The amyloid precursor protein interacts with neutral lipids
Liposomes and monolayer studies
Raghda Lahdo
1
, Ste
´
phane Coillet-Matillon
1
, Jean-Paul Chauvet
2
and Laurence de La Fournie
`
re-Bessueille
1
1
Laboratoire de Physico-Chimie Biologique, Universite
´
Claude Bernard, Lyon, France;
2
IFOS, Equipe Bioinge
´
nierie et Reconnaissance
Ge
´
ne
´
tique, Ecully, France
The amyloid protein precursor (APP) was incorporated into
liposomes or phospholipid monolayers. APP insertion into
liposomes required neutral lipids, such as
L

observed are not due to major secondary structural changes
in APP. These observations may be related to the parti-
tioning of APP into membrane microdomains.
Keywords: amyloid precursor protein; liposomes; monolayer;
phospholipids; protein–lipid interactions.
Limited proteolysis of the amyloid precursor protein (APP)
generates the amyloid-b-protein (Ab), which is a major
component of brain senile plaques in Alzheimer’s disease
(AD) [1,2]. APP occurs in neural and non-neural tissues as
several membrane-associated glycoproteins of 110–135 kDa
[3]. It is a N- and O-glycosylated single-chain molecule
consisting of 770 amino acid residues, with an isoelectric
point of 4–5. The APP gene is expressed in brain and in
several peripheral tissues, but the physiological functions of
APP and its role in the disease are still poorly understood. A
recent report proposed that APP normally behaves in the
brain as a cell surface signalling molecule, and that an
alteration of this function is one of the possible causes of the
neurodegeneration and consequent dementia in AD [4]. The
Ab peptide is produced in the endosomal compartment and
in the endoplasmic reticulum or Golgi complex [5,6]
through the sequential action of b-andc-secretases [7–12].
APPcouldalsobecleavedbyana-secretase, within the Ab
sequence, thus preventing amyloidogenesis, and results in
the secretion of the larger soluble amino-terminal product
(sAPP; reviewed [13]). The molecular mechanism involved
in APP cleavage and Ab production has still to be resolved.
Minor changes of the membrane lipid composition could
affect the stability of APP as well as its processing, or alter
the function of secretases within the membrane and their

´
Claude Bernard, Lyon I, 43 bd du 11
novembre 1918, 69622 Villeurbanne cedex, France.
Fax:+33472431543,Tel.:+33472448324,
E-mail:
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid peptide; APP,
amyloid precursor protein; b-OG, n-octyl b-
D
-glucopyranoside; LUV,
large unilamellar vesicles; PtdCho, phosphatidylcholine; PtdEtn,
phosphatidylethanolamine; PtdSer, phosphatidylserine.
(Received 10 January 2002, revised 11 March 2002,
accepted 15 March 2002)
Eur. J. Biochem. 269, 2238–2246 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02882.x
show that at least some of the physiological effects of Ab
may involve direct interactions between the peptide and the
membrane lipids. However, the mechanisms of Ab–mem-
brane interaction are still unclear. As no data exist on the
binding of APP itself to the lipid membrane, it is of great
interest to examine the interactions of APP with lipids. In
the present study, we have investigated the ability of APP to
interact with lipids. The potential interactions of the protein
with lipid membranes were characterized using two approa-
ches. In the first, the insertion of APP into liposomes was
analysed via a detergent-mediated reconstitution procedure.
In the second, the interaction of the protein with a
monomolecular lipid film was characterized using the
Langmuir technique. The role of electrostatic and hydro-
phobic effects in APP association with lipids was investi-
gated in more detail under various conditions involving

quently removed by a further 3–6 h evaporation period
under vacuum. The remaining lipid film was then hydrated
in 20 m
M
Tris/HCl, 150 m
M
NaCl pH 7.4 at a higher
temperature (room temperature) than the phase transition
temperature of the corresponding lipid and dispersed
vigorously by vortexing. LUV were formed using six fast
freeze–thaw cycles. They were subsequently extruded 19
times through two polycarbonate membranes (400 and
200 nm pore size), using a mini extruder (Avanti Polar). The
final phospholipid concentration was 20 mgÆmL
)1
.
Preparation of phospholipid–protein complexes
The incorporation of APP into liposomes was performed
using the detergent-mediated procedure described by Le
´
vy
et al. [33]. Briefly, the complexes of phospholipid and
protein were prepared at a phospholipid/protein ratio of
500 : 1 (w/w). The lipid/protein mixture containing trace
amounts of
125
I-labeled APP was incubated with the desired
concentration of b-OG. The excess of detergent was then
removed by extensive dialysis against 20 m
M

