From the a Adolf-Butenandt Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Schillerstrasse 44, Ludwig-Maximilians University, 80336 Munich, Germany, the c ZMBH-Center for Molecular Biology, University of Heidelberg, Im Neuenheimer Feld 282, Germany, the e Department of Postgenomics and Diseases, Division of Psychiatry and Behavioral Proteomics, Osaka University Graduate School of Medicine, 565-0871 Osaka, Japan, and the f Max-Planck Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany
Received for publication, November 11, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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The Because of the increasing mean life expectancy, there is
considerable interest in the understanding of the molecular and
biochemical mechanisms of age-related diseases. By far the most
frequent age-related neurological disorder is Alzheimer's disease
(AD).1 During the aging
process the patients accumulate insoluble amyloid BACE and Cell Culture, cDNAs, and Transfection--
HEK293 were
cultured as described (9). The cell lines stably overexpressing wild
type Antibodies, Metabolic Labeling, Immunoprecipitation, and
Immunoblotting--
Antibodies 7520 (43, 44) and 7524 (9)
directed against the respective C termini of BACE or BACE-2 and
antibody 3926 (45) against the A BACE Activity Assay--
The fluorometric BACE activity assay
was carried out as described previously (46). To selectively inhibit
BACE activity GL189 was used as described previously (46). Soluble BACE
(sBACE) was isolated and incubated with synthetic A Mass Spectrometry/MALDI-TOF--
Cells were
grown on 10-cm dishes and incubated with 4 ml of Dulbecco's modified
Eagle's medium high glucose (DMEM; PAA Laboratories) supplemented with
10% fetal calf serum (PAA Laboratories) and penicillin/streptomycin
for 24 h. Subsequently the samples were prepared for mass
spectrometry as described previously (49-51). Samples were analyzed on
MALDI-target plates by matrix-assisted laser desorption ionization
(Brucker Reflex III).
In order to analyze the A-amyloid precursor protein (
APP) is
proteolytically processed by two secretase activities to produce the
pathogenic amyