
enhancement, r = (F
F0)/F0, is plotted as a
function of L/P. Fluorescence intensity at 466 nm in the presence and
absence of LUVs are denoted by F and
F0, respectively. Lipid composition was as
follows: GM1/cholesterol/SM (40:30:30) (
), PC (
), PC/cholesterol
(2:1) (
), PS (
). For PS preparations, CaCl2 in the
buffer was replaced by 1 mM EDTA. S.D. values for 2-4
preparations are shown by error bars.
Binding--
The effects of lipid
composition on peptide binding to GM1-containing DIG-mimicking
membranes were examined in detail. Fig. 4A shows the R
value as a function of GM1/DAC-A
ratio instead of L/P, because GM1
seems to constitute a "binding site" for the peptide. The binding
was strongly dependent on GM1 as well as cholesterol contents. For the
cholesterol-rich systems, a decrease in GM1 content from 40 to 20%
significantly reduced DAC-A
binding (Fig. 4, open
circles versus open
triangles). Surprisingly, in the case of the GM1-poor
systems, a decrease in the cholesterol/SM ratio from 1 to 0.25 markedly
reduced DAC-A
binding (Fig. 4, open versus
closed triangles).
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Fig. 4.
Binding of DAC-A to
GM1-containing membranes. A, R
versus GM1/DAC-A
ratio plots are shown for
GM1/cholesterol/SM (40:30:30) (
), GM1/cholesterol/SM (40:12:48)
(
), GM1/cholesterol/SM (20:40:40) (
), and GM1/cholesterol/SM
(20:16:64) (
). S.D. values for 2-4 preparations are shown by
error bars. B, binding isotherms were
estimated from the data in A. The bound peptide per
exofacial GM1, x, is plotted as a function of free peptide
concentration, cf. The traces are the best fit
binding isotherms using Equation 4 and the parameters shown in Table
I.
/GM1 plots
(DAC-A
/GM1
0) and are summarized in Table
I. In the case of the GM1-poor
cholesterol-poor bilayers, the Rmax value was
assumed to be the same as that of the GM1-poor cholesterol-rich system
because data close to saturation could not be obtained (Fig.
4A). This assumption was reasonable because the
Rmax values for the other systems were similar.
The R/Rmax ratio gives the bound
fraction of the peptide at each data point. Fig. 4B shows binding isotherms, i.e. bound DAC-A
per exofacial GM1
(x) versus free DAC-A
concentration
(cf) plots. GM1 molecules on the outer leaflets
(50% of total GM1) were assumed to be available for DAC-A
binding.
The two isotherms of the GM1-rich systems were sigmoidal, implying
cooperative binding. Therefore, the curves were analyzed by Equation 4
(Fowler's equation) instead of the conventional Langmuir equation
(26).
The maximal x values, xmax, were
estimated by linear extrapolation of x versus
1/cf plots (1/cf
(Eq. 4)
0). The binding constant and lateral interaction parameter are denoted by
K (M
1) and
, respectively. The
binding isotherms at the lower GM1 content could be well explained by
the Langmuir equation (i.e.
= 0 in Equation 4). The
obtained binding parameters are summarized in Table I. Interestingly,
the binding affinities (K) were very similar (2-3 × 106 M
1), whereas the binding
capacities (xmax) were highly dependent on GM1
as well as cholesterol contents.
Parameters for binding of DAC-A to GM1-containing membranes
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Fig. 5.
Detection of GM1 clustering.
Fluorescence emission spectra of BODIPY-GM1 (1 µM) doped
in various LUVs were recorded at an excitation wavelength of 480 nm.
The spectra are normalized to the monomer peaks (~520 nm). Lipid
composition was as follows: BODIPY-GM1/GM1/cholesterol/SM (10:30:30:30)
(trace 1), BODIPY-GM1/PC (10:90)
(trace 2), BODIPY-GM1/GM1/cholesterol/SM
(5:15:40:40) (trace 3),
BODIPY-GM1/GM1/cholesterol/SM (5:15:16:64) (trace
4), BODIPY-GM1/PC (5:95) (trace
5).
GM1-poor
cholesterol-rich > GM1-poor cholesterol-poor, consistent with the
order of the xmax values (Table I). The excimer
formation in BODIPY-GM1/GM1/cholesterol/SM (10:30:12:48) bilayers
corresponding to the GM1-rich cholesterol-poor system was also
examined. The excimer fluorescence was even larger than that of trace 1 (data not shown), but the r value was much smaller (0.064).