protein was performed at a constant surface area either in a
small Teflon dish (diameter, 1.6 cm) with a subphase
volume of 4 mL or with a larger Teflon dish (diameter,
3.4 cm) with a subphase volume of 19 mL. The surface
pressure was measured as a function of time by the
Wilhelmy plate method using plates cut from filter paper
(Whatman no. 1) and a computer-controlled transducer
readout. The surface activity of the protein was recorded at
a free water interface and characterized in the presence of a
preformed lipid monolayer. Lipid monolayers were spread
at the air–water interface from a chloroform solution to give
an initial surface pressure (p
i
). Ten minutes after the
formation of the monolayer, a desired volume of APP in
20 m
M
Tris/HCl, 150 m
M
NaCl pH 7.4 or in Tris/maleate
20 m
M
,150 m
M
NaCl pH 7, was injected below the surface.
The subphase was stirred continuously with a Teflon-coated
stirring bar and a magnetic stirrer. In similar experiments,
we examined the effect of pH or NaCl concentrations in the
subphase buffer on the adsorption characteristics of APP in
the absence or in the presence of a phospholipid monolayer.

mined. Control experiments showed that all the radioactiv-
ity associated with pure APP in the absence of liposomes
was recovered at the bottom of the gradient (25% sucrose),
while pure phospholipid liposomes were collected at a
sucrose concentration of 15%. After a 20-h dialysis period
of the reconstitution mixture composed of APP, PtdCho
liposomes and b-OG, we observed a comigration of
phospholipid and protein over a relatively narrow density
range, at % 15% sucrose. A small fraction of the protein was
associated with the lipid, and % 80%freeproteinwas
recovered at the bottom of the gradient (Fig. 1). The
presence of the free protein indicates a low incorporation
efficiency of APP during the reconstitution procedure. The
influence of lipid/protein ratio on efficiency of protein
incorporation into liposomes during reconstitution was
examined. Proteoliposomes samples containing APP and
PtdCho liposomes were incubated in the presence of b-OG
at initial phospholipid/protein ratios of 1000 : 1, 500 : 1 or
200 : 1 (w/w). Most of the protein migrated at the bottom
of the gradient (Fig. 1), which indicated incomplete incor-
poration of APP into liposomes whatever the lipid/protein
ratio. Therefore, the following experiments were carried out
with phospholipid/protein ratio of 500 : 1. Reconstitution
experiments performed with ionic or zwitterionic detergents
were less efficient and less reproducible. The role of different
phospholipids in the insertion of APP into lipid bilayers was
examined (Fig. 2). Vesicles containing PtdEtn/PtdCho
(molar ratio 62.5 : 37.5) did not change the incorporation
rate of APP into the membrane. However, PtdSer-contain-
ing liposomes totally prevented the binding of APP to the

pressure are shown in Fig. 3A. The results showed that the
final surface pressure increased with APP concentration in
the subphase, an indication that the interface was not
saturated by the protein, for concentrations up to
1 lgÆmL
)1
. Moreover, the surface pressure increase does
not occur immediately after the injection of the protein into
the subphase (Fig. 3A). For protein concentrations of 0.35,
0.5, 1 and 2 lgÆmL
)1
the surface pressure started to increase
after 90, 50, 20 and 10 min, respectively. For the lowest APP
concentration used, the observed lag time reached several
hours. The sigmoidal shape of the p vs. time curve suggests
the occurrence of a co-operative process. These results
indicate that the adsorption of APP at the air–water
interface is both concentration- and time-dependent. The
process of the protein adsorption can be analysed by a first
order equation:
Fig. 1. Density gradient centrifugation profiles for APP liposomes
reconstituted from b-OG. Liposomes (1 mg) were incubated with b-OG
(16 m
M
) and APP. The detergent was removed by dialysis, the samples
were then submitted to a flotation on discontinuous sucrose gradients.
Fractions (0.5 mL) were collected from the bottom of the gradient and
APP content was measured by assaying for
125
I-labeled APP. (- - -,

