Alzheimer's disease is characterized
pathologically by the presence of neurofibrillary tangles and amyloid
plaques. The principal component of the plaque is the
-amyloid
peptide (A
), a 39-43-residue peptide. The conformational change
required for the conversion of soluble peptide into amyloid fibrils is
modulated by pH, A
concentration, addition of kinetic and
thermodynamic enhancers, and alterations in the primary sequence of
A
. We report here the ability of gangliosides to induce an
-helical structure in A
and thereby diminish fibrillogenesis.
Circular dichroism and a fluorescence dye release assay data indicate
that gangliosides interact with and induce
-helix formation in A
.
We find that the sialic acid moiety of gangliosides is necessary for
the induction of
-helical structure. Differences in the amount and
the position of the sialic acid on the carbohydrate backbone also
affect the conformational switch. The A
-ganglioside interaction at
pH 7.0, monitored by CD, is stable over time and resistant to high
concentrations of NaCl. The induction of
-helical structure is
greater with A
1-40 than A
1-42. The ability of gangliosides to
sequester A
from fibril formation was also evaluated by electron
microscopy.
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INTRODUCTION |
Alzheimer's disease is characterized by the presence of amyloid
plaques surrounded by dead and dying neurons in the brain (1, 2). The
principal component of the plaque is the
-amyloid (A
)1 peptide, a
39-43-residue peptide found in normal human tissue and generated as a
cleavage product from the larger amyloid precursor protein (3-6). A
in the plaque is in the form of an amyloid fibril, approximately 100 Å in diameter and several microns long (7). The conformational changes
required for the conversion of soluble A
into amyloid fibrils have
been demonstrated to be a nucleation-dependent process (8,
9) which is modulated by pH, A
-peptide concentrations, and presence
of nucleation seeds, such as proteoglycans and apolipoproteins
(10-14). Alterations in the primary sequence of A
peptides greatly
affect amyloid fibril formation (15). The E22Q mutation in A
, found
in hereditary cerebral hemorrhage with amyloidosis of the dutch type
(16), yields a peptide with increased ability to form amyloid fibrils (17). On the other hand, the single mutation of V18A increases the
apparent
-helical content of A
and diminishes fibrillogenesis (18). We have found that the interaction of A
40 and A
42 with mixed ganglioside preparations and GM1 increases the
-helical content, and we speculated that this interaction may prevent amyloid fibril formation (19).
It has been well documented that amyloid fibrils are intimately
associated with neuronal, microglial, and endothethial membranes. Cell
processes are extended out into close proximity of amyloid deposits
(20, 21), and cell culture studies have demonstrated that
overexpression of A
results in cell surface ruffling (22-24). These
results, as well as the studies on A
-membrane interactions (19,
25-31), suggested that the interaction of A
on cellular membranes
may be a mechanism leading to cell death. In addition, cell culture
experiments have demonstrated that A
neurotoxicity is dependent on
the formation of
-structure and fibrils (12, 32, 33). Therefore, it
is of interest to investigate possible A
interactions that decrease
-structure transitions, fibril formation, and subsequent
neurotoxicity.
We report here the investigation of the ability of various gangliosides
to induce an
-helical structure in A
40 and A
42. It was our aim
to investigate whether a specific ganglioside could reproduce the
structural transition seen with mixed gangliosides and whether this was
a function of the ceramide backbone or variation in carbohydrate and
sialic acid content. We also investigated the effect of mixed
gangliosides on A
40 and A
42 fibril formation using electron
microscopy.
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MATERIALS AND METHODS |
A
Peptides--
A
40 and A
42 were purchased from U.S.
Peptide Inc. (Rancho Cucamonga, CA). To minimize the presence of
endogenous fibril nucleation seeds, peptides were initially dissolved
in 0.5 ml of 100% trifluoroacetic acid (Aldrich), diluted in distilled
H2O, and immediately lyophilized. The lyophilized peptide
was then dissolved in 40% trifluoroethanol (TFE, Aldrich) in
H2O and stored at
20 °C until use.
Vesicle Preparation--
Pure lipids or mixtures of lipids
consisting of bovine brain phosphatidylcholine (PC), bovine brain mixed
ganglioside preparation, the monosialogangliosides GM1, GM2, GM3,
disialoganglioside GD1a, trisialoganglioside GT1b, ceramide, sulfatide,
and sphingomyelin were dissolved at a concentration of 5 mg/ml in
either chloroform or chloroform:methanol (2:1). Bovine brain PC was
purchased from Avanti Polar Lipids; all other lipids were purchased
from Sigma. Mixed ganglioside preparations are composed of
predominantly GM1, and the remainder consists of the GM, GD, and GT
series of gangliosides as well as other minor gangliosides. An aliquot
of each lipid was dried under a stream of nitrogen, lyophilized
overnight, and suspended in PBS, pH 7.0, in the presence or absence of
a saturated solution of 5-(and -6)-carboxy-2',7'-dichlorofluorescein
(Molecular Probes Inc., Eugene, OR). Lipid suspensions were carried
through 10 cycles of freeze-thaw in a acetone:dry ice bath. Unilamellar vesicles were then prepared by sonication for 30 min or until the
solution cleared in a bath sonicator (Branson Ultrasonic Corp., Danbury, CT). Column chromatography on Sepharose 4B (Sigma) (1.5 × 12 cm) was used to separate free dye from dye-loaded vesicles.
Sucrose Density Gradient Centrifugation--
Discontinuous
sucrose gradients were prepared by layering 10 and 40% sucrose
solutions. To the top of each tube, 100 µl of sample was layered, and
the gradients were centrifuged at 40,000 rpm for 18 h at 4 °C
in an SW 55Ti rotor in a Beckman LA70 Ultracentrifuge. A total of 20 fractions were collected using an ISCO Density Gradient Fractionator
model 640. The lipid vesicles were detected using the Bartlett assay
for phosphorus (34), and the peptide was detected by Tyr absorbance at
275 nm. Fractions exhibiting Tyr absorbance were confirmed to contain
A
peptide by dot-blot analysis using a polyclonal anti-A
antibody
(Boehringer Mannheim) and ECL detection.
Membrane Disruption Assay--
Dye-loaded vesicles were diluted
in PBS, pH 6.0 or pH 7.0, to a final lipid concentration of 20 µM (35). After 50 s incubation (with continual
stirring), A
peptide samples, bee venom mellitin (Sigma), or solvent
controls were added at a 1:20 peptide:lipid ratio. The final peptide
concentration was 1.0 µM. To monitor dye leakage, we used
a Photon Technology International Fluorimeter (London, Ontario,
Canada). Excitation was at 506 nm, and emission was monitored at 524 nm
with a 4-nm bandwidth. Data was collected at a rate of one data point
every 5 s. At the end of the experiment, 10% Triton X-100 was
added to a final concentration of 0.3% to obtain complete dye release
and measure total fluorescence. Vesicles were not used unless the total
fluorescence was greater than 200% of the initial fluorescence and the
spontaneous diffusion of dye was less than 10% of the total
fluorescence. The percentage of total dye release was defined as
indicated in Equation 1.
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(Eq. 1)
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Circular Dichroism--
CD spectra were recorded on an Aviv
Circular Dichroism Spectrometer model 62DS (Lakewood, NJ) at 25 °C.
Spectra were obtained from 200 to 260 nm, with a 0.5-nm step, 1-nm
bandwidth, and 20-s collection time per step. Peptide:lipid ratios were
maintained at 1:20 with a final peptide concentration of 10 µM. The effect of various lipids on peptide conformation
was determined by adding an aliquot of stock peptide solutions to lipid
vesicles suspended in PBS with continuous stirring. The contribution of
lipid vesicles to the CD signal was removed by subtracting the CD
spectra of pure lipid vesicles from the CD spectra of peptide:lipid
suspensions. Peptide conformations in 40% TFE:H2O were
determined under the same conditions. When ellipticity at either 218 or
222 nm was measured, data were collected at a rate of 1 s/point for 300 data points.
Electron Microscopy--
For negative staining, carbon-coated
pioloform grids were floated on aqueous solutions of peptides (30 µg/ml) in the presence and absence of lipids (1:20, peptide:lipid
ratio). After grids were blotted and air-dried, the samples were
stained with 1% (w/v) phosphotungstic acid. The peptide
assemblies were observed in a Hitachi 7000 operated at 75 kV.
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RESULTS |
A
-ganglioside Binding Studies--
We have used synthetic lipid
vesicles as a model system to investigate the interactions of A
with
ganglioside containing cellular membranes. Gangliosides alone are
unable to form stable vesicles (36); consequently, the various
gangliosides were mixed with PC in a 1:1 ratio (by weight) to produce
stable vesicles. We have previously demonstrated that A
40 and A
42
are unable to cause membrane disruption and aggregation of PC vesicles
(19, 28). The ability of A
40 and A
42 to bind PC, mixed
gangliosides, and GM1 was investigated using a binding assay that
utilized sucrose density gradient centrifugation.
