(Received for publication, August 22, 1996)
From the Polymerization of Alzheimer amyloid The Alzheimer amyloid Several studies suggest that Sf9 cells were grown in
Grace's supplemented insect medium with 10% fetal bovine serum (Life
Technologies, Inc.) to a density of 106 cells/ml (in a
total volume of 500 ml in a 1,000-ml spinner flask). The cells were
then infected with recombinant baculovirus containing the C100 gene
(generously provided by Dr. Rachael Neve, Harvard University. Four days
after infection, the cells were centrifuged at 1,000 × g for 10 min. The pellet was washed with phosphate buffer,
pH 6.2, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged again. The harvested cells were stored at
Infected and
harvested cells (one vial, 108 cells) were homogenized in a
Dounce homogenizer with 2 ml of 5 mM Hepes buffer, pH 7.4, containing 1 mM PMSF. The homogenate was centrifuged at 500 × g for 5 min. The supernatant was loaded on a
10-30% sucrose gradient with 5 mM Hepes, pH 7.4, and
centrifuged in a swinging bucket rotor (Beckman SW 40 Ti) at
200,000 × g for 16 h. Twelve fractions, 800 µl
each, were aspirated from the top of the tubing. A 100-µl aliquot of
each fraction was subjected to SDS-PAGE and immunoblotting.
Infected and harvested cells (two
vials, 2 × 108 cells) were homogenized in a Dounce
homogenizer with 2 ml of 5 mM Hepes buffer containing 1 mM PMSF. The homogenate was centrifuged at 20,000 × g for 10 min. The pellet was triturated in 1 ml of 5 mM Hepes buffer with 1 mM PMSF. A 23%
sucrose/5 mM Hepes solution was underlaid the homogenized
pellet. Centrifugation was performed at 125,000 × g
for 1.5 h in a fixed angle rotor (Beckman TLA 45). The resulting pellet was extracted with 300 µl of methanol/200 µl of chloroform for 1 h. After addition of 100 µl of methanol, the sample was centrifuged for 30 min at 125,000 × g. The pellet was
extracted overnight with 200 µl of 70% formic acid with continuous
stirring. The sample was centrifuged for 30 min at 125,000 × g, and the supernatant was injected on a Superose 12 size
exclusion column and eluted with 70% formic acid. Fractions were
collected and analyzed by SDS-PAGE and immunoblotting using 6E10 and
369 as primary antibodies. The fractions with the highest content of C100 were pooled and aliquoted prior to evaporation of the mobile phase
in a vacuum centrifuge.
Purified C100 was
incubated in Tris-buffered saline (TBS) containing 150 mM
NaCl and 50 mM Tris-HCl, pH 7.4, at 37 °C for 96 h.
The incubated fractions were digested with 0, 10, 100, or 1000 µg of
proteinase K/ml for 5 h. Purified C100 was also incubated for 0, 4, 48, and 170 h prior to digestion with proteinase K. In the same
experiment, polymerized C100 (48 h incubation) was dissolved in 70%
formic acid with continuous stirring for 2 h, lyophilized, and
digested with proteinase K (100 µg/ml). The polymerized and digested
samples were centrifuged at 22,000 × g, and 170 µl of the supernatant was replaced with an equal volume of 2 mM PMSF, 9 M urea, and 50 mM Tris,
pH 10. After 3 days with continuous stirring, the samples were analyzed
by SDS-PAGE and immunoblotting.
The
fractions from the gradient centrifugation with the highest C100
content, corresponding to 23-30% sucrose, were diluted with TBS, pH
7.4, and centrifuged at 100,000 × g for 1 h to
remove the sucrose. The pellet was suspended in the same buffer,
aliquoted, and incubated at 37 °C in a shaking water bath overnight.
Two volumes of either trypsin (150 µg/ml in 50 mM
ammonium bicarbonate buffer, pH 7.8), or proteinase K (50 µg/ml in 50 mM TBS, pH 7.4) were added to the samples. After 5 h
at 37 °C, the samples were analyzed by SDS-PAGE and immunoblotting.
