From the Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486
Received for publication, September 25, 2002
, and in revised form, March 27, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In cells, the activity of BACE and -secretase to process APP has
been observed in the membrane fractions, particularly in the trans-Golgi
network (1,
8,
9,
10,
11). Production of A
has
also been detected in both the endoplasmic reticulum and the trans-Golgi
network (12,
13,
14,
15). Several lines of
experimental evidence suggest that cellular APP processing is sequential and
that cleavage of APP by
- or
-secretase is a prerequisite for
-secretase-mediated processing. First, only two species of APP
N-terminal products cleaved by endogenous secretases were reportedly detected
in cells: the BACE-cleaved product sAPP
and the
-secretase-cleaved product sAPP
(1,
2,
3,
4,
5,
6,
7). No
-secretase
cleaved N-terminal APP, or so-called sAPP
, without prior
- or
-secretase cleavage, was ever detected or reported. Second, in an in
vitro cell-free assay system, the N-terminally truncated form of APP,
CT99 (or CT100), has been demonstrated as an efficient substrate for
-secretase (16,
17). In contrast, the
full-length APP is a very poor substrate for
-secretase.2
Therefore, removal of the N-terminal region of APP seems to be essential for
-secretase-mediated cleavage.
In the present study, we found that BACE-mediated cleavage at the
A-34 site is dependent on the
-secretase activity. BACE cleaves
the A
-34 site in vitro only within A
1 40 peptide,
a product from
-secretase, not the site in the non-cleaved CT99
fragment. Moreover, the cellular production of A
134 species
induced by overexpression of BACE1 or BACE2 was blocked by known
-secretase inhibitors in a concentration-dependent manner, whereas the
-secretase inhibitors had no effect on enzymatic activity of BACE1 or
BACE2 in vitro. Our results suggest that the cleavage activity of
BACE at the A
-34 site, both in vitro and in cells, is dependent
on
-secretase activity. Therefore, BACE and
-secretase activity
can be mutually dependent.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BACE Activity in Vitro AssayA 1 40 was
purchased from Enzyme Systems Products (Livermore, CA). Human cathepsin D was
purchased from Calbiochem. The production of recombinant APP-CT100-Flag was
described previously (16).
Treatment of CT100-Flag with aminopeptidase (Sigma) resulted in a CT99-Flag
with the amino acid residue Asp at its N terminus. CT99-flag was confirmed by
mass spectrometric analysis and by its reactivity with a neo-epitope-specific
antibody, FCA-18, purchased from Dr. F. Checler
(20). Assays were performed
for the indicated time in the presence of 50 mM ammonium acetate,
0.15 M NaCl, pH 4.5 or 5.0, and 0.1 mg/ml BSA at 37 °C. The
reaction was terminated by heating samples at 75 °C for 5 min. Samples
were analyzed by mass spectrometry or/and by reversed-phase high pressure
liquid chromatography.
BACE1 or BACE2 activity in vitro was also assessed by cleavage of
a FRET substrate encompassing P8-P4' of the APPsw site,
TAMRA-5-CO-EEISEVNLDAEF-NH-QSY7, similar to the one described by Ermolieff
et al. (21). The
reaction condition is the same as described above and the cleavage product was
measured by a LJL Analyst AD instrument (LJL BioSystems, Sunnyvale, CA) with
excitation at 530 nm and emission at 580 nm.
Cell Culture, Transfection, and Analysis of APP Processing
ProductsHEK293T cells stably expressing human APP695 were seeded
in 100-mm dishes and transfected with either BACE1 or BACE2 DNA as described
above. Transfection was performed using LipofectAMINE reagent (Invitrogen)
according to the manufacturer's instructions. Media were changed on the next
day, and either Me2SO control or compound-1 was added to the new
media. After an additional 24 h of incubation at 37° C, the media and
cells were harvested. A level in media was assayed by Origen system, as
described previously, using biotinylated 6E10 antibody (Senetek, Maryland
Heights, MO) and ruthenylated G210 antibody
(16) licensed from the
University of Heidelberg.
Mass Spectrometric Analysis of BACE-cleaved ProductsMALDI
mass analysis was performed with a surface-enhanced laser
desorption/ionizationtime-of-flight (SELDI-TOF) mass spectrometer from
Ciphergen Biosystems (Fremont, CA) and a Voyager DE/RP MALDI-TOF from Applied
Biosystems. Typically, 1 µl of reaction mixture was mixed with 1 µl of a
saturated MALDI matrix solution (-cyanohydroxycinnamic acid in 1:1
water/acetonitrile with 0.1% trifluoroacetic acid) and was then spotted onto
Ciphergen NP2 MALDI chips. After drying and crystal formation, sample spots
were typically analyzed with 100 laser shots.
