From the Mayo Clinic, Jacksonville, Florida 32224
Received for publication, July 7, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Alzheimer's disease is characterized by the
deposits of the 4-kDa amyloid The A The secretases that ultimately cleave APP to A The final step in A Materials--
All buffers and reagents were obtained from VWR
Scientific, Sigma, Life Technologies, Inc., or Fisher Scientific unless
otherwise stated. Guinea pig brain and cow brain were obtained from
Cocalico Biologicals Inc. Fetal calf serum and newborn calf serum were obtained from HyClone. Bis-Tris precast gels, MES buffer concentrate, and molecular weight markers were from Invitrogen Corp.
Maleimide-activated keyhole limpet hemocyanin (KLH), Supersignal West
Pico substrate, the BCA protein assay kit, and Brij 35 were from
Pierce. The previously reported substrate-based difluoroketone
inhibitor
(t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2,2-difluoropentanoyl-Val-Ile-OMe) of Antibodies, Peptides, and Standards--
Antibodies against
marker proteins syntaxin-6 (43) (Transduction Laboratories) and
synaptophysin (44) (Roche Molecular Biochemicals) were kind gifts from
Drs. Sevlever (Mayo Clinic, Jacksonville, FL) and Lahiri (Indiana
University, IN). The anti-cross-reacting determinant (anti-CRD)
antibody (Oxford Glycosciences) reacts with a neoepitope generated
after the cleavage of GPI-anchored proteins by PI-PLC. Pf998, a
rabbit polyclonal antibody against the juxtamembrane cytoplasmic domain
of APP (726-744 of APP770) and the synthetic C-terminal 57 residues of
APP (CTF
The rabbit antibody O443 was raised against a maleimide-activated
KLH-conjugated synthetic peptide (CKMQQNGYENPTYKFFEQMQN) prepared at
the Mayo Clinic Protein Core Facility, which corresponds to the
C-terminal 20 residues of APP. The animal care, injections, and bleeds
were carried out by Cocalico Biologicals, Inc. (Animal approval number
A3669-01), and the sera were characterized in our laboratory. The
antibody detects less than 0.1 fmol (0.6 pg) of synthetic CTF
The LC99 construct in the pCEP4 vector consisting of the signal
sequence of APP fused to its C-terminal 99 residues, starting at the
A Preparation and Incubation of Active Membrane
Fractions--
Brains were minced and homogenized for 1 min on ice in
10 volumes of buffer A (50 mM HEPES, 150 mM
NaCl, and 5 mM EDTA, pH 7.4). All subsequent steps were
carried out at 4 °C unless otherwise indicated. Homogenates were
sequentially fractionated by centrifugation at 2500 × g for 15 min to collect unbroken cells and nuclei (F1P), and
the post-nuclear supernatant was spun at 10,000 × g
for 15 min (F2P) followed by 100,000 × g for 1 h
(F3P). The supernatant (F3S) was discarded, and the three pellets were
washed once in buffer A and resuspended in 200 µl/g initial brain
weight of buffer B (50 mM HEPES buffer, 150 mM
NaCl, pH 7.0). For A
To determine the pH optimum of fragment generation, aliquots of F2P
were resuspended and incubated either in 50 mM sodium citrate, 150 mM NaCl, pH 5.0-6.5, buffer B, pH 7.0-7.5,
or 50 mM Tris, 150 mM NaCl, pH 8.0-9.0. To
make sure that the alternative buffers were not interfering with the
assay, we also carried out the pH optimum analysis in buffer B adjusted
to the various pH levels and essentially obtained the same results
(data not shown).
Sucrose Density Gradient Fractionation of F2P--
Step
gradients were set up using 25, 27.5, 30, 32.5, 35, 37.5, and 40%
sucrose in buffer A containing 5 mM PNT in 7 layers of 0.6 ml each. The F2P obtained from 0.75 g of guinea pig brain was
resuspended in buffer A containing 20% sucrose, overlaid onto the
gradient, and centrifuged for 20 h at 48,000 rpm in a Beckman SW
50.1 rotor. Eight fractions (0.6 ml) were collected from the top of the
gradient and diluted 5-fold in buffer A. Membrane pellets were
collected by centrifugation at 100,000 × g for 1 h and then resuspended in equal volumes of buffer B. The protein
concentration peaked in fractions 5 and 8. Fractions 1 and 2 containing
low levels of protein were pooled for further analysis. The fractions were incubated, and the supernatants were analyzed for the release of
CTF
The membrane pellets from the fractions were analyzed for the presence
of marker proteins. Markers of the Golgi, syntaxin-6 (31 kDa), synaptic
vesicles and synaptophysin (38 kDa), were readily identified by their
size. The anti-CRD antibody detects multiple GPI-anchored proteins as
broad patches of ~38 and 62 kDa. The bands were considered as
specific as they were only detected after PI-PLC treatment (data not shown).
Immunoassays--
Western blotting and immunoprecipitation were
carried out as described previously (46), except that Hammerstein grade
casein (1%) in 25 mM Na2HPO4, 0.2 M NaCl, pH 7.0 was used as a blocking agent for some of the
antibodies, and Bis-Tris gels with the MES running buffer system was
used for protein separation. Where relevant, scanned images were
quantified using the ImageQuant software (Molecular Dynamics). For
comparison of treatments between experiments, treated samples were
compared with controls adjusted to 100%. A Generation of a Novel C-terminal APP Fragment from Guinea Pig Brain
Membranes--
To assay for
We also measured the levels of A
To determine the stability of CTF
We attempted to determine the cleavage site(s) that generate CTF Caspase Inhibitor Does Not Reduce CTF CTF
The active F2P was further fractionated by centrifugation to
equilibrium in a sucrose step gradient ranging from 20 to 40% sucrose
as described above. The eight fractions were examined for Solubilization of the Active The current study reports a novel An in vitro assay for Subcellular fractionation studies based on the in vitro
generation of CTF Although high levels of A Recent reports show that PS1 specifically binds a Although presenilins have been identified as being essential for
Since the generation of CTF peptide (A
). The A
protein
precursor (APP) is cleaved by
-secretase to generate a C-terminal
fragment, CTF
, which in turn is cleaved by
-secretase to generate
A
. Alternative cleavage of the APP by
-secretase at A
16/17
generates the C-terminal fragment, CTF
. In addition to A
,
endoproteolytic cleavage of CTF
and CTF
by
-secretase should
yield a C-terminal fragment of 57-59 residues (CTF
). However,
CTF
has not yet been reported in either brain or cell lysates,
presumably due to its instability in vivo. We detected the
in vitro generation of A
as well as an ~6-kDa fragment
from guinea pig brain membranes. We have provided biochemical and
pharmacological evidence that this 6-kDa fragment is the elusive
CTF
, and we describe an in vitro assay for
-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF
fragment. Three compounds previously
identified as
-secretase inhibitors, pepstatin-A, MG132, and a
substrate-based difluoroketone
(t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2,2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF
, providing additional evidence that the fragment arises from
-secretase cleavage. Consistent with reports that presenilins are the elusive
-secretases, subcellular fractionation studies showed that presenilin-1, CTF
, and CTF
are
enriched in the CTF
-generating fractions. The in vitro
-secretase assay described here will be useful for the detailed
characterization of the enzyme and to screen for
-secretase inhibitors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 that is
invariably deposited in Alzheimer's disease (AD) is a 38-43-residue
peptide derived from a larger precursor, APP, as summarized below (Fig.
1) (1-6). Normal neuronal cells constitutively secrete A
, which is
detected in cerebrospinal fluid and blood (7, 8). The length of most of
the secreted A
molecules is 40 residues (A
40) (9-11), but a
small fraction (~10%) is 42 residues long (A
42) (12). Mutations
in APP (12), presenilin-1 (PS1), and presenilin-2 (PS2) (13-15) linked
to familial AD (FAD) invariably increase the levels of either A
42
alone or both A
40 and A
42, providing evidence that it plays an
important causative role in the pathogenesis of AD (16, 17).
Furthermore, there is evidence that in typical late onset AD, as in
FAD, there are genetic determinants that increase levels of A
42
(18). To generate A
, APP is sequentially cleaved by proteases
referred to as
- and
-secretase as described below.
-Secretase
cleaves at the N-terminal end of the A
sequence, producing a
secreted derivative, sAPP
(19), and the A
-bearing C-terminal
fragment of 99 residues, CTF
, which is subsequently cleaved by
-secretase to release A
(2). An alternative activity,
-secretase, cleaves APP within A
(between residues 16 and 17) to
the larger secreted derivative, sAPP
(20, 21), and
membrane-associated 83-residue fragment, CTF
(22). Cleavage of
CTF
and CTF
by
-secretase generates A
and a smaller
fragment of 24-26 residues called P3, respectively (4). In addition,
-secretase cleavage should theoretically yield an ~6-kDa fragment
of 57-59 residues, CTF
(Fig. 1). Although the focus of the field
has been on A
, it is important to recognize that A
42 is naturally
linked to CTF
-57. Expression of CTF
was shown to be toxic to
neurons (23, 24), and more recently, it was suggested that the 31 C-terminal residues of APP are responsible for at least part of the
toxicity (25). Since CTF
can also serve as a precursor for this
toxic 31-residue peptide, its production and turnover are important
parameters that can influence the course of AD.
and CTF
have been
the subject of intense research as potential targets for the treatment
of AD. A novel pepstatin A-insensitive aspartyl protease, BACE/Asp2,
was recently identified as the
-secretase that cleaves at positions
1 and 11 of the A
sequence (26-29). In addition to BACE,
thimet oligopeptidase, a metalloprotease, was shown to be
involved in
-secretase activity in COS7 cells (30). We recently
showed that GPI-anchored proteins play an important role in
-secretase activity (31). Recently, a family of disintegrin
metalloproteases, the adamalysins such as tumor necrosis factor
-converting enzyme and ADAM 10, has been implicated in
-secretase
cleavage of APP (32-34). However, tumor necrosis factor
-converting
enzyme was shown to be specific for the phorbol ester-induced
-secretase activity (33). Although ADAM 10 appeared to be involved
in both constitutive and inducible pathways, inhibition of this
activity by a dominant-negative mutation indicated that it also plays a
more important role in the inducible
-secretase pathway (34).
biogenesis is
-secretase cleavage and is the
activity responsible for generating CTF
in vivo. This cleavage is particularly interesting as the scissile bond lies within
the transmembrane domain (35, 36). Since the discovery that a PS1
knockout mutant mouse was deficient in
-secretase activity, the
possibility was raised that PS1 and PS2 are the elusive
-secretases
(37). The finding that PS1 and PS2 show conserved and essential
aspartate residues within their respective transmembrane domains led to
the suggestion that they are unusual aspartyl proteases with active
sites within or close to the membrane (38). Recently, an in
vitro assay for
-secretase was described, and PS1 was found to
be a part of the active protease (39). By using this assay, a
biotinylated inhibitor was cross-linked to PS1 and PS2, showing that
the presenilins are indeed the active subunits of
-secretase (40,
41). However, the active enzyme was shown to be a large complex,
presumably with many unidentified subunits, of which some may be also
essential for
-secretase activity. To understand this interesting
cleavage event, it is important to tease out each component of the
-secretase and examine its individual role in detail by a
combination of biochemical and genetic methods. To address these
issues, it is necessary to have a robust in vitro
-secretase assay in both the membrane-associated and soluble states.
To develop such an assay, we incubated membranes from guinea pig and
cow brains as well as from cultured cells to look for production of
A
and CTF
. The CTF
fragment was detected in all systems
tested, but guinea pig brain was used because the entire APP sequence
is known, and the sequence of A
is identical to human allowing the
use of human-specific reagents for its analysis. After appropriate
fractionation, we observed a consistent time-dependent generation of a putative CTF
fragment from these membranes. In addition to describing a useful robust cell-free assay for
-secretase, this study presents the initial characterization of
CTF
from brain membranes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase that we are calling Wolfe-1 (42) was synthesized by Dr. Ziani-Cherif at the Mayo Clinic Chemical Core Facility. Additional reagents such as pepstatin A (Peptides International), 1,10-phenanthroline (PNT; J. T. Baker Co.), thiorphan (ICN),
phosphatidylinositol-specific phospholipase C (PI-PLC; Molecular
Probes), human recombinant caspase-3,
Z-Val-Ala-Asp(OMe)-CH2F (Z-VAD-FMK), MG132,
phenylmethylsulfonyl fluoride, E64, leupeptin,
N-acetyl-Leu-Leu-Nle-CHO (ALLN), CHAPSO, and Big CHAP
(Calbiochem) were purchased as indicated. The Chinese hamster ovary
(CHO) cell line, G9PLAP, expressing the human placental alkaline
phosphatase (PLAP) cDNA, a kind gift from Dr. Victoria Stevens
(Emory University, Atlanta, GA), was maintained in F12HAMS medium
supplemented with 10% fetal calf serum and 200 µg/ml geneticin (31).
-57) were kind donations from Pfizer. CTF
-57 includes 10 residues from the transmembrane domain and the entire 47-residue
cytoplasmic domain of APP and corresponds to one of the predicted
CTF
fragments. The monoclonal antibodies BNT77, BA27, and BC05
utilized for the quantification of A
40 and A
42 were kind gifts of
Dr. Nobu Suzuki and have been described previously (12).
by
Western blot analysis. The anti-PS1 antibody was a copy of that
reported by Duff et al. (45) and was prepared against a
KLH-conjugated synthetic peptide corresponding to residues 2-13 (CRKTELPAPLSYF).
sequence, was transfected into CHO cells (CHO C99) for use as a
standard for CTF
.
measurements, the F2P fraction was incubated in
buffer B supplemented with 5 mM EDTA, 5 mM PNT,
and 1 mM thiorphan. The G9PLAP cell line was similarly
prepared, except that the initial homogenate was directly centrifuged
at 10,000 × g for 15 min to obtain a F2P pellet that included membranes from F1P as well. For most assays, fraction F2P was
rehomogenized for 10 s and incubated at 37 °C for the indicated
time. The reactions were stopped by chilling on ice, and the membranes
were removed by centrifugation at 10,000 × g for 15 min unless otherwise specified. Finally, the supernatants were analyzed
for A
and the released C-terminal fragments of APP.
as described earlier.
was measured as reported
earlier by a specific and sensitive sandwich ELISA using a monoclonal
antibody BNT77 against A
11-28 for capture and horseradish
peroxidase-labeled end-specific antibodies BA27 (A
40) or BC05
(A
42) for detection (12, 31).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase activity, guinea pig brains
were homogenized and fractionated as described under "Experimental
Procedures" into F1P, F2P, and F3P and resuspended in equal volumes
of buffer B. Since the yield of membranes from CHO cells was low, these membranes were only fractionated into 10,000 and 100,000 × g pellets (F2P and F3P). Equal volumes of each fraction were
incubated for 0 and 2 h at 37 °C, chilled on ice, and
centrifuged at 100,000 × g to remove the membranes.
Supernatants were examined by Western blotting with the O443 antibody
raised against the C-terminal 20 residues of APP (Fig.
1). Western blots detected a soluble C-terminal fragment of ~6 kDa from the F1P and F2P of guinea pig brain and the F2P fraction of CHO cells, which increased considerably after 2 h at 37 °C (Fig.
2A). The fragment was not
detected upon incubation of F3P (Fig. 2A). For further
characterization, we immunoprecipitated the supernatant obtained from
the F2P fraction of guinea pig brain with Pf988, an antibody
against the juxtamembrane domain of APP (residues 726-744; Fig. 1),
and we analyzed the recovered APP C-terminal fragments by Western
blotting with O443 as above. The capacity to immunoprecipitate the
putative CTF
with Pf998 shows that it includes epitopes from
the juxtamembrane region on the cytoplasmic domain of APP (Fig.
2B).
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Fig. 1.
Major APP processing pathways.
The APP holoprotein is a type I integral membrane protein with a large
N-terminal extracellular domain, a single transmembrane domain, and a
short cytoplasmic tail. A depicted as a violet box is
partly extracellular (28 residues) and partly embedded within the
membrane (12-14 residues). Antibodies O443 and Pf998 are
against the final 20 residues and 19 juxtamembrane residues,
respectively. APP is cleaved to sAPP
and CTF
by
-secretase or
sAPP
and CTF
by
-secretase. The membrane-bound CTF
and
CTF
are further cleaved within the transmembrane domain by
-secretase (green arrow) to generate P3 and A
,
respectively. Most (90%) A
and P3 end at residue 40 and a small
fraction (5-10%) after residue 42 of the A
sequence. In addition,
-secretase cleavage should yield a C-terminal fragment, CTF
, of
57-59 residues.
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Fig. 2.
A 6-kDa C-terminal fragment of APP is
released on incubation of membrane fractions. Guinea pig brain was
fractionated into 2,500 × g (F1P), 10,000 × g (F2P), and 100,000 × g
(F3P) fractions, as described under "Experimental
Procedures." CHO cells were processed into two fractions of 10,000 × g (F2P) and 100,000 × g
(F3P). Fractions were incubated for 0 or 2 h in the
presence of 5 mM PNT, and the membranes from the incubation
mixture were removed by centrifugation. Supernatants were directly
analyzed on Western blots using O443 antibody (A).
B, the incubated guinea pig brain F2P fraction was
immunoprecipitated using Pf998 and then analyzed by Western
blotting with O443. Lysates from CHO cells expressing LC99
(C99) mark the positions of the CTF and the 83-residue
CTF
fragments.
in the supernatant using a well
characterized sandwich ELISA (12). The data obtained from 10 independently processed brain samples in three experiments detected
moderate levels of A
40 (62.3 ± 3.3 pM; Fig.
3). A
42 was also detected at low
levels of 5.4 ± 1 pM consistent with the in
vivo findings that A
42 constitutes 5-10% of the secreted A
(12). The increase in A
40 was highly significant (p = 4.7 × 10
12) but A
42 was not
(p = 0.18), presumably due to the large variance in
background levels (two-tailed t test).
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Fig. 3.
A 40 is generated
during the incubation of the F2P membrane fraction. F2P membrane
fractions were incubated for 0 h (squares) and 2 h
(triangles) in the presence of 5 mM PNT, 5 mM EDTA, and 1 mM thiorphan. Membranes from the
incubation mix were removed by centrifugation, and A
40 in the
supernatant was quantified by sandwich ELISA as described under
"Experimental Procedures." The bar at each time point
indicates the mean values of 24.8 ± 2.1 pM (0 h) and
87.2 ± 3.5 pM (2 h).
, the supernatant obtained after
removal of membranes from the incubation mix was incubated further for
2 and 4 h in the absence of membranes. The CTF
fragment was
degraded by over 50% after 2 h and by over 90% after 4 h at 37 °C. Addition of PNT, a metalloprotease inhibitor, partially protected the fragment from degradation after 4 h (Fig.
4). In addition, PNT increased the yield
of the C-terminal fragment by over 2-fold, suggesting that one or more
metalloproteases in the preparation degrade the fragment and/or the
enzyme responsible for its generation (Fig. 4). Although several
protease inhibitors including phenylmethylsulfonyl fluoride, E64,
leupeptin, and ALLN were tested, protection was only seen by EDTA and
PNT, making metalloproteases the only identified class important for
degrading the released CTF
in vitro (data not shown). The
pH optimum of the observed
-secretase activity was between pH 7 and
pH 7.5, but activity was detected in a broad range from pH 5 to pH 9 (Fig. 5). This pH optimum is similar to
that recently reported for
-secretase activity using membranes
purified from HeLa cells (39).
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Fig. 4.