Mixed ganglioside:PC, GM1:PC, and PC vesicles were incubated with
either A
40 or A
42 (Fig. 1). The
A
-bound vesicles were separated from unbound vesicles by sucrose
density gradient centrifugation in a 10-40% discontinuous sucrose
gradient. The presence of peptide in the various fractions was detected
by Tyr absorbance and dot-blot analyses with an anti-A
polyclonal
antibody; lipid was detected by phosphorus assay. When samples
containing A
40 and PC vesicles were examined, all the peptide was
recovered near the top of the gradient, although the vesicles were
found near the bottom of the gradient (Fig. 1A). Similar
results were seen for A
42 incubated with PC vesicles (data not
shown).

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Fig. 1.
Sucrose density gradient of A 40 in the
presence of lipid vesicles consisting of either PC (A) or
mixed ganglioside:PC (B). A 40 was detected by Tyr
absorbance at 275 nm (dashed line). Lipid vesicles were
detected by the Bartlett assay for phosphorus content (solid
line). Dot-blot analysis of fractions containing Tyr absorbance
confirmed the presence of A 40.
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However, when vesicles were made with mixed gangliosides and PC, the
A
40 peptide was recovered further down the sucrose gradient coincident with fractions containing vesicles (Fig. 1B).
Similar results were seen for A
42-mixed ganglioside:PC vesicles and
when both peptides were incubated with GM1:PC vesicles (data not
shown). These results demonstrate that although A
40 and A
42 do
not bind to PC vesicles, they bind ganglioside:PC vesicles, suggesting that any effects observed with ganglioside:PC vesicles will be solely
the result of A
-ganglioside interactions.
A
-induced Dye Release from Lipid Vesicles--
To investigate
further the A
-ganglioside interaction, we employed a dye release
assay. 5-(and 6-)Carboxy-2',7'-dichlorocarboxyfluorescein is
incorporated into various lipid vesicles at a concentration that
induces self-quenching. The addition of a membrane disrupting molecule
results in the release of dye and an increase in fluorescence. The
integrity of the vesicles was determined by monitoring the spontaneous
diffusion of dye over time. If the spontaneous dye release exceeded
10% of the total release or if bee venom mellitin did not induce 100%
dye release, then the vesicles were not used in this study. The
interaction of peptides with lipid vesicles may induce dye leakage as a
result of peptide surface binding that disrupts vesicle integrity,
penetration of the lipid membrane, or micellar fusion (37). We
investigated the ability of A
40 and A
42 to induce dye leakage
from lipid vesicles at pH 7.0 (Table I).
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Table I
Percentage dye release
A peptides 1-40 and 1-42 (dissolved in 40% TFE) were added to
lipid vesicles in a 1:20 peptide to lipid ratio with a final peptide
concentration of 1 µM. Dye release was measured at pH 7.0. Mellitin was used as a positive control, and 40% TFE was used as
a negative solvent control for dye release. Data points were collected
continuously for 10 min, and the data are reported as the maximal
percent dye release. S.D. were calculated from at least five
experiments for each lipid system. Mixed, mixed ganglioside
preparation. Sph, sphingomyelin.
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We have previously demonstrated that A
40 is able to induce dye
release from mixed ganglioside:PC and GM1:PC vesicles at pH 7.0, whereas A
42 could not (19). To determine if this is a general
phenomenon associated with all gangliosides or if there is a dependence
on the carbohydrate and sialic acid content, we investigated the
ability of A
40 and A
42 to induce dye leakage from vesicles
containing GM2, GM3, GD1a, and GT1b (Table I). A
40 was able to
induce dye leakage from GM2 containing vesicles but not GM3, GD1a, and
GT1b containing vesicles. These results indicate that the position and
amount of carbohydrate affect the A
40-ganglioside interaction. The
inability of A
40 to induce leakage from ceramide containing vesicles
confirms that the ganglioside head group, which is missing in ceramide,
is critical for A
-ganglioside interaction. A
42 was unable to
induce dye leakage from all ganglioside containing vesicles. On the
glycosphingolipid synthetic pathway, ceramide is a precursor of
gangliosides, cerebrosides, or sphingomyelin. We found that A
40 and
A
42 were able to cause dye leakage from sphingomyelin vesicles
(Table I). These results are surprising since the head group of
sphingomyelin is either phosphorylethanolamine or phosphorylcholine,
and their physical properties are very similar to
phosphatidylethanolamine (PE) and phosphatidylcholine. Previously, we
have shown that A
40 and A
42 do not disrupt PE or PC vesicles (28). The difference between sphingomyelin and PC:PE is the fatty acyl
composition, the former containing one fatty acyl chain and one
hydrocarbon chain of sphingosine, whereas the latter contains 2 fatty
acyl chains. Variation in the dye release assay may result from the
inability of the sphingosine group to pack efficiently in vesicle
systems, thereby allowing access to the hydrophobic core of the
lipid.