In all figures containing MALDI mass spectrometric data, the generic
notation A X-X refers to the length of the A
fragment, such as
A
1 40 or A
134. This notation is used to denote the
A
fragment corresponding to the mass of the singly charged peptide ion
(M+H)+. In addition, in cases in which the doubly charged ion
(M+2H)2+ is visible, as in Fig.
2, the generic notation (A
X-X + 2H)2+, denotes
the A
species corresponding to the mass of the doubly charged peptide
ion.
|
Mass Spectrometric Analysis of APP Processing Products in
Media For analysis of APP processing species secreted into media,
the collected media samples were immunoprecipitated with 6E10 antibody and
protein G beads (Protein G UltraLink; Pierce Chemical). After incubation at
room temperature for 1 h, the supernatants were removed, and the
immunoprecipitation beads were first washed three times with a buffer
containing 150 mM NaCl, 10 mM Tris, 2 mM
EDTA, 0.1% Triton X-100, and 0.1% Igepal CA630, pH 7.8, then washed twice with
a buffer consisting of 500 mM NaCl, 10 mM Tris, 2
mM EDTA, 0.1% Triton X-100, and 0.1% Igepal CA630, pH 7.8, and
finally washed three times with 100 mM
NH4HCO3, pH 7.8. After the final wash, the captured
products were eluted from the beads with a minimal volume of a saturated
solution of -cyano-4-hydroxycinnamic acid dissolved in 50%
acetonitrile/0.1% trifluoroacetic acid. One microliter of the bead eluate was
then spotted on to the an NP-2 SELDI sample target (Ciphergen Biosystems) or a
stainless steel target for an ABI Voyager and allowed to air dry. The dried
targets were placed into the SELDI-TOF and Voyager mass spectrometers and
matrix/analyte coprecipitates were desorbed with N2 laser at 337
nm. The laser power was attenuated and the detector sensitivity manipulated
such that spectra, which were the average of 100 laser shots, provided a
qualitative assessment of the immunoprecipitation-captured cleavage
products.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When BACE1 or BACE2 was incubated with a synthetic A peptide 1-40
in vitro, a cleavage site located between A
residues 34 and 35
was clearly identified through mass spectrometric analysis as shown in
Fig. 1, B1, B3, and
B4. The theoretical mass value for A
134 is
3786.9 and the observed mass values for the cleaved 134 products were
3785.6 and 3786.6 for BACE1 and BACE2, respectively. However, when A
1 40 peptide was incubated with cathepsin D, A
species cleaved at
residues between 19 and 20 were observed by mass spectrometric analysis, but
not the A
134 product (Fig.
1B, 1 and 2). The absence of A
134
species in cathepsin D-treated sample is not the result of a
secondary cleavage of A
134, because a shorter incubation time
and with less enzyme showed similar results (data not shown). In contrast,
both purified full-length human BACE1 (Fig.
1B, 3) and BACE2 (Fig.
1B, 4) processed the A
-34 site. Thus, the cleavage
of A
-34 site is specific for BACE1 and BACE2 not for cathepsin D.
|
BACE1 Lacking the Transmembrane and Cytoplasmic Regions Cleaves the
A-34 Site in VitroOne of the major differences
between BACE and cathepsin D is that cathepsin D does not contain a
transmembrane domain and cytoplasmic tail. To investigate whether the
transmembrane and cytoplasmic regions of BACE contribute to its specific
activity of processing the A
-34 site, we tested the activities of
truncated forms of BACE1 lacking both the transmembrane and cytoplasmic
domains using the A
1 40 substrate. Results from mass
spectrometric analysis showed that truncated BACE1, both the pro-(amino acids
22 460) and the mature (amino acids 46460) forms
(19), cleaves the A
-34
site in vitro (Fig.
2). Therefore, the differential cleavage of A
-34 site
between BACE and cathepsin D is probably because of the difference in their
enzyme active sites. It is interesting to note that the truncated forms of
BACE1 did not cleave the A
-20 site as seen with the full-length
enzyme.