PNT increases the yield of the putative
CTF and partially protects it from
degradation. Western blots probed with O443 (A)
show F2P membranes incubated in the absence of PNT for 0 h
(lane 1) or 2 h (lane 2) or in the presence
of PNT for 0 h (lane 11) and 2 h (lane
12). Supernatants generated after a 2-h incubation without PNT
were incubated further in the absence (lanes 3-6) or
presence of PNT (lanes 7-10) for an additional 2 (lanes 3, 4, 7, and 8) and 4 h (lanes
5, 6, 9, and 10). Note the higher CTF
recovery after
4 h in the presence of PNT (lanes 9 and 10)
than in its absence (lanes 5 and 6). Also note
the 3-fold higher yield of CTF
with PNT (lane 12) than
without PNT (lane 2). The graphical data shown in
B represent the mean quantified by densitometry ± S.D.
from three independent experiments.
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Fig. 5.
The yield of CTF is
optimal at close to neutral pH. The generation of CTF
at each
pH was determined as an increase in the fragment yield as detected by
Western blotting with O443 after a 2-h incubation compared with 0 h as blank.
in
collaboration with Dr. Rong Wang (Rockefeller University, New York).
Presumably due to its low levels and unfavorable flying characteristics, the exact size of CTF
could not be detected by
matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)
mass spectroscopy. However, the fragment comigrates with a 57-residue
synthetic peptide corresponding to one of the predicted CTF
fragments on polyacrylamide gels (Fig.
6). Judging from the separation of CTF
from CTF
and the molecular weight markers, small changes of over
five residues (0.5 kDa) should be readily detected in this size range.
Thus, the cleavage size is likely to be close to the C-terminal end of
the A
sequence and occur within the transmembrane domain of APP as
predicted for
-secretase activity (Fig. 6).
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Fig. 6.
The 6-kDa fragment migrates with synthetic
CTF -57 and is degraded by caspase-3.
Western blot probed with O443 shows F2P from guinea pig brain incubated
at 0 (lane 1) and 2 h (lanes 2-5).
Z-VAD-FMK was included in lanes 3 and 5, and
caspase-3 was included in lanes 4 and 5. Note
that caspase-3 reduces the yield of CTF
in lane 4, but
the yield is recovered when Z-VAD-FMK is added in lane 5. The synthetic CTF
-57 (lane 6) migrates with the fragment
generated from brain membranes (lanes 2-5). The synthetic
CTF
is degraded by caspase-3 (lane 7), which is protected
by Z-VAD-FMK (lane 8).
Production--
Previous
studies have shown that caspase can cleave within the cytoplasmic
domain of full-length APP in cells undergoing apoptosis (25, 47-49).
This cleavage resulted in an ~3-kDa fragment and was inhibited by a
broad spectrum caspase inhibitor, Z-VAD-FMK (49). It is unlikely that
the observed 6-kDa fragments are generated by caspase, as the fragment
is much larger than reported, and this cleavage occurs in the membrane
pellet fraction after washing away the cytoplasm. However, we cannot
rule out that some caspase is bound to the membrane pellet. To rule out
the possibility that the CTF
-like fragment is a product of caspase
cleavage, we included Z-VAD-FMK, a broad spectrum caspase inhibitor, in
our incubations. Z-VAD-FMK did not inhibit the generation of the
putative CTF
(Fig. 6). We also incubated the 57-residue synthetic
CTF
as well as the brain membrane fraction with caspase ± Z-VAD-FMK. Caspase-3 cleaved the synthetic CTF
as shown by the
reduction in band intensity, and Z-VAD-FMK blocked this reduction,
indicating that the inhibitor was active under the conditions used
(Fig. 6). Similarly, reduction in the yield of the 6-kDa CTF
was
also observed when brain membranes were spiked with purified caspase,
which was restored in the presence of Z-VAD-FMK (Fig. 6).
-Secretase Inhibitors Reduce CTF
--
A number of protease
inhibitors were tested to determine the class of proteases involved in
the generation of the putative CTF
and to understand the role of
-secretase in generating this fragment. Previous reports have
already identified several inhibitors that lower
-secretase activity
as defined by a reduction in the production of A
and by the increase
in CTF
and CTF
levels in cell lysates (2). Three of these
inhibitors are pepstatin-A, MG132, and Wolfe-1 (a
-secretase
substrate-based difluoroketone inhibitor). F2P membranes treated with
the inhibitors pepstatin-A (Fig.
7A, lanes 5-8), Wolfe-1 ((42)
Fig. 7A, lanes 9-12), and MG132 (Fig. 7B, lanes
7-12) showed a dose-dependent reduction in the yield
of the 6-kDa fragment generated from brain membranes in
vitro. These data provide additional evidence, indicating that the
putative CTF
fragment is a product of
-secretase cleavage.
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Fig. 7.
Known
-secretase inhibitors reduce the yield of
CTF
. F2P from guinea pig brain was
incubated in the presence of 5 mM PNT and the indicated
protease inhibitors and supernatants were analyzed on Western blots
with the O443 antibody as described under "Experimental
Procedures." A compares CTF
in PNT alone for 0 (C-0) and 2 h (C-2) with PNT + 10 µM (P-1) and 100 µM
(P-2) pepstatin-A, or 0.2 mM (W-1)
and 1 mM (W-2) Wolfe-1. B compares
samples incubated in the presence of PNT alone for 0 (C-0)
and 2 h (C-2), and PNT + 50 µM
(M-1) or 200 µM (M-2) MG132 for
2 h. The relative yield of CTF
from the various conditions is
summarized in C, with each bar representing the mean and
S.E. of 8-10 independent incubations from three separate
experiments.