A synthetic peptide corresponding to amino acids 48-99
in C100 was a gift from Dr. Andrew J. Czernik (Rockefeller University). The peptide was incubated at 100 µM in TBS, pH 7.4, for 1 week. Proteinase K was added to final a concentration of 50 µg/ml.
After 5 h at 37 °C, the samples were analyzed by SDS-PAGE and
immunoblotting.
Gradient gels, 5-18%, were
run in a Tris-Tricine buffer system (17). Laemmli sample buffer
containing 6 M urea was added to the samples. After boiling
for 5 min, 80 µl of sample was applied to the gel. After 3.5 h
at 70 mA, the gel was either stained with Coomassie Brilliant Blue or
electroblotted overnight to a nitrocellulose membrane (0.2-µm pore
size, Schleicher & Schuell). The membranes were incubated with
monoclonal antibodies 6E10 and 4G8 (mouse), directed against residues
5-10 and 18-21 in A To determine the intracellular
localization of C100, infected cells were fixed in 2%
formaldehyde/0.1% glutaraldehyde in phosphate-buffered saline, pH 7.3, dehydrated in graded ethanol, and embedded in LR White. Thin sections
were immunogold stained with 369 as primary antibody and anti-rabbit
IgG conjugated to 10 nm colloidal gold particles as secondary antibody.
Purified C100 was incubated (96 h) as above. One sample was digested
with proteinase K (50 µg/ml) for 5 h, and one was not digested.
The samples were centrifuged for 1 h at 125,000 × g, and the pellet was triturated in water (Milli-Q, Waters).
A 5-µl sample of this suspension was placed on a grid covered by a
carbon-stabilized Formvar film. After 0.5-1 min, excess fluid was
adsorbed on a filter paper, and the grids were air dried. Negative
staining was made with 2% uranyl acetate in water. For immunostaining,
the grids were incubated on droplets of Tris-buffered saline, pH 7.4, containing 0.1% bovine serum albumin to block nonspecific binding. The
samples were then exposed to primary antibodies 6E10 and 369, washed,
and exposed to secondary antibodies conjugated to colloidal gold
particles of different sizes (anti-mouse IgG conjugated to 10 nm of
colloidal gold particles and anti-rabbit IgG conjugated to 20 nm of
colloidal gold particles). Both primary and secondary antibodies were
dissolved in TBS, pH 7.4, with 0.1% bovine serum albumin, which also
was used to wash the samples after exposure to antibodies.
CHO cells were stably transfected with
Previously, it was shown
that polymerization of synthetic A Purified C100 was allowed to polymerize by incubation in TBS for
96 h before the addition of proteinase K, an enzyme capable of
cleaving most peptide bonds (19). The samples were then subjected to
SDS-PAGE, followed by immunoblot analysis using 6E10 or 369 as primary
antibody. The 6E10 epitope is located in the N terminus (amino acids
5-10), and the 369 epitopes are located in the cytoplasmic domain
(amino acids 70-75 and 90-95) of C100. Polymerized C100 could not be
completely dissolved under the conditions used, although the bulk of
the material migrated as a monomer at ~12 kDa (calculated molecular
mass, 11.3 kDa). In addition to this band, a number of high molecular
mass species, corresponding to polymers of various sizes, were seen
(Fig. 1, lane 1). The antibodies used bind
with higher avidity to polymers than to monomers, and the amount of polymers is therefore likely to be overrepresented in the
immunoblots.3 Proteinase K (10 µg/ml)
treatment of polymerized C100 cleaved the protein and generated several
fragments with molecular masses lower than that of native C100 (Fig. 1,
lane 2). Addition of higher concentrations of proteinase K
yielded a 6E10-reacting species with a molecular mass indistinguishable
from that of synthetic A
In
the next series of experiments, it was investigated whether
polymerization of C100 is required for the generation of A
Purified and polymerized C100, digested with proteinase K
or nondigested, were adsorbed to grids, negatively stained with uranyl
acetate, and examined by electron microscopy. Like A
The intracellular localization of C100 in Sf9 cells infected with
recombinant baculovirus containing DNA coding for C100 was examined
with immunogold staining of thin sections of fixed and embedded cells.
The bulk of the immunoreactivity was confined to a complex system of
membranes found in both the cytoplasm and the nucleus (Fig.
3E), indicating that recombinant C100 is indeed inserted in
membranes.
Like polymerized synthetic A
It was then examined whether C100 inserted in the membrane of
transfected cells can serve as a substrate for the generation of A
To investigate the role of the cytoplasmic domain of C100
in fibril formation, a synthetic peptide corresponding to this part of
C100 was incubated under the same conditions as purified C100. When the
incubated peptide was analyzed by electron microscopy, fibrils similar
to those generated from A To investigate whether C100
generated from full-length
Here, we have presented evidence that A C100, a metabolic fragment generated through Polymerization of synthetic A
It was also demonstrated that most of the C100 produced by
An important and obvious question is whether A Apparently, nonspecific protease activity cleaving any peptide bond can
serve as The mechanism for A
Laboratory of Biochemistry and Molecular
Pharmacology,
Department of
Cell and Molecular Biology, Medical Nobel Institute, Karolinska
Institute, S-171 77 Stockholm, Sweden and ** Department of Neurology
and Neuroscience, Cornell University Medical College,
New York, New York 10021
peptide
(A
) into amyloid fibrils is associated with resistance to
proteolysis and tissue deposition. Here, it was investigated whether
A
might be generated as a protease-resistant core from a polymerized
precursor. A 100-amino acid C-terminal fragment of the Alzheimer
-amyloid precursor protein (C100), containing the A
and
cytoplasmic domains, polymerized both when inserted into membranes and
after purification. When subjected to digestion using the nonspecific
enzyme proteinase K, the cytoplasmic domain of C100 was degraded,
whereas the A
domain remained intact. In contrast, dissociated C100
polymers were almost completely degraded by proteinase K. Mammalian
cells transfected with the human Alzheimer
-amyloid precursor
gene contained a fragment corresponding to C100, which needed similar harsh conditions to be dissolved, as did polymers formed by purified C100. Hence, it was concluded that C100 polymers are formed in mammalian cells. These results suggest that the C terminus of A
can
be generated by nonspecific proteases, acting on a polymerized substrate, rather than a specific
-secretase. This offers an explanation of how the A
peptide can be formed in organelles containing proteases capable of cleaving most peptide bonds.
peptide
(A
)1 is the primary constituent of the
amyloid deposited in the brain parenchyma and blood vessels of the
brain in association with Alzheimer's disease (AD) (1-4). A
is
generated through proteolytic processing of Alzheimer
-amyloid
precursor protein (
-APP), a transmembrane protein expressed in most
mammalian cells (5). Much interest has been focused on the metabolic
mechanisms generating A
(6).
-APP can be metabolically processed
by at least two pathways. The
-secretase pathway cleaves
-APP in
the central section of the A
domain (7, 8) and thereby precludes
amyloidogenesis, whereas the
-secretase pathway generates the free N
terminus of intact A
(6). The C-terminal
-APP fragment containing
the intact A
and cytoplasmic domains will hereafter be referred to
as C100 (9). Subsequently, both pathways converge, and the
-APP
metabolites are cleaved by a protease activity, tentatively named
-secretase, generating the free C terminus of A
and a related,
nonamyloidogenic fragment, p3 (10, 11). The enzyme or enzymes
catalyzing
-secretase cleavage have not yet been identified. The
enzymatic characteristics of
-secretase activity include a certain
degree of nonspecificity, since the
-APP fragment can be cleaved
after either amino acid 40 or amino acid 42 of the A
domain (6). In
either case, the cleavage site is located within the predicted
transmembrane region of
-APP (5) and should therefore be protected
by the phospholipid bilayer, raising the possibility that this cleavage
may not occur on the C100 substrate as long as it is
membrane-inserted.