A
40-induced dye release from mixed ganglioside and GM1 containing
vesicles plateaus before 100% dye release; this may be the result of
multiple vesicle types present in our system, one of which is
accessible to disruption and another which is inaccessible. Alternatively, A
40 may have a strong interaction with a limited number of lipid molecules where it becomes sequestered, thereby leaving
a number of vesicles unaffected. To differentiate between these two
possibilities, the dependence of dye release on peptide concentration
was examined. Incremental increases in concentrations of A
40
resulted in a stepwise increase in dye release from both mixed
ganglioside:PC and GM1:PC vesicles (Fig.
2A). The step-shaped A
40-ganglioside dye release curves suggested that binding of A
40
to mixed ganglioside or GM1 containing vesicles is effectively irreversible. Increasing concentrations of A
42 failed to induce dye
leakage from either mixed ganglioside:PC or GM1:PC vesicles (Fig.
2B). The data from the dye release and the sucrose gradient assays suggest that although A
42 is able to bind gangliosides, its
interaction with the ganglioside head group does not induce disruption
of the bilayer.

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Fig. 2.
The concentration dependence of A 40
(A) and A 42 (B) membrane interaction was
investigated by means of the dye release assay. The initial lipid
concentration was held constant at 20 µM. The initial
peptide concentration was 1 µM. Incremental amounts of
peptide were added to mixed ganglioside:PC (dashed line) or
GM1:PC (solid line) vesicles when the dye release had stabilized. Equal volumes of 40% trifluoroethanol (dotted
line) were included to monitor vesicle stability. These results
are representative of at least three experiments.
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Ganglioside-induced A
Structural Transitions--
The
conformation of A
40 and A
42 in the presence of GM2, GM3, GD1a,
GT1b containing vesicles was investigated by CD spectroscopy. As
reported previously, A
40 (Fig.
3A) and A
42 (Fig.
4A) in 40% trifluoroethanol
are partly
-helical (19, 38). Upon dilution into PBS, at pH 6.0 or
pH 7.0, both A
40 and A
42 adopt a random structure at early time
points (Figs. 3A and 4A). We have previously demonstrated that the interaction of A
40 and A
42 with mixed ganglioside:PC (Figs. 3B and 4B) and GM1:PC
vesicles results in a
/
conformation at pH 7.0 but a
-structured conformation at pH 6.0 (19). Although A
40 interacts
with GM2:PC vesicles, as demonstrated by the dye release assay, this is
not accompanied by a significant change in the CD spectrum at either pH
6.0 or pH 7.0 (Fig. 3, C and D). GM3:PC vesicles
were also unable to induce CD changes. Similarly, A
42 did not
undergo significant changes in CD in the presence of GM2:PC or GM3:PC
vesicles at both pH 6.0 and pH 7.0 (Fig. 4, C and
D).

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Fig. 3.
Circular dichroism spectra of A 40 in the
presence or absence of various lipids. Lipids were present at a
1:20 peptide to lipid ratio with a final peptide concentration of 10 µM. CD spectra of A 40 in PBS, pH 6.0 (solid
line), PBS, pH 7.0 (dotted line), and 40% TFE
(dashed line) are shown in A. CD spectra of A 40 in the presence of mixed ganglioside:PC (B), GM2:PC
(C), GM3:PC (D), GD1a:PC (E), and
GT1b:PC (F) at both pH 6.0 (solid line) and pH
7.0 (dotted line).
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Fig. 4.
Circular dichroism spectra of A 42 in the
presence or absence of various lipids. Lipids were present at a
1:20 peptide to lipid ratio with a final peptide concentration of 10 µM. CD spectra of A 42 in PBS, pH 6.0 (solid
line), PBS, pH 7.0 (dotted line), and 40% TFE
(dashed line) are shown in A. CD spectra of A 42 in the presence of mixed ganglioside:PC (B), GM2:PC
(C), GM3:PC (D), GD1a:PC (E), and
GT1b:PC (F) at both pH 6.0 (solid line) and pH
7.0 (dotted line).
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The interaction of A
40 with GD1a containing vesicles does not induce
a structural transition at either pH (Fig. 3E). Although A
40 did not induce dye release from GT1b:PC vesicles, the
interaction with GT1b containing vesicles induces
-structure at pH
6.0 (Fig. 3F). A
42 was unable to induce membrane
disruption of GD1a:PC and GT1b:PC vesicles, but these interactions
induce
-structure formation at both pH 6.0 and pH 7.0 (Fig. 4,
E and F). It is interesting that the extent of
-structure is identical at pH 6.0 and pH 7.0; this is similar to the
structural transition of A
42 in the presence of phosphatidylinositol
vesicles (28). These results imply that A
42 is interacting with the
ganglioside head group and that induction of
-structure does not
affect membrane integrity.
We have previously demonstrated that asialo-GM1:PC vesicles were unable
to induce the structural transitions of both A
40 and A
42,
implying a critical role for the sialic acid moiety (19). Neither
ceramide nor sialic acid alone could induce the partly helical
structure seen in A
40- and A
42-mixed ganglioside-PC interactions
at pH 7.0 (Fig. 5). Similarly, these
components could not induce
-structure in A
40 at pH 6.0. These
results imply that although the sialic acid is critical for structure
formation, its association with the carbohydrate backbone is also
necessary. Both ceramide and sialic acid induced a small change in the
structure of A
42 at pH 6.0. The ability of ceramide alone to induce
a structural change may be the result of the presence of both a
hydrogen acceptor (amide carbonyl) and a hydrogen donor (hydroxyl
group) on ceramide, which we have previously shown to be necessary for
-structural transitions (28).

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Fig. 5.
Circular dichroism spectra of A 40
(A, B, and C) and A 42
(D, E, and F) in the presence of
ceramide (A and D), sialic acid (B
and E) and sphingomyelin (C and
F). Lipids and sialic acid were present at a 1:20
peptide:reagent ratio with a final peptide concentration of 10 µM. CD spectra were obtained in PBS at pH 6.0 (solid line) and pH 7.0 (dashed line).
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As previously noted, ceramide is a biosynthetic precursor to
gangliosides, sphingomyelin and cerebrosides. To determine if the
ability of A
40 and A
42 to induce dye release from sphingomyelin vesicles at pH 7.0 has a structural correlate, we investigated the
ability of sphingomyelin to induce structure in A
40 and A
42 (Fig.
5, C and F).
-Structure was evident in A
42
in the presence of sphingomyelin at pH 6.0 but not pH 7.0. The ability
of sulfated cerebrosides (sulfatides) to induce
-structure in A
40
and A
42 was investigated in light of the importance of sulfated
glycosaminoglycans to enhance A
-fibrillogenesis (10, 11, 39). We
were unable to detect a structural change in either A
40 or A
42 in
the presence of sulfatide containing vesicles (data not shown). These
results suggest that sulfated cerebrosides do not function in a similar manner to sulfated glycosaminoglycans during fibrillogenesis.
Effect of Relative Ganglioside Concentration on A
-Ganglioside
Interactions--
The relative concentration of ganglioside required
to induce the structural transitions in A
40 was determined at both
pH 7.0 and pH 6.0 (Fig. 6). The
ganglioside content was decreased by decreasing the ganglioside:PC
ratio. The ability of mixed gangliosides or GM1 to induce
-helical
structure was monitored at 222 nm. Decreasing the GM1:PC ratio of the
lipid vesicles, from 0.5 to 0.33, eliminated the A
40 structural
transition to
-helix at pH 7.0 (Fig. 6A). On the other
hand, decreasing the mixed ganglioside:PC ratio from 0.5 to 0.1 reduced
helical content by only 36% (Fig. 6B). These results may
suggest that there is an unidentified ganglioside that induces more
stable helix formation than GM1, or alternatively, mixed gangliosides
can segregate within PC vesicles to a higher extent than GM1 enabling
the necessary environment for the structural transition (36). Similar
to the GM1 results, when the mixed ganglioside:PC ratio is decreased
from 0.5 to 0.3, the ability to induce
-structure in A
40 at pH
6.0 is eliminated (Fig. 6C). The monotonic relationship
between
-structure and ganglioside content suggests that the
structural transition does not involve the formation of a discrete
peptide-ganglioside complex but is an interaction between the peptide
and negatively charged regions of the membrane. These results are
consistent with the
-structural transitions induced by acidic
phospholipids (28).

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Fig. 6.
The dependence of circular dichroism at 222 and 218 nm of A 40:lipid mixtures on ganglioside:PC ratios. The
total lipid concentration was at a 1:20 peptide:lipid ratio with a
final peptide concentration of 10 µM. The ganglioside:PC
ratio was varied by increasing concentrations of PC. The -helical
content of A 40 in the presence of GM1 (A) and mixed
gangliosides (B) was monitored at pH 7.0, whereas the amount
of -structure in the presence of mixed gangliosides (C)
was monitored at pH 6.0. Data points are reported as the mean of 300 data points.
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Ionic Strength Dependence of A
-Ganglioside
Interactions--
The interaction of A
40 with mixed ganglioside
containing vesicles may be the result of electrostatic and/or
hydrophobic interactions. The ability of increasing concentrations of
NaCl to shield charges on both A
and lipid vesicles can give an
indication of the contribution of electrostatic interactions to the
stability of the A
-ganglioside complex. The
-helical content of
A
40 in the presence of increasing concentrations of NaCl was
monitored at 222 nm, and
-structure was monitored at 218 nm. At pH
7.0, the helical structure was retained even at 1.0 M NaCl
(Fig. 7A). These results imply
that the
-helical structure is not stabilized by charge
interactions. At pH 6.0, the amount of
-structure present in A
40
decreased by one-third at 120 mM NaCl and did not change
significantly up to 1 M NaCl (Fig. 7B). These
results indicate that the
-structure seen in A
40-ganglioside
interactions is stabilized by both electrostatic and hydrophobic
interactions. When A
42-ganglioside interactions were monitored in
NaCl concentrations up to 1 M and at pH 6.0, the amount of
-structure decreased. These results suggest that electrostatic
interactions are involved in stabilizing the
-conformation of
A
42.

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Fig. 7.
The dependence of circular dichroism at 222 and 218 nm of A 40:lipid mixtures on ionic strength. The ability
of mixed ganglioside:PC vesicles to induce -helix (A) at
pH 7.0 and -structure (B) at pH 6.0 was monitored at 222 and 218 nm, respectively. Data points are reported as the mean of 300 data points.
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Time Dependence of A
-Ganglioside Interactions--
The
stability of the A
40 and A
42-mixed ganglioside-PC interaction
over a period of 96 h was also investigated (Fig.
8). It is well documented that A
alone
or in the presence of kinetic enhancers undergoes a structural
transition from random coil to
-structure that is necessary for
fibril formation. In the absence of lipid vesicles and at pH 7.0, A
40 was randomly structured for up to 72 h. At 96 h,
-structure could be detected in A
40 (Fig. 8C). On the
other hand, A
40 remained
-helical in the presence of mixed
ganglioside:PC vesicles for the entire 96-h time course (Fig. 8). The
magnitude of
222 nm, which is indicative of helical
content, did not vary over time indicating that A
40 was not lost due
to aggregation or precipitation. These results suggest that when
monomeric A
40 interacts with gangliosides it forms a stable
-helical structure that does not progress to the
-sheet structure
of amyloid fibrils.

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Fig. 8.
The stability of A 40-mixed ganglioside
interactions over a 96-h period, monitored by circular dichroism
spectroscopy. A 40 alone (dashed line) and in the
presence of mixed ganglioside:PC vesicles (solid line) were
incubated at 10 µM peptide concentration in PBS, pH 7.0. A 40 was unstructured at t = 0 h (A)
and remained unstructured for up to 48 h (B), whereas
at 96 h (C) -structure was apparent. In contrast,
A 40 in the presence of mixed ganglioside:PC vesicles remained
-helical over the entire 96 h.
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In the absence of lipid vesicles and at pH 7.0, A
42 was randomly
structured when diluted into PBS. At 48 h,
-structure could be
detected in A
42 (data not shown). The amount of CD signal at 218 nm
decreased significantly over the 96-h period indicating that
aggregation was occurring. The
-helical content of A
42 in the
presence of mixed ganglioside:PC vesicles decreased steadily over the
96 h. CD signal could be detected at 218 nm indicating a shift to
-structure (data not shown). These results suggest that although
A
42-ganglioside interactions initially form an
-helical
structure, this may proceed to the
-structure over time.
A
-Fibril Formation--
The structural characteristics of
A
40 and A
42 fibrils in the presence and absence of gangliosides
were examined by means of electron microscopy. Negative stain
preparations examined by electron microscopy indicated that A
40 and
A
42 exhibited distinct abilities to assemble into fibrous structures
in the presence of gangliosides. Initial studies involved the
incubation of unseeded A
peptides in the presence of PC and mixed
ganglioside:PC vesicles. Negative stain electron microscopy
demonstrated that A
40 and A
42 formed short, indistinct fibers
that were highly aggregated when incubated alone or in the presence of
PC and mixed ganglioside:PC vesicles (data not shown). All fibers were
detected in areas devoid of vesicles.