Therefore, this system could not be directly compared with the other systems.
-(1-40) were
estimated from CD spectra. Fig. 6 shows
data of the GM1-rich cholesterol-rich system. The spectrum in buffer
had a minimum at 197 nm, characteristic of unordered structures
(trace 1). At lower GM1/A
ratios, the spectra
exhibited shallow minima around 218 nm, reminiscent of
-sheets (Fig.
6, traces 2 and 3). In contrast, the
peptide adopted helical structures at higher GM1/A
ratios, as
suggested by double minima around 209 and 222 nm (Fig. 6,
traces 4 and 5). As estimated from the
ellipticity at 222 nm (
13,000 degrees cm2
dmol
1), the helicity at the largest GM1/A
value
investigated was ~40% (29). The absence of an isodichroic point
indicated that the helix-to-sheet transition is not a simple two-state
process. The GM1-rich cholesterol-poor system showed very similar CD
spectra (data not shown).
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Fig. 6.
Secondary structures of native human
A -(1-40). CD spectra of the peptide (15 µM) were recorded in the absence and presence of
GM1/cholesterol/SM (40:30:30) SUVs at 30 °C. GM1/A
ratio was as
follows: 0 (trace 1), 10 (trace
2); 20 (trace 3); 40 (trace
4); 80 (trace 5). The spectra are the
averages for two preparations, and the S.E. values were within 700 degrees cm2 dmol
1 at 220 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptides with slight chemical
modifications, such as 125I (30-32) and Trp labeling (14),
have been widely used in many studies and provided valuable
information. The DAC moiety employed in this study was as small as a
single aromatic amino acid and was attached to the N terminus of A
.
Indeed, DAC-A
behaved very similarly to the native peptide. First,
DAC-A
shared the common binding site with native A
(Fig.
2B). Second, the interfacial location of the DAC moiety in
membranes is fully compatible with the observation that native A
lies on the surface of GM1-containing membranes (13). Third, DAC-A
showed lipid specificity identical to that of the native peptide, as
described below.
showed no affinity for
phospholipids PC or PS but high affinities for GM1-containing membranes
at physiological ionic strength (Fig. 3), consistent with previous
studies using native (11, 13) and Trp-labeled A
(14). Our study
clearly indicated that A
does not bind cholesterol, because (i)
DAC-A
was not bound to PC/cholesterol (2:1) liposomes (Fig. 3) and
(ii) The cholesterol content in DIG-mimicking membranes did not
correlate with A
-binding activity (Fig. 4). Wood's group (33)
reported that aggregated but not freshly dissolved A
binds
cholesterol, in accordance with our results.
obviously recognizes GM1 or more accurately gangliosides (10-14).
Therefore, the binding isotherms were analyzed by the cooperative
binding model (Equation 4). The binding affinities, K, were
practically the same (2-3 × 106
M
1) for the four DIG-mimicking systems
investigated (Table I). The competitive binding experiments (Fig.
2B) suggested that the K value of native A
is
40-fold smaller (~ 6 × 104
M
1), which corresponds to a difference in a
Gibbs free energy of 2 kcal/mol. This value is a reasonable one for
transfer of the DAC moiety from water to membrane interface (25). A
binding affinity of 7 × 105
M-1 was reported for the Y10W-A
-(1-40)-GM1
system (14). The Tyr-to-Trp substitution enhances membrane binding by
~1 kcal/mol (25), which corresponds to a 5-fold increase in affinity.
Therefore, taken together with the present estimation, the affinity of
native A
for GM1 is estimated to be ~105
M
1. Even in the absence of specific molecular
recognition, binding isotherms are fitted by the Langmuir equation. The
K values for magainin 1-PS (37) and
calcitonin/dimyristoylphosphatidylglycerol (38) systems were reported
to be 3.8 × 105 and 1 × 105
M
1), respectively, which were comparable with
the estimated affinity of native A
.
, the
binding isotherms (Fig. 4B) would be independent of lipid composition. However, the binding capacity increased quadratically with
intramembrane GM1 content in the cholesterol-rich matrix (Fig.
4B and Table I), suggesting that some cooperative
interactions between GM1 molecules generate the binding site. In
accordance with this view, the membrane binding of native A
-(1-40)
also requires a threshold GM1 content dependent on the lipid
composition of the host matrix (12, 13). The most straightforward
interpretation is that A
recognizes not monomeric but clustered GM1
or that in a GM1-enriched microdomain (39), the formation of which is regulated by cholesterol content. Indeed, a correlation was observed between binding capacity (xmax in Table I) and
excimer formation of BODIPY-GM1 as a measure of clustering (Fig. 5).