-secretase activity is localized to an
endosomal or lysosomal compartment (12-14). Lysosomes contain a
multitude of proteolytic enzymes (15) and therefore it seems unlikely
that C100 in endosomes or lysosomes will encounter protease activities
with defined and narrow substrate specificities (i.e. a
specific
-secretase). Instead, C100 will be present in an
environment of enzymes cleaving many or most peptide bonds. In an
earlier article, it was reported that A
acquires protease resistance
in association with polymerization (16). It is therefore possible that
the A
domains of several C100 molecules can interact and generate a
"protease-resistant core," capable of withstanding the nonspecific
proteolytic environment of endosomes or lysosomes. In the present study
we have investigated whether purified and membrane-associated C100
polymers can serve as a substrate for the generation of A
by
nonspecific proteolysis, using recombinant C100 and proteases with
various substrate specificities.
Expression of C100 in Insect Cells
80 °C in aliquots of 108 cells/vial.
, respectively, and polyclonal antibody 369 (rabbit), recognizing epitopes located in the C terminus of
C100.2 Horseradish peroxidase-coupled
secondary antibodies and chemiluminescence detection (Amersham) were
used to visualize bound primary antibodies.
-APP751SW (18). Approximately 40 × 106
cells were homogenized in a Dounce homogenizer with 1 ml of 0.3 M sucrose in 20 mM Tris, pH 7.3. The homogenate
was centrifuged for 5 min at 500 × g. The supernatant
was centrifuged for 1.5 h at 125,000 × g in a
Beckman TLA rotor. The resulting pellet was suspended in 1% SDS in 20 mM Tris, 7.4, and the centrifugation was repeated. The
supernatant was lyophilized, and the pellet was delipidated with
methanol/chloroform. A solution of 9 M urea/100 mM Tris, pH 10, was added to the pellet. After 1 day with
continuous stirring, Laemmli sample buffer was added to the sample. The
lyophilized SDS extract was dissolved in Laemmli sample buffer
supplemented with urea. The samples were then subjected to SDS-PAGE
followed by immunoblot analysis with 6E10 or 369 as the primary
antibody.
Proteolytic Digestion of Polymerized C100 Yields Peptides
Indistinguishable from Synthetic A
into fibrils leads to resistance
to proteolytic degradation (16). We therefore investigated whether
purified C100, which readily polymerizes and forms fibrils (9, 28),
also develops protease resistance in association with fibril
formation.
(Fig. 1, compare lanes 3 and
4 with 5). The A
-specific antibody 4G8,
recognizing amino acids 18-21 in A
, yielded an essentially identical pattern (data not shown). Analysis with antibody 369 showed
that all material corresponding to the cytoplasmic domain of C100 had
been degraded by the proteinase K (100 and 1000 µg/ml) treatment
(data not shown).
Fig. 1.
Generation of A-like peptide from
polymerized C100 through nonspecific proteolysis. Identical
amounts of polymerized C100 were subjected to digestion with the
indicated concentrations of proteinase K (Prot. K) for
5 h at 37 °C before termination of the reactions with PMSF (1 mM) and urea (9 M). Synthetic A
1-40 was
loaded on lane 5 as a reference. The positions of the molecular mass standards (kDa) are shown to the left.
[View Larger Version of this Image (57K GIF file)]
in a
reaction catalyzed by a nonspecific protease. Purified C100 was
incubated in TBS at 37 °C for the indicated periods (Fig. 2). To determine whether the polymerization and protease
resistance is a reversible reaction, one polymerized aliquot was
treated with formic acid, which dissociates the fibrils, and
lyophilized prior to digestion. After addition of proteinase K, the
samples were incubated for 5 h. The reaction was then stopped, and
the samples were dissolved and analyzed by immunoblotting (Fig. 2). The
nondigested sample showed a smearlike pattern, indicating that the
fibrils had not been completely dissolved (Fig. 2, lane 1).