Is Generated in a Fraction Enriched in CTF
, CTF
, and
Presenilin--
CTF
and CTF
are the postulated substrates for
-secretase activity, and PS1/PS2 are reported to be an integral part
of purified
-secretase (39). Furthermore, reports show that PS1
binds APP and its C-terminal fragments, consistent with its role in
-secretase activity (50-52). To examine the role of PS1 in the
generation of CTF
, we analyzed brain membrane fractions. As
described earlier, we initially obtained three membrane pellet
fractions F1P, F2P, and F3P generated after sequential centrifugation.
The fractions were probed with antibodies against APP and PS1 and
marker proteins as described under "Experimental Procedures". The
cell-surface GPI-anchored proteins (53) and the Golgi-marker syntaxin-6
(43) were preferentially found in F3P, which also had the highest
concentration of protein (Fig. 8). In
contrast, most of the
-secretase activity together with CTF
and
CTF
was in F2P, suggesting that
-secretase activity was not
enriched in the plasma membrane or in Golgi vesicles. Although the
N-terminal fragment of PS1 was observed in all three fractions, its
mobility was somewhat retarded in F3P. The reason for this shift in
mobility is not known but may be due to post-translational modification
such as phosphorylation of PS1 (54).
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Fig. 8.
CTF is generated in
a fraction distinct from the cell surface and the Golgi apparatus.
Guinea pig brain fractions F1P, F2P, and F3P were analyzed on Western
blots stained using reagents shown on the right side of each
panel. Note that full-length APP is enriched in F3P, whereas CTF
,
CTF
, and some of the CTF
are enriched in F2P. The N-terminal
fragment of PS1 at ~30 kDa was seen in all fractions. However, the
fragment was slightly shifted up in F3P. The anti-CRD antibody against
PI-PLC-cleaved GPI-anchored proteins detected two major groups of bands
in the 38- and 62-kDa range primarily in F3P. Each of these groups
presumably represents several GPI-anchored proteins in these size
ranges. The Golgi marker, syntaxin-6 at 31 kDa, was also primarily in
F3P. The synaptic vesicle marker, synaptophysin, was primarily detected
in F1P and F2P indicating that most of it is pelleted at 10,000 × g, although a sizable quantity is also seen in F3P. A
total protein stain (Ponceau S) detected the strongest signal in F3P,
although Fig. 2 shows that most of the CTF
is generated in
F2P.
-secretase
activity by incubating membrane pellets as described earlier. In
addition, the membranes were analyzed on Western blots for APP, CTF
,
CTF
, and PS1 (Fig. 9). The peak of
-secretase activity as determined by the relative intensity of the
CTF
fragment generated was in fraction 5. Interestingly, PS1,
CTF
, and CTF
are all enriched in this fraction. However, it is
important to note that PS1 was distributed in several fractions with
low
-secretase activity (Figs. 8 and 9). Recent findings show that
most of the PS1 is in the endoplasmic reticulum and intermediate
compartments with small amounts in the Golgi and cell surface (55). PS1
was also detected in detergent-resistant glycosphingolipid-enriched membranes (DIGs (56)), which was also enriched in cell-associated A
(57). Our preliminary
observations2 show that PS1,
BACE, and the ADAM 10 protease (Kuzbanian) are all enriched in
detergent-insoluble glycosphingolipid-enriched membranes isolated from
guinea pig brain.3 Since we
did not detect
-secretase activity in detergent-insoluble glycosphingolipid-enriched membranes prepared by flotation of Triton
X-100-extracted membranes, we homogenized guinea pig brain in carbonate
buffer to strip peripheral proteins, and generated a membrane fraction
enriched in caveolin as described by Lisanti and coworkers (58). The
-secretase activity was enriched in the caveolin-rich fractions
suggesting that it may be present in DIG-related
membranes.3
View larger version (41K):
[in a new window]
Fig. 9.
The peak of PS1,
CTF , and CTF
coincide
with the peak of CTF
generation. F2P from
guinea pig brain was further fractionated on sucrose density gradients,
and eight fractions were collected from the top of the tube as
described under "Experimental Procedures," and equal volumes of
each fraction were analyzed by Western blotting with O443 (top
three panels) and anti-PS1 N terminus (bottom panel).
Fractions 1 and 2 were mixed and loaded in the 1st lane, and
the remaining fractions were loaded in individual lanes. The top
panel shows full-length APP, the 2nd panel CTF
, and
the 3rd panel CTF
generated after incubation for 2 h. CTF
can be detected as a faint band above CTF
in the 2nd
panel. The bottom panel shows the ~30-kDa PS1-NTF.
Note that PS1-NTF, APP, CTF
, CTF
, and generation of CTF
peak
in fraction 5.