BACE1 or BACE2 Does Not Cleave A-34 Site Using CT99 as
SubstrateTo further investigate BACE in vitro cleavage
specificity, we tested BACE activity using a recombinant CT99 protein
substrate. CT99 or CT100 has been described previously as an efficient in
vitro substrate for
-secretase
(16,
17). Control mass
spectrometric analysis of CT99 alone (Fig.
3A) at mass range 10005000 Da did not show any
notable species (Fig.
3B). To our surprise, when CT99 was incubated with BACE1,
cleavage products of CT99 at either the A
-20 site or the A
-34 site
were not observed (Fig.
3C). This was true for both full-length and truncated
forms of BACE1 (data not shown). The absence of BACE1-mediated cleavage at
A
-20 or A
-34 sites in CT99 was not caused by substrate depletion,
because the substrate peak was still observed by mass spectrometry at the end
of the reaction (data not shown), nor was it caused by enzyme inactivation,
because that addition of a small peptide substrate encompassing the
-scissile bond at the end of the CT99 reaction yielded an appropriate
cleavage product (data not shown). Likewise, BACE2 also did not cleave CT99 at
the A
-34 site, although it cleaved at the A
-19 and -20 sites
(Fig. 3D). Because the
CT99 used here is in solution and not embedded in the membrane, the
hydrophobic domain of the CT99 might be "abnormally" folded in the
buffer. To test whether addition of a limited amount of detergent could expose
the A
-34 site in CT99 for processing, the in vitro reaction
with CT99 was also performed in the presence of 0.05% Triton X-100. Both BACE1
and BACE2 were fully functional in the presence of 0.05% Triton X-100 to
cleave the
site of APP (data not shown)
(3). However, neither of them
showed any cleavage of A
-34 site in CT99 (data not shown). Therefore,
unlike A
1 40 peptide, CT99 is not a substrate for A
-34
site cleavage by either BACE1 or BACE2 in vitro.
|
Overexpression of BACE1 or BACE2 in Cells Increases A
134 ProductionTo evaluate the effect of BACE1 or BACE2
on cellular A
134 production, HEK293T cells stably expressing
APP695 (HEK293T/APP695) were either mock transfected or transfected with BACE1
or BACE2 cDNA. A
1 40 level secreted in the media by these cells
was determined by a modified ELISA assay using antibodies 6E10 and G210
(16) against A
1
40 (Fig. 4A). Consistent with
previous reports, overexpression of BACE1 moderately increased the A
1 40 production (Fig. 4A,
2) (4), and
overexpression of BACE2 abolished the generation of A
1 40
(Fig. 4A, 3)
(22). When the conditioned
media were subjected to immunoprecipitation and mass spectrometric analysis
(see "Materials and Methods"), results showed that HEK293T/APP695
cells produced few A
134 species
(Fig. 4B, 1);
Transfection of BACE1 increased the relative amount of A
134
species, in addition to A
120 and A
1 40
(Fig. 4B, 2);
transfection of BACE2 similarly increased A
134 level, despite
abolishing A
1 40 (Fig.
4B, 3). This suggests that in cells, BACE2 prefers
A
-19, -20, or -34 sites over the A
-1 site. This result not only
agrees well with BACE2 activity data in vitro
(18,
23) but also provides a
plausible explanation for the negative effect of BACE2 on cellular A
1 40 production. In summary, our results indicate that overexpression
of BACE1 or BACE2 in cells increases the production of A
134.
|
-Secretase Inhibitors Block the Production of A
134 in CellsTo evaluate the effect of
-secretase
inhibitors on the cellular production of A
134 species,
HEK293T/APP695 cells, either mock-transfected or transfected with BACE1 or
BACE2, were treated with either Me2SO or with 10 µM
concentrations of each of the two known
-secretase inhibitors, compound
1 and compound 2 (24,
25). As expected, both
compounds inhibited A
1 40 production in HEK293TAPP695 cells
(Fig. 5A, 13)
and the cells transfected with BACE1 (Fig.
5A, 46). The effect of compounds on the cellular
production of A
1 40 was further confirmed by mass spectrometric
analysis of the A
species in the conditioned media of these cells
(Fig. 5B). Notably, in
BACE1 overexpressing cells, these
-secretase inhibitors blocked the
production of not only A
1 40 species but also A
134
species (Fig. 5B,
46). Likewise, in BACE2 overexpressing cells, the
increased generation of A
134 species was also inhibited by these
compounds (Fig. 5B,
79). In contrast, the generation of other A
species such as 116, 119, or 120 were largely unblocked
(Fig. 5B).
|
Inhibition of A 134 Production by
-Secretase Inhibitor Is Concentration-dependent and Coincides with
the Blockade of A
1 40 ProductionTo further
evaluate the effect of
-secretase inhibitors on the cellular production
of A
134 species, another known
-secretase inhibitor with
a different structural class, compound 3
(17,
26), was tested in cells at
various concentrations (1 and 10 µM). Results from the modified
A
ELISA assay revealed a concentration-dependent inhibition of A
1 40 production in both HEK293T/APP695 cells
(Fig. 6A, 13)
and cells transfected with BACE1 (Fig.