The structural characteristics of fibrils formed from seeded A
in
the presence and absence of gangliosides was also determined using
negative stain electron microscopy. A
42 incubated alone produces
short aggregated fibers (data not shown). When seeded A
42 was
incubated in the presence of PC vesicles, no fibers were seen in
association with lipid vesicles. Some fibers could be detected in areas
that were devoid of lipid and were indistinguishable from those of
A
42 incubated alone. The mixed ganglioside:PC vesicles present with
A
42 were starting to collapse but were similar to the structure of
mixed ganglioside:PC vesicles incubated alone (Fig.
9A). At initial times, no
fibrils could be detected when A
42 was added to mixed ganglioside:PC
vesicles (Fig. 9B). When A
42 was incubated for longer
periods in the presence of mixed ganglioside:PC vesicles, many long
fibers could be seen arranged along the surface of the lipid vesicles
(Fig. 9C). The A
42 fibrils had some lateral aggregation
present. The mixed ganglioside:PC vesicles are no longer small
sonicated vesicles but seem to have coalesced into larger vesicles with
increased surface area. These results suggest that the glycolipids are
creating a support for the fibrils to extend across.

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Fig. 9.
Negative stain electron microscopy of A 42
and in the presence of mixed ganglioside:PC vesicles. Mixed
ganglioside:PC preparations contained both unilamellar
(arrows) and multilamellar vesicles (arrowheads).
A 42 incubated in buffer alone demonstrates small thin fibers. When
A 42 was incubated in the presence of PC vesicles (B) no
fibers could be detected although many long aggregated fibers were
arranged along the surface of mixed gangliosides:PC vesicles
(C). Magnification, 87,000 (A and B)
and 116,000 × (C).
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When A
40 was incubated alone, it also produced short aggregated
fibers (Fig. 10A). In the
presence of PC vesicles, many fibers could be seen arranged along the
surface of the vesicles (Fig. 10C). Fibrils appeared to have
a lot of lateral aggregation present. These results suggest that once
A
40 is seeded then it will form fibrils across the lipid membrane
surface as a result of hydrophobic interactions. When A
40 was
incubated in the presence of mixed ganglioside:PC vesicles, a few long
fibers could be detected (Fig. 10B). The fibrils were
present as singular fibers that transversed the lipids rather than
staying at the edge of the membrane as seen with PC vesicles alone. As
detected with A
42, the mixed ganglioside:PC vesicles appeared to
have coalesced into large structures with an increased surface area.
These results are consistent with gangliosides acting as an inhibitor
of nucleation, since a lower number of fibrils were present and they
were of greater length (9).

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Fig. 10.
Negative stain electron microscopy of A 40
in the presence of PC and mixed ganglioside:PC vesicles. A 40
incubated in buffer alone (A) demonstrates short thin
fibers. When seeded A 40 was incubated in the presence of PC vesicles
(C) long fibers were present along the surface of the
vesicles, probably as a result of hydrophobic interactions.
Alternatively, only a few thin fibers could be detected in the presence
of mixed ganglioside:PC vesicles (B). Magnification, 116,000 (A) and 87,000 × (B and C).
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DISCUSSION |
A
peptides have been reported to exert many effects on cell
function. It has been proposed that A
exerts these effects by initially disrupting the membrane lipid environment that in turn would
affect the activity of various proteins involved in signal transduction
and ion fluxes (40). A
peptides may alter the neuronal membrane
lipid environment by inducing lipid peroxidation and/or changing lipid
fluidity. It has been shown previously that A
40 increased both
annular and bulk fluidity of synaptic plasma membranes (29). In
contrast, A
40 also was shown to decrease the bulk fluidity of
lymphocyte membranes and of membranes isolated from cortex,
hippocampus, striatum, and cerebellum (41). The disparity between these
systems may originate from differences in the lipid composition of the
membranes. The synaptic plasma membranes were isolated directly,
whereas membranes isolated from various regions of the brain are
homogenates containing plasma membranes, intracellular membranes, and
various organelles. With particular relevance to the results reported
here, synaptic plasma membranes have a relatively high concentration of
gangliosides with respect to other cellular membranes.
We have shown that A
40 is able to induce dye leakage from
ganglioside containing vesicles and requires the presence of a single
sialic acid molecule and a carbohydrate backbone. If the vesicles
contain more complex gangliosides (i.e. GM3, GD1b, and GT1a)
then A
40 is unable to induce membrane disruption. The
A
40-ganglioside interaction forms a stable, specific complex of
A
40 with a limited number of gangliosides. We have shown that mixed
gangliosides and GM1 are able to form this complex. In contrast, we
found that A
42 was unable to induce dye release from all ganglioside
containing vesicles. These results suggested that A
42 bound to the
surface of gangliosides and does not affect bilayer integrity; however, the possibility that it affects membrane fluidity cannot be ruled out.
The interaction of A
with gangliosides in the neuronal membrane may
represent one mechanism by which A
disrupts the membrane fluidity
and/or integrity thus explaining the increased permeability to ions and
dysregulation of signal transduction systems seen in the presence of
A
(40).
The high binding potential of gangliosides has been attributed to the
general surfactant character of the gangliosides and the direct
involvement of the ceramide or oligosaccharide portions of the
molecules in binding (42). Whereas certain toxins, interferons, and
hormones interact primarily with the oligosaccharides of gangliosides, certain antibiotics and membrane proteins interact with the ceramide group. The carbohydrate moiety of gangliosides is a low affinity receptor for the hormone, thyrotropin, and it induces the
conformational change necessary for thyrotropin's insertion into the
lipid bilayer (43). The antibiotic, valinomycin, interacts with the
fatty acyl chains of gangliosides and fits into a cavity created by the
ganglioside packing in the hydrophobic region of the lipid (44). This
interaction does not disrupt the lipid bilayer organization.
Our results demonstrate that both A
40 and A
42 bind gangliosides.
The interactions of A
40 and A
42 with mixed gangliosides may
explain some of the different effects of these peptides in vitro and in vivo. The binding of A
40 to mixed
gangliosides or GM1 containing vesicles induces an
-helical
structure at pH 7.0 and
-structure at pH 6.0. The helical structure
was stable over time and resistant to large changes in ionic strengths
but required proper positioning of sialic acid on the carbohydrate
moiety. These results suggest that stable
-helical structure is
stabilized by both polar and hydrophobic interactions. Gangliosides
could not induce the
-helical structure if the A
was already
aggregated in a
-structured form. Conversely, the helical
A
40-ganglioside complex does not proceed to
-structure, at least
over a period of 4 days. These results were confirmed using electron
microscopy in that unseeded A
40 did not progress to form fibrils,
whereas some fibrils formed if seeded A
40 was present with mixed
ganglioside:PC vesicles. The decreased number but increased length of
A
40 fibrils in the presence of mixed gangliosides:PC vesicles in
comparison to A
40 alone suggests that mixed gangliosides:PC vesicles
are acting as an inhibitor of the fibril nucleation step.
The interaction of A
42 with gangliosides also induces an increased
amount of
-helical structure at pH 7.0 and
-structure at pH 6.0. Yanagisawa and co-workers (45) reported that A
42 was isolated in
association with GM1 from human brain that had a high proportion of
diffuse plaques. They speculated that this may be a specific form of
A
42 that led to formation of diffuse plaques. The presence of
A
42-ganglioside complexes with an increased
-helical content may
help explain the lack of cell death associated with diffuse plaques.
Previous studies have demonstrated that A
neurotoxicity is
associated with the formation of
-structure, A
aggregation, and
fibril formation (12, 32, 33). The
-helical structure of A
bound
to gangliosides may therefore be a non-toxic conformation.
It is well documented that A
42 has a greater propensity to form
-structure than A
40 (46). The ability of gangliosides to induce
-structure in A
42 at both pH 6.0 and pH 7.0 is proportional to
the number of negative charges. Increasing the number of sialic acid
residues on the carbohydrate backbone (i.e. with GD1a and GT1b) resulted in formation of
-structure at pH 7.0. The presence of
a high net negative surface charge on the lipid vesicles will favor a
-structured transition and prevent the formation of the
-helical
structure. The requirement for hydrophobic interactions to stabilize
the helical conformation and the inherent properties of A
42 alone
suggest that formation of the helical A
42-ganglioside complex will
be disfavored when there is an elevation of A
42 concentrations, as
reported in early onset Alzheimer's cases (47).
We examined the ability of A
42 to form fibrils similar to amyloid.
When A
42 was incubated in the presence of mixed ganglioside:PC vesicles, many fibrils could be detected that lie along the surface of
the lipid vesicles. These data are consistent with the ability of
gangliosides to affect both the induction of a structural transition in
and the lack of membrane disruption by A
42. The ability of A
to
form fibrils with a high degree of lateral aggregation on the surface
of the vesicles may help explain association of plaques to cellular
plasma membrane. Finally, although we have only discussed the
interaction of A
with plasma membranes, the A
-ganglioside interaction may also be important in the Golgi apparatus or the transport vesicles. Recently, it has been shown that A
can be generated in the trans Golgi network in the absence of transport vesicles (48, 49); this is also the site of ganglioside biosynthesis (42).