Nonpolymerized C100 was almost completely digested, and no A
-like
species were detected (Fig. 2, lane 3), indicating that monomeric C100 cannot serve as a substrate for the generation of A
by nonspecific proteolysis. This is in agreement with previous results
using monomeric A
as a substrate (16). Polymerized C100 that had
been treated with formic acid to dissociate the fibrils was not totally
digested, and a faint band in the A
region (~4 kDa) was detected
(Fig. 2, lane 2). The difference between this sample and the
nonpolymerized sample may be due to the fact that the fibrils formed
were not completely dissociated in formic acid. The polymerized and
proteinase K-treated samples gave a strong signal in the A
region,
i.e. ~4 kDa (Fig. 2, lanes 4 and 5).
Fig. 2.
Effect of C100 polymerization on the
generation of an A-like peptide. Purified C100 was allowed to
polymerize at 37 °C for the indicated periods. One sample was not
digested (lane 1). One sample was treated with formic acid
to dissociate the fibrils prior to digestion (lane 2). This
sample, a nonpolymerized sample (lane 3), and polymerized
samples (lanes 4 and 5) were treated with
proteinase K (100 µg/ml) for 5 h at 37 °C. The positions of
the molecular mass standards (kDa) are shown to the
left.
[View Larger Version of this Image (72K GIF file)]
, C100 formed
fibrils with a diameter of about 5-8 nm, typically arranged in tight
bundles (Fig. 3, A and B).
Immunogold staining, with 6E10 and 369 as primary antibodies, revealed
that the fibrils contained epitopes located both in the A
and the
cytoplasmic domains of C100 (Fig. 3D). Thus, it was
concluded that the fibrils consisted of intact C100. Electron
microscopic examination of the proteinase K-treated C100 fibrils
revealed a fibrillar structure, indistinguishable from that of genuine
A
(Fig. 3, compare A and C). Moreover,
immunogold staining revealed that the proteinase K-treated C100 fibrils
remained reactive with 6E10 but not with 369 (data not shown). Hence,
it is concluded that nonspecific proteolysis of C100 can generate an
A
species electrophoretically, immunologically, and morphologically
equivalent to purified A
.
Fig. 3.
Electron microscopic examination of fibrils
and infected Sf9 cells. A, fibrils of A purified from
amyloid extracted from brain tissue, negatively stained with uranyl
acetate. B, polymerized C100, stained as in A.
C, polymerized, proteinase K-digested C100, stained as in
A. D, immunogold staining of C100 fibrils with
the A
-specific antibody 6E10 (10 nm gold) and antibody 369, which is
specific for the C terminus of C100, (20 nm gold). E,
infected Sf9 cells. Sf9 cells infected with recombinant baculovirus containing the C100 DNA-fragment were fixed in 2% formaldehyde/0.1% glutaraldehyde in phosphate-buffered saline, pH 7.3, dehydrated in
graded ethanol, and embedded in LR White. Thin sections were immunostained using 369 as the primary antibody and anti-rabbit IgG
conjugated to 10 nm colloidal gold particles as the secondary antibody.
Labeled membrane vesicles and fragments are seen in a cell in an early
lytic phase of virus infection. Bars, 100 nm.
[View Larger Version of this Image (110K GIF file)]
(16), the A
region
of polymerized C100 was apparently partly resistant to proteolytic
degradation. An intriguing possibility was therefore that C100 inserted
into cell membranes can aggregate and thereby develop protease
resistance. It was therefore investigated whether membrane-inserted
C100 can polymerize, similar to the purified protein. For this purpose, a membrane preparation from Sf9 cells, infected with
baculovirus-containing C100 DNA, was subjected to SDS-PAGE and
immunoblot analysis with antibodies 6E10 and 369. Purified, monomeric
C100 was loaded on the gel as a reference. The C100 protein from a
crude membrane preparation displayed a smearlike pattern, strongly
suggesting that C100 had formed polymers of different sizes (Fig.
4, lane 1), whereas the purified protein
migrated as a single species with an apparent molecular mass of ~12
kDa (calculated molecular mass, 11.3 kDa) (Fig. 4, lane 2).
It is unlikely that the polymers were formed after the addition of
sample buffer, which contained a highly denaturing chaotrope (urea) and
detergent (SDS), since purified monomeric C100 did not form polymers
when it was analyzed under the same conditions. Hence, it is concluded
that C100 can polymerize while still inserted in cell membranes.