6A, 46). Furthermore, the mass spectrometric
analysis of A
species in conditioned media of these cells showed that
both the production of A
1 40 and A
134 species were
blocked by compound-3 in a dose-dependent manner
(Fig. 6B). Notably, in
BACE1 overexpressing cells, the extent of A
134 inhibition
coincided with the blockade of A
1 40 production
(Fig. 6B, 46).
In BACE2 overexpressing cells, the generation of A
134 was
similarly inhibited by compound 3 in a dose-dependent manner
(Fig. 6B,
79).
|
Because both BACE and -secretase are aspartyl proteases
(2,
3,
16,
17), we performed experiments
to determine whether any of the
-secretase inhibitors used in our
cellular study blocked the activities of BACE1 or BACE2 in vitro.
When BACE1 or BACE2 were incubated in vitro with a peptide substrate
containing the APPsw
site sequence, a steady formation of the cleavage
products was observed with time (Fig.
7). The known BACE inhibitor StatV
(3) inhibited the cleavage
activities of both BACE1 and BACE2. However, no inhibitory effect was observed
with any of the
-secretase inhibitors
(Fig. 7), whereas at the same
concentration (10 µM), all three compounds completely blocked
-secretase activity in cells (Figs.
5 and
6).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
First, we show that the A-34 site is cleaved by BACE both in
vitro and in cells; second, we demonstrate that
-secretase
activity is required for BACE cleavage at the A
-34 site both in
vitro and in cells.
In vitro, human BACE, both the full-length form and the truncated
form lacking transmembrane and cytoplasmic domains, cleaved the A-34
site within A
1 40 peptide substrate. However, cathepsin D, a
closely related aspartyl protease, did not cleave the A
-34 site,
although it shares many of the cleavage sites in APP with BACE, including the
-scissile bond3
and the A
-19 or -20 sites (Fig.
1). Thus, the A
-34 site seems to be rather specific for
BACE. Notably, the sequence around the A
34 site, IIGL-MVGG, shows a
high homology to a reported in vitro "optimized sequence"
for BACE1, EIDL-MVLD (27).
In cells, overexpression of BACE1 or BACE2 increased the generation of
A 134 species (Fig.
3), suggesting that cellular production of A
134 is
dependent on BACE levels. The A
134 species has also been
observed in cells stably overexpressing BACE1
(10,
28). However, in all these
cases, it is difficult to completely rule out the possibility that
overexpressed BACE1 increases the cleavage of the A
-1 site, and other
enzymes, such as
-secretase, could be responsible for the cleavage at
the A
-34 site in cells. In this aspect, our data from BACE2
overexpression studies is particularly noteworthy. The overexpression of BACE2
similarly increased A
134 level, but abolished A
1
40 production (Fig. 4B,
3). This indicates that two different enzymes must be
responsible for the cleavage of A
-34 and A
-40 sites. In fact, we
and others have observed that BACE1 prefers the A
-1 site, whereas BACE2
prefers the internal cleavage sites within A
1 40, such as the
A
-19 or -20 sites and the A
-34 site
(18). These
"internal" cleavage activities by BACE2 would lead to a decrease
in A
1 40 level and, we expect, in A
134 level if
the A
-34 site were indeed processed by
-secretase. Thus, taken
together, our data indicate that in cells, the A
34 site is cleaved by
BACE1 or BACE2 not the
-secretase.
Several lines of evidence indicate that BACE cleavage of the A-34
site is dependent on
-secretase activity. In vitro, BACE
cleaves only the A
-34 site within the A
1 40 peptide, the
product of
-secretase cleavage; BACE did not process the A
-34
site in CT99, the substrate for the
-secretase. In contrast, BACE2
cleavage of A
-19 and -20 sites occurred with both A
1 40
and CT99 substrates. We were unable to observe any BACE1 cleavage product
using CT99 as substrate, although the enzyme was active toward the
-scissile bond cleavage. One possible explanation is that other
BACE1-mediated cleavages within CT99 could also be dependent on
-secretase activity. More experiments are needed to test this
hypothesis. In cells, cleavage of the A
-34 site induced by BACE1 or
BACE2 is blocked by several different classes of
-secretase inhibitors.