-Secretase--
The in
vitro
-secretase activity is lost when either F2P or membrane
fraction 5 purified by sucrose density gradients is extracted with
several detergents such as Triton X-100, methyl
-cyclodextrin,
digitonin, Nonidet P-40, CHAPS, and octyl
-glucoside (data not
shown). The data presented compare
-secretase activity in fraction 5 in the presence of Brij 35, Big CHAP, and CHAPSO (Fig.
10A).
-Secretase
activity, determined as an increase in CTF
production, appeared
equivalent to the detergent-free control in CHAPSO, reduced in Brij 35, and almost absent in Big CHAP. Thus, the detergent extraction profile
of this in vitro activity is similar to the in
vitro assay recently reported by Li et al. (39). Since
dissolving the activity is essential for its further purification and
characterization, we removed the CHAPSO-insoluble membranes by
centrifugation at 100,000 × g for 60 min and compared
-secretase activity in the supernatant with activity in the mixture (Fig. 10B). Our data indicate that most of the activity was
extracted from the sucrose gradient purified fraction 5 in this
detergent, as judged by the approximately similar intensities of the
CTF
band obtained by incubation of equivalent amounts of the
CHAPSO-solubilized supernatant alone and the CHAPSO-treated membrane
mix. Solubilization of the activity is necessary for further
biochemical analysis of
-secretase in vitro.
View larger version (19K):
[in a new window]
Fig. 10.
The in vitro
-secretase activity can be solubilized in select
detergents. Membranes from fraction 5 shown in Fig. 9 were diluted
and collected by centrifugation as described under "Experimental
Procedures." A, the fraction was incubated in pairs in the
absence (lanes 1 and 2) and presence (lanes
3-14) of the indicated detergents for 0 and 2 h. Activity
was recognized as an increase in the CTF
level at 2 h
(even lanes) over 0 h (odd lanes) for each
condition. Note that CTF
levels did not increase in the presence of
Big CHAP (lanes 7-10). B shows that CTF
is
generated in the CHAPSO-soluble fraction. CHAPSO-resistant membranes
were removed by spinning at 100,000 × g for 1 h,
and the supernatants were incubated for 0 (lanes 1 and
4) and 2 h (lanes 2 and 5). The
CHAPSO-treated membranes were incubated for 2 h (lanes
3 and 6) as in A. Note that removal of
CHAPSO-resistant membranes did not reduce the yield of CTF
. The
CHAPSO-resistant membrane pellet did not generate any CTF
(data not
shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase assay from brain
membranes and is the initial description of CTF
, a previously undescribed fragment predicted as a product of
-secretase cleavage of APP. The CTF
fragment is normally not detected in cell lysates and brain homogenates, probably due to its instability in
vivo. A possible exception is a report of a faint 5.8-kDa CTF in
human brain homogenates (22). However, CTF
is readily detected in an
in vitro
-secretase assay based on fractionated brain
membranes or cell homogenates. The in vitro detection of
CTF
strongly suggests that
-secretase cleavage is due to a
specific endoproteolytic cleavage and not random degradation of the
cytoplasmic tail domain of APP as suggested previously (59).
-secretase has been recently
reported by looking for A
as a product using a purified substrate
consisting of the C-terminal 99 residues of APP (39). In the presence
of thiorphan, an inhibitor of A
degradation in the brain (60), we
were also able to detect the generation of A
40 (Fig. 3) as well as
A
42 in our in vitro assay. Since a pool of CTF
is not already present in brain or cell lysates at detectable levels, the low
background allows the characterization of
-secretase activity from
fractionated membranes more easily than the measurement of A
. Based
on the reports that
-secretase accounts for <10% of the secretory
processing of APP (22), using CTF
as an end point for
-secretase
activity may be 10 times more sensitive than the measurement of A
.
In addition, the measurement of CTF
is not complicated by
aggregation like A
or by the presence of alternative fragments such
as P3 (Fig. 1).
suggest that
-secretase activity is localized in fractions enriched in CTF
and CTF
. PS1 is apparently enriched in these fractions, consistent with the currently favored hypothesis that presenilins include the active site of
secretase. However, high levels of PS1 are still seen in fractions showing little in
vitro
-secretase activity. Thus, if PS1 is indeed the active subunit of
-secretase as suggested, only a small subset of it, presumably in a special compartment, is involved in
-secretase activity. The restriction of the activity may be due to either the
observed enrichment of the substrate CTF
and CTF
in this compartment, limitations in essential components of the
-secretase complex other than presenilins, or unfavorable conditions in the isolated organelle.
are secreted by several cell lines in
culture, CTF
is not readily detected in cell lysates, indicating that it is either rapidly degraded in the cytoplasm or sequestered in a
manner that prevents its detection. Similar fragments from other
membrane proteins such as Notch and SREBP are transported into the
nucleus where they act as transcription factors, are rapidly degraded,
and are difficult to detect (61, 62). The C-terminal tail of APP
includes a sequence (KFFEQ) that resembles a motif present in soluble
proteins that are rapidly degraded in lysosomes (63). It will be useful
to determine whether this sequence is responsible for rapid CTF
turnover in cells and to identify alternative cellular pathways for its
degradation, if any.