e
À p
0

¼À
t
s
ðEqn 1Þ
where p
e
, p, p
0
, are the surface pressure values at steady-
state conditions, at time t and at time t ¼ 0, respectively,
and s is the relaxation time.
In order to evaluate the parameters that control the
successive steps of the adsorption of APP at the air/water
interface, the plots of ln(1–p/p
e
) vs. time were obtained
according to Eqn 1. The curves (insert in Fig. 3A) present at
least two linear parts. According to Graham & Phillips’
work [34], the relaxation time s
1
, corresponding to the first
linear part, is corelated with the protein adsorption and
perhaps unfolding at the surface layer, while s
2
, defined by
the second linear part, can be related to a subsequent

The presence of 5 m
M
NaCl in the subphase buffer
decreased the surface activity to 4.5 mNÆm
)1
with a 2-h lag
time (Fig. 3B). The surface activity of the protein in the
absence of lipids was also studied on subphase buffers of
different pH (Fig. 3C). Lowering the pH to 6 did not
promote changes on the final surface pressure of the protein
monolayer, but increased the lag time threefold. At a pH
below 6, longer lag times and lower surface activities for
APP were observed (p
e
¼ 10 mNÆm
)1
at pH 5, and
p
e
¼ 2mNÆm
)1
at pH 4) (Fig. 3C). This result may be
explained by the progressive reduction of the global charge
of the protein as the pH approaches the isoelectric point,
giving a less hydrophilic character to the APP molecule.
Interactions of APP with phospholipid monolayers.
Increase in surface pressure (Dp) vs. time curves (Fig. 4A)
observed for APP adsorption into films of PtdCho (initial
p
i

monolayers of various phospholipids were studied at a low
concentration of APP. Fig. 5A shows typical records of the
Fig. 3. Kinetics recording the surface behaviour of APP at the air–water
interface. (A) p–t curves corresponding to the penetration of APP into
the air–water interface under different bulk concentrations of the
protein (buffer: Tris/HCl 20 m
M
, NaCl 150 m
M
,pH7;Teflontrough
19 mL, 9 cm
2
). Insert: Ln (1-p/pe)) vs. t plots for the adsorption of
APP into the air–water interface. Protein concentrations in the bulk
are 0.35 lgÆmL
)1
(a) and 1 lgÆmL
)1
(b). (B) p–t curves corresponding
to the penetration of APP into the air–water interface in the presence
of various salt concentrations (buffer: Tris/maleate 20 m
M
,pH7;
Teflon trough 4 mL, 2 cm
2
). (C) p–t curves corresponding to the
penetration of APP into the air–water interface under various pH
(buffer: Tris/maleate 20 m
M
,NaCl150m

Comparison of the results for p
i
¼ 5mNÆm
)1
with those for
higher p
i
are illustrated in Fig. 5B [Dp ¼ f(p
i
)]. The data
suggest that the increase in the surface pressure Dp due to
the adsorption of APP on the phospholipid monolayers is
dependent on the initial surface pressure of the lipid film.
The similarity of the Dp vs. time profiles for the phosphol-
ipids PtdCho and PtdEtn (Fig. 5A) confirmed the observa-
tion that APP did preferentially interact with either one of
the two phospholipids during adsorption at the air–water
interface. When APP was injected below monolayers of
PtdCho, PtdEtn or PtdSer formed at p
i
above 23, 19 and
11 mNÆm
)1
, respectively, the protein was no longer able to
induce any increase of the surface pressure (Fig. 5B). These
critical surface pressures for APP penetration (p
c
) corres-
pond to the extrapolated initial surface pressures beyond
which no increase in the surface pressure occurred.