Moreover, the inhibition of A
-34 site processing is dose-dependent and
coincides with the blockade of A
-40 site cleavage. None of the
-secretase inhibitors tested here showed any inhibitory effect on
enzymatic activity of BACE1 or BACE2 in vitro. Therefore, the most
likely explanation would be that
-secretase cleavage of CT99 in cells
is a prerequisite for BACE-mediated processing of A
34 site.
Additionally, two recent studies also reported that
-secretase
inhibitors blocked the production of the A
134 species in cells
(28,
29). During the manuscript
revision for this article, Fluhrer et al. published a study with
similar findings using presenilin dominant-negative mutants to inhibit
cellular
-secretase activity
(30).
A question that remains is how the activity of -secretase affects
the BACE cleavage of the A
34 site. Results from the in
vitro experiments using either A
1 40 or CT99 substrates
suggest that certain features in CT99 fragment interfere with BACE cleavage of
A
-34 site. One possibility is that the hydrophobic domain of the CT99,
normally embedded in the membrane, might be misfolded and therefore prevented
the access of BACE. However, addition of a limited amount of detergent, 0.05%
Triton X-100, did not promote the processing. Alternatively, CT99 fragment
could still possess an "unfit" conformation or contain an
inhibitory element downstream from the
-secretase processing sites. In
any case, CT99 needs to be truncated at the
-secretase cleavage sites
for BACE to cleave the A
-34 site. In vitro, such a requirement
is fulfilled by directly using the
-secretase-cleaved product and a
synthetic A
140 peptide as substrate; in cells, this is
accomplished through the cellular
-secretase activities. A
-secretase inhibitor(s) blocked the "truncation" of CT99
and therefore inhibited BACE cleavage at the A
34 site in
cells.
In summary, we show that BACE, either BACE1 or BACE2, cleaves A-34
site in vitro and in cells, but only after
-secretase-mediated
processing of APP. It is interesting to note that N-terminal truncation of APP
by BACE or
-secretase is a prerequisite for
-secretase cleavage
around A
-40 sites. And it is, in turn, as shown in the present study,
that
-secretase cleavage at the C terminus of A
peptide is a
prerequisite for BACE-mediated processing at the A
-34 site. Our data
indicate a sequential and mutual dependence of BACE and
-secretase
activities in APP processing. We and others have observed and reported that
BACE cleaves at least four sites within a stretch of 50 amino acid residues of
APP, around and within the A
region, approximately every 10 amino acid
residues (18). Results
described in this study further support such a complex picture in APP
metabolism: product from one enzyme can become substrate for the other and
verse visa.
The biological role of the A 134 species is still unclear.
Although A
134 species, either in soluble or insoluble form, has
not been reported in AD brain, its presence in human cerebrospinal fluid has
been observed (31). The
contribution of A
134 species to AD pathogenesis remains to be
determined. The discovery that BACE cleavage at the A
-34 site is
dependent on
-secretase activity could facilitate our understanding of
APP processing by these two critical enzymes.
![]() |
FOOTNOTES |
---|
Present address: Dept. of Chemistry, Washington State University, Pullman,
WA 99164-4630.
¶ Present address: RRL-617A, Box 459, Memorial Sloan-Kettering Cancer Center,
1275 York Ave., New York, NY 10021.
To whom correspondence should be addressed: WP16-205, Dept. of Biological
Chemistry, Merck Research Laboratories, West Point, PA 19486. Tel.:
215-652-3622; Fax: 215-652-0264; E-mail:
xiao-ping_shi{at}merck.com.
1 The abbreviations used are: A,
amyloid peptide; AD,
Alzheimer's disease; BACE,
site APP-cleaving enzyme; APP, amyloid
precursor protein; CT99, C-terminal; HEK, human embryonic kidney; MALDI,
matrix-assisted laser desorption/ionization; SELDI, surface-enhanced laser
desorption/ionization; TOF, time of flight; ELISA, enzyme-linked immunosorbent
assay.
2 Y.-M. Li, M. Xu, Q. Huang, and S. J. Gardell, unpublished results.
3 X.-P. Shi, K. Tugusheva, J. E. Bruce, A. Lucka, M. K. Sardana, and D. J.
Hazuda, unpublished results.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|