However, the relationship between xmax and
excimer fluorescence was semiquantitative. The
xmax value of the GM1-poor cholesterol-poor
system was very small, whereas significant excimer fluorescence was
observed (Fig. 5, trace 4), probably because the
introduction of relatively high amounts of BODIPY-GM 1 (25% of total
GM1) slightly affected domain formation. The excimer experiments also
suggested that DIG-like environments play a crucial role in GM1
clustering. Ferraretto et al. reported that GM1 as well as
cholesterol form GM1- and cholesterol-enriched domains in SM bilayers
(39). It is therefore plausible that at lower GM1 contents
(e.g. 20%) an increase in cholesterol content, through
segregation of cholesterol molecules by the cholesterol-rich domain
formation, enhances local GM1 concentration, which leads to GM1
clustering (Fig. 4). In contrast, a GM1 content of 40% appears to be
sufficient for effective formation of GM1-rich domains, and thus
cholesterol content would have no further effect on GM1 clustering. In
the PC matrix, a high level of excimer fluorescence was never observed
despite its lower rigidity (Fig. 5, traces 2 and
5). An electron microscopic study also indicated that GM1 is
molecularly dispersed in fluid phosphatidylcholine bilayers at lower
GM1 contents (40).
-helix-rich conformations at lower x
values to
-sheet-rich conformations at higher x values
(Fig. 6). The formation of the latter structures can involve
interpeptide interactions. Terzi et al. (41) reported
similar structural transition in phosphatidylglycerol bilayers at low
ionic strength. The presence of
/
structures was also found in
ganglioside-containing membranes (10). The conformational
transition of the N-terminal region (residues 10-24) from
-helix to
-strand was reported to facilitate amyloid formation
(42).
generation, is significantly modulated by the content of
cellular cholesterol (43-45). It is particularly of interest that a
recent study indicated that a unique A
species with seeding ability
was generated by cultured cells in a cholesterol-dependent
manner (46). Even with this information we are still far from
understanding how cholesterol is involved in the development of AD,
especially in the initiation of amyloid fibril formation.
, one of the candidates as an endogenous seed for
Alzheimer amyloid. With regard to the alteration of cholesterol in
neuronal membranes, recent studies by Wood and co-workers (47, 48) are
very informative. They reported that the content of cholesterol in the
exofacial leaflets of synaptic plasma membrane is increased during
aging (47) and by apolipoprotein E deficiency (48). Taken together with
the results of present study, these observations strongly suggest that
alterations in the content of cholesterol in neuronal membranes
underlie abnormal aggregation of A
in the AD brain.
forms amyloid fibrils via seeded polymerization in the
brain with AD, then the seed could be a target for therapeutic and
preventive treatment regimens for AD. Indeed, we have recently found
that GM1/A
formed in DIG-like membranes works as a seed for fibril
formation.2 To generate a
compound that specifically recognizes GM1/A
and inhibits its seeding
ability, it will be necessary to clarify the molecular processes
underlying alterations of the secondary structures of A
via binding
to and accumulation in GM1 "clusters."
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FOOTNOTES |
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* 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.
¶ Supported by Grants-in-aid for Scientific Research on Priority Areas C, Advanced Brain Science Project, from the Ministry of Education, Science, Sports and Culture, Japan, and Research Grant for Longevity Sciences 11-C01 from the Ministry of Health and Welfare.
Supported by Grants-in-aid for Scientific Research on Priority
Areas B-12140202.
§§ To whom correspondence should be addressed: Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81 75 753 4574; Fax: 81 75 761 2698; E-mail: katsumim@ pharm.kyoto-u.ac.jp.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M100252200
2 H. Hayashi, K. Hasegawa, H. Yamaguchi, K. Matsuzaki, H. Naiki, and K. Yanagisawa, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid
-protein;
BODIPY-GM1, BODIPY®FL
C5-GM1;
DAC, 7-diethylaminocoumarin-3-carbonyl;
DIG, detergent-insoluble glycolipid-rich domain;
LUV, large unilamellar
vesicle;
PC, phosphatidylcholine;
PS, phosphatidylserine;
SM, sphingomyelin;
L/P, lipid/peptide ratio;
GM1, monosialoganglioside
GM1.
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