Fig. 4.
C100 can polymerize in cell membranes. A
membrane preparation from Sf9 cells infected with recombinant
baculovirus containing C100 DNA (lane 1) and C100 purified
by size exclusion chromatography (lane 2) were dissolved in
Laemmli sample buffer and subjected to SDS-PAGE and immunoblot analysis
with antibody 6E10. The positions of the molecular mass standards (kDa)
are shown to the left.
[View Larger Version of this Image (30K GIF file)]
,
analogous to purified and polymerized C100. A membrane fraction from
Sf9 cells infected with recombinant baculovirus containing C100 DNA was
incubated overnight in TBS with or without the zwitterionic detergent
CHAPS. CHAPS was added to dissolve potential membrane vesicles, thereby
making both the N and C termini of C100 accessible to the protease. The
CHAPS molecules act by hydrophobic interaction with the transmembrane
region of C100, possibly substituting for the phospholipid bilayer, and
the transmembrane region is thereby still shielded from proteases.
After digestion with proteinase K for 5 h, the samples were
analyzed by immunoblotting (Fig. 5). As seen in Fig. 5,
lane 3, a fragment with a slightly higher apparent molecular
mass than A
, ~6 kDa, was formed. The addition of CHAPS did not
alter the apparent molecular mass of the proteolytic fragment generated
by proteinase K (Fig. 5, lanes 2 and 4). A
cleavage at lysine 53 (which is the first amino acid residue C-terminal
to the predicted transmembrane region of C100) would generate a 5.9-kDa
fragment. The detected species corresponds in molecular mass and
immunoreactivity to such a fragment, indicating that the A
domain of
C100 can form a protease-resistant core while still inserted in
membranes, and that the transmembrane region is shielded from
proteolysis by phospholipids and CHAPS. The experiment was repeated
using trypsin instead of proteinase K. Trypsin cleaves C-terminally to
lysine and arginine residues, a putative cleavage site being lysine 53. A cleavage after this residue should generate a 5.9-kDa fragment,
provided that putative tryptic cleavage sites within the A
domain of
C100 (i.e. Arg5, Lys16, and
Lys28) were protease resistant. A 6E10 immunoreactive
fragment with the same apparent molecular mass as the fragment
generated by proteinase K was detected (data not shown). It is
therefore concluded that C100 inserted in membranes can polymerize and
develop protease resistance, and that dissolution of the phospholipid
bilayer is necessary to generate the free C terminus of A
.
Fig. 5.
A can be generated from C100 inserted in
membranes. Membranes from Sf9 cells infected with recombinant
baculovirus containing the C100 gene were subjected to treatment with
proteinase K (50 µg/ml) for 5 h at 37 °C in the presence or
absence of CHAPS (1%) and thereafter analyzed by SDS-PAGE and
immunoblotting with antibody 6E10. The positions of the molecular mass
standards (kDa) are shown to the left.
[View Larger Version of this Image (28K GIF file)]
and C100 were found (data not shown).
Thus, the cytoplasmic domain of C100 may be involved in the
polymerization of C100, since this region can form fibrils per
se. After digestion with proteinase K, no immunoreactivity was
found when the sample was analyzed by SDS-PAGE followed by immunoblotting with 369 as a primary antibody. Apparently, the fibrils
formed from the cytoplasmic domain of C100 were, in agreement with our
other experiments, not protease-resistant.
-APP751SW Generate
SDS-insoluble C100 Fragments
-APP exists in an aggregated form, CHO
cells were stably transfected with
-APP751SW (18). The
Swedish mutation was chosen, since it produces approximately five times
more A
than the wild-type gene and, according to the present
hypothesis, therefore should produce more C100 polymers. A postnuclear
supernatant from CHO cells, stably transfected with
-APP751SW, was centrifuged at 125,000 × g for 1 h. The resulting pellet was extracted with 1% SDS in TBS and centrifuged once more. The supernatant was lyophilized, and the pellet was dissolved in 9 M urea at pH 10 for
24 h. After addition of urea to the SDS-extracted sample and SDS
to the urea-extracted sample, Laemmli sample buffer was added, and the
samples were subjected to SDS-PAGE. Immunoblot analysis, using 6E10 or
369 as primary antibody, showed a remarkable difference between the urea-extracted sample and the SDS extract (Fig. 6). No
immunoreactivity could be detected in the SDS extract, either with 6E10
or with 369 as the primary antibody. The urea treated sample displayed, with 6E10 as the primary antibody, one intense band in the C100 region.
The same band was detected with 369, suggesting that the detected
species corresponds to C100. A fragment with slightly lower molecular
mass was also detected with antibody 369, probably corresponding to the
C-terminal fragment of
-APP generated by
-secretase cleavage, the
Esch fragment (7). The absence of immunoreactive fragments in the SDS
extract indicates that C100 generated from
-APP751SW is
tightly bound and, in accordance with our earlier results, probably
occurs as a polymer.
Fig. 6.
Extraction of C100-like material from
mammalian cells. Left panel, CHO cell membranes extracted
with urea or SDS as described under "Experimental Procedures" and
purified C100 were separated by SDS-PAGE. Following electroblotting,
immunoreactive material was visualized using the A region-specific
antibody 6E10. Right panel, the same samples as in
A, but here immunoreactivity was visualized using antibody
369, which is specific for the cytoplasmic region of
-APP.
Filled arrowhead, C100; open arrowhead, the Esch fragment.
[View Larger Version of this Image (29K GIF file)]
can be generated from
polymerized, but not from nonpolymerized, C100 by the nonspecific proteolytic activity of proteinase K.
-secretase cleavage of
-APP (13, 20-23), can polymerize and form fibrils similar to those
of A
(24). In our experiments, polymerization occurred both when the
protein was inserted in cell membranes and after purification of the
protein. A reasonable explanation is that C100 polymerizes through
interaction between A
domains, since A
readily forms fibrils
(25). The cytoplasmic domain of C100 also forms fibrils per
se, and this interaction may enhance the ability of C100 to form
fibrils.
is associated with increased
resistance of the molecule to proteolytic enzymes with varying substrate specificities in vitro (16). It has also been
shown that aggregates of synthetic A
1-42 internalized by cultured human skin fibroblasts are stable for several days and co-localizes with late endosomes and lysosomes (26). Therefore, we wanted to
investigate whether the A
domains in C100 could form
"protease-resistant cores" through a similar mechanism.
Intracellular proteolysis in endosomes and lysosomes involves several
endoproteases and exoproteases with various substrate specificities
(15, 27). Since it would be difficult to mimic this combination of
enzymes in a reconstituted system, we decided to use proteinase K, a
bacterial protease with a wide substrate specificity, cleaving all
natural peptide bonds (19). This enzyme has previously been suggested to be capable of generating A
-like peptides from
-APP (28). Digestion of polymerized C100 with proteinase K rendered a fragment chromatographically, immunologically, and morphologically
indistinguishable from that of synthetic A
. These results
demonstrate that: (i) polymerization of C100 is associated with
formation of a protease-resistant core of tightly bound A
domains;
and (ii) an enzyme cleaving all natural peptide bonds can substitute
for the putative
-secretase. When C100 was digested in cell
membranes, in the presence or absence of detergent, a species with
slightly higher molecular mass than A
was formed. The transmembrane
domain of C100 is apparently shielded from proteolysis by phospholipids
or detergent. However, the phospholipid membrane can be degraded in
lysosomes, exposing the transmembrane domain of the protein to
proteases. The proposed model is described schematically in Fig.
7.
Fig. 7.
Proposed model of generation of the A
peptide in cells. When
-APP is cleaved by
-secretase, en
route to or at the plasma membrane, C100 is generated (1). A
fraction of these molecules bind to each other by interactions between
their A
domains and possibly also between their cytoplasmic domains.
Thereby, protease-resistant A
cores are formed (2). Bound
and free C100 molecules are transported to an organelle, in which they
are exposed to a multitude of proteolytic enzymes (3).
Nonresistant parts of the molecules are degraded (4),
whereas the protease-resistant A
cores remain intact and subsequently are secreted from the cells (5).
[View Larger Version of this Image (45K GIF file)]
-APP751SW-transfected CHO cells was SDS-insoluble. The
C100 produced by these cells could, like C100 polymers formed from
purified C100 or C100 in Sf9 cells, be dissolved in 9 M
urea at pH 10. The SDS insolubility is also characteristic for the A
fibrils found in Alzheimer's disease brains (29). These findings
indicate that the C100 molecules produced by the
-APP751SW-transfected cells are tightly associated,
probably in polymeric form. If present in human brain cells, such
polymers could be partially degraded, leaving a protease-resistant core
of polymeric or oligomeric A
.
can be formed through
nonspecific proteolysis not only in the artificial systems described
here but also in intact cells. A recent study (30) demonstrated that
A
secreted from cells was present as soluble oligomers of various
size. The oligomers were detectable after separation under denaturing
conditions in SDS-PAGE, indicating that they were joined by strong
bonds. It can be speculated that the oligomers had been formed prior to
-secretase cleavage and secretion in a reaction similar to the one
described here, rather than from monomeric A
in the cell media. When
-APP-transfected human kidney cells are treated with aggregated A
1-42, the accumulation of stable, insoluble amyloidogenic aggregates
is stimulated (31). One possible explanation to this finding is that
the A
domains in endogenously produced C100 bind to internalized
A
1-42 and thereby form protease-resistant cores. The fact that an
intact fragment corresponding to C100 lacking the A
domain has yet
not been detected in cells may also be an argument against the idea that the
-secretase is a specific enzyme (see Ref. 21).
-secretase, if the substrate has the right higher order
structure. This model suggests that A
can be formed in organelles
such as endosomes and lysosomes, containing a multitude of proteases
covering a broad spectrum of substrate specificities (15, 27). Low pH
promotes formation of A
fibrils in vitro (32). Through a
similar mechanism, the acidic environment in late endosomes and
lysosomes may enhance the formation of C100 fibrils with a
protease-resistant core corresponding to A
.
formation proposed here may have pharmacological
implications. It has been suggested that protease inhibitors capable of
inhibiting
- and
-secretase activity may be used to reduce A
production in vivo and, therefore, are candidate drugs for
the treatment of Alzheimer's disease-associated amyloidosis (3).
Considering the present model, it would not be possible to find a
selective
-secretase inhibitor. Alternatively, a molecule capable of
antagonizing C100 polymerization would prevent the generation of a
protease-resistant core of A
, thereby preventing amyloidogenesis in
Alzheimer's disease.
*
This study was supported in part by grants from the Swedish
Medical Research Council (to J. T. and L. T.) and the Swedish Heart
Lung Foundation (to J. T.), USPHS Grant AG11508 (to S. E. G.), and
the King Gustaf V 80th Birthday Fund (to J. T.). 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.
Recipient of fellowships from the Axel Margret Ax:son Johnson
Foundation, the Jeansson Foundation, the Sandoz Foundation, and the
Nicholson Foundation.
¶
Supported in part by a fellowship from The Swedish Society for
Medical Research. Present address: Laboratory of Molecular and Cellular
Neuroscience The Rockefeller University 1230 York Ave., New York, NY
10021.
§
To whom correspondence should be addressed. Tel.: 46-8-729-46-02;
Fax: 46-8-34-19-39; E-mail: tjern{at}ian.ks.se.
1
The abbreviations used are: A, Alzheimer
amyloid
-peptide;
-APP, Alzheimer
-amyloid precursor protein;
C100, C-terminal 100 amino acids of
-APP; TBS, Tris-buffered saline;
PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel
electrophoresis; CHO, Chinese hamster ovary; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2
F. Lindqvist, J. Georgieva, L. O. Tjernberg, C. Lilliehöök, S. E. Gandy, L. Terenius, and C. Nordstedt,
manuscript in preparation.
3
L. O. Tjernberg J. Näslund, and C. Nordstedt, unpublished observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.