Fig. 4. Kinetics recording the surface behaviour of APP at the phos-
phatidylcholine–water interface. (A) p–t curves of APP inserted into the
PtdCho monolayer under different bulk concentrations of the protein
(buffer:Tris/HCl20m
M
, NaCl 150 m
M
,pH7;Teflontrough19mL,
9cm
2
). (B) p–t curves of APP inserted into the PtdCho monolayer
under different salt concentration (buffer: Tris/maleate 20 m
M
,pH7;
Teflontrough4mL,2cm
2
). (C) p–t curves of APP inserted into the
PtdCho monolayer under different pH (buffer: Tris/maleate 20 m
M
,
NaCl 150 m
M
; Teflon trough 4 mL, 2 cm
2
).
2242 R. Lahdo et al. (Eur. J. Biochem. 269) Ó FEBS 2002
of APP in 10 m
M
NaH
2

DISCUSSION
In order to get insight into the role of lipids in APP
processing we have characterized its association with
artificial lipid vesicles or lipid monolayers. APP was able
to interact with the membranes of liposomes, although
incomplete APP incorporation into liposomes could be
observed with b-OG-mediated reconstitution. The nonionic
detergent b-OG was used because it is a nondenaturating
detergent with a high critical micellar concentration
(20–25 m
M
). Therefore, it can be rapidly removed by
dialysis [33,36]. The low incorporation rates observed in
b-OG-mediated reconstitution were, however, in the same
range as those observed by Lin et al.[26]forAb incorpor-
ated into liposomes using the sonification technique. The
ability of APP to insert into neutral lipid vesicles was
dependent on pH. Indeed, the binding was greater at low
pH, when the net charge of the protein is close to a nil value,
than at neutral pH, where APP is in an anionic form. It is
more likely that a low pH is required for the protonation of
the negatively charged carboxyl groups of aspartic or
glutamic acids, whose pK values are near 4. The protona-
tion of the carboxyl functions reduce significantly the
repulsion between the negatively charged groups on the
membrane and the negatively charged amino acids present
on APP. This requirement for a low pH suggests that
electrostatic repulsion prevents the protein–membrane
association. Furthermore, no incorporation was obtained
in the presence of negatively charged lipids in the mem-

tions used in this study, the interaction of APP with a
neutral lipid monolayer is not dependent on the bulk pH, as
it was observed for binding experiments into liposomes.
These results may be explained by an effect of local
interfacial pH [41]. Therefore, alteration of the charge of the
protein could be induced at the vicinity of the monolayer. A
difference in the orientation of the protein moiety during the
insertion process into the monolayer film or into the bilayer
containing the surfactant is also possible. The lower surface
activity obtained with the PtdSer monolayers could be the
result, as presented before, of an electrostatic repulsion
effect between lipids and the protein at physiological pH.
Anionic lipids, which represent % 20% of biological
membrane lipids, provide either a source of electrostatic
Fig. 6. CD of APP. (A) CD spectra of APP in phosphate buffer 10 m
M
pH 7 as a function of salt concentration (—–, No NaCl; – ) –150m
M
NaCl;- ,500m
M
NaCl). (B) CD spectra of APP in phosphate buffer
10 m
M
NaCl 150 m
M
asafunctionofpH(—–,pH7;––––,pH6,
– ) –, pH 5; - - -, pH 4).
Ó FEBS 2002 Interaction of amyloid precursor protein with lipids (Eur. J. Biochem. 269) 2243
attractions for the binding of proteins to membranes or a
source of repulsive effects [42–44].

at pH 4 suggested an aggregation and/or precipitation of
the protein. The possibility that the lower surface activity of
APP observed at pH 4 could be due to a lower available
amount of the protein cannot be ruled out. No loss of
tertiary structure, as measured by intrinsic fluorescence,
indicates that the protein does not undergo major unfolding
during acidification or upon variation of ionic strength
(data not shown). Whether minor conformational modifi-
cations of APP, such as local variations in secondary or
tertiary structures in defined regions of the protein, are
associated with its lipid binding ability remains to be
determined. Finally, we have considered the possibility that
the pH-induced conformational transition may lead to
changes in the protein quaternary structure. We have used
gel filtration chromatography to test this hypothesis, and
have obtained no evidence for a different structure at
pH 4–7 (data not shown). The APP is apparently organized
as a trimer in the conditions used for these studies. It was
recently reported that cellular APP can form noncovalent
homodimers and tetramers [45].
The molecular events which guide protein interaction at
the membrane surface or insertion into lipid vesicles are
important in order to shed light on the toxicity mechanism
of Ab which may be based in part on perturbations of the
lipid–water interface. In vivo,productionofAb is believed to
occur through sequential cleavage of APP by b-and
c-secretases [46]. Cell biological studies suggest three
potential locations for intracellular b-secretase activity:
endosomal compartments at mildly acidic pH, Golgi-
derived vesicles and endoplasmic reticulum/intermediate

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