-secretase
inhibitor indicating that the active site of
-secretase lies within
the transmembrane domains of PS1 and PS2 (40, 41). It was proposed that
presenilins are unusual aspartyl proteases, but this is not definitive
as the aspartyl protease inhibitor, pepstatin A, can inhibit other
classes of proteases at high concentrations (64). In addition, other
-secretase inhibitors are known to inhibit several classes of
proteases including serine proteases, cysteine proteases, and the
proteasome (2). The data showing
-secretase activity in fractions
enriched in PS1 agree with studies using knockout mice deficient in
PS1/PS2 that fail to generate A
and accumulate CTF
and CTF
(37, 65, 66). The detection of CTF
provides another powerful tool
for the analysis of
-secretase activity in vitro and for
identifying mechanisms involved in its regulation and for developing
drugs that block this activity.
-secretase activity and are shown to contain the active sites, it is
probably not sufficient, as it has not been possible to reconstitute
the activity with pure PS1/PS2 and purified
-secretase is a
multisubunit complex (39). The mechanism of the endoproteolytic cleavage of the membrane-spanning domains of proteins may identify the
role of each component. For example, it has been suggested that the
cleavage is initiated by the sliding of the peptide out of the membrane
into the cytoplasm (67). However, the energy required for this sliding
reaction is likely to be very high. It is also likely that the reaction
is mediated by a water molecule within the active site of the enzyme
for the hydrolytic reaction. Thus, a water molecule may be held between
the two transmembrane aspartate residues on PS1/PS2 that serve to
hydrolyze within the transmembrane domain. It is possible that some of
the unidentified subunits are involved in either transport of water or
the APP for hydrolysis. The mechanisms for maintaining a supply of the water molecules in the membrane for the hydrolysis may provide useful
insights into the mechanisms of
-secretase cleavage and identify
additional therapeutic targets for inhibiting this activity. A similar
intramembrane proteolytic cleavage has been described for the sterol
regulatory element binding protein (SREBP). The protease that cleaves
SREBP has been identified as S2P by genetic complementation of a mutant
cell line. The predicted active site of this enzyme resembles that of a
metalloprotease with the exception that hydrophobic residues flank the
domain, suggesting its intramembranous location (62). The development
of an in vitro assay for S2P may be useful for comparison
with
-secretase and also provide useful insights into intramembrane proteolysis.
is closely tied to the generation of
A
, the mutations that increase the production of A
42 should also
increase the 57-residue CTF
starting at residue 43 of the A
sequence. Since FAD mutations that increase A
by preventing its
degradation have not been reported, the correlation between CTF
-57
and AD is as strong as A
42. In addition, CTF
includes a peptide
sequence that is reported to induce apoptosis in neurons as discussed
in the Introduction. The localization of the enzyme, the biological
activity of the fragment, the cleavage site involved in its production,
the mechanism of its turnover, and its role in AD are important
unanswered questions that will be facilitated by this initial
description of CTF
.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Daniel Sevlever of the Mayo
Clinic and Dr. Debomoy Lahiri for several antibodies used in this study
and Pfizer Corp. for synthetic CTF57 and antibody PF998. We also
thank Meera Parasuraman, Dr. Daniel Sevlever, Dr. Debomoy Lahiri, and
Dr. John Hardy for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This study was supported by grants from ISOA, Bayer Corp., Axonyx Corp., and Smith fellowship (to K. S.).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.
Current address: Brandeis University, Waltham, MA 02454.
§ To whom correspondence should be addressed: Mayo Clinic, 4500 San Pablo Rd., Jacksonville, FL 32224. Tel.: 904-953-7383; Fax: 904-953-7370; E-mail: samba@mayo.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005968200
2 I. Pinnix, U. Musunuru, H. Tun, A. Sridharan, T. Golde, C. Eckman, C. Ziani-Cherif, L. Onstead, and K. Sambamurti, unpublished observations.
3 H. Tun, I. Pinnix, and K. Sambamurti, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A, Alzheimer's
amyloid
protein;
AD, Alzheimer's disease;
FAD, familial AD;
A
40, A
ending at residue 40;
A
42, A
ending at residue 42;
APP, A
protein precursor;
sAPP, secreted APP derived from APP by
proteolytic cleavage;
sAPP
, sAPP product of
secretase cleavage;
sAPP
, sAPP product of
secretase cleavage;
CTF
, C-terminal
membrane associated product of
secretase cleavage;
CTF
, C-terminal membrane associated product of
secretase cleavage;
CTF
, C-terminal soluble product of
secretase cleavage;
ADAM, adamalysin, a new class of disintegrin metalloproteases;
PS1, presenilin-1;
PS2, presenilin-2;
PNT, 1,10-phenanthroline;
CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonic
acid;
Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
MES, 4-morpholineethanesulfonic acid;
PLAP, placental alkaline phosphatase;
CHO, Chinese hamster ovary;
Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2F;
PI-PLC, phosphatidylinositol-specific phospholipase C;
KLH, keyhole limpet
hemocyanin;
ELISA, enzyme-linked immunosorbent assay;
SREBP, sterol
regulatory element-binding protein;
GPI, glycosylphosphatidylinositol;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
![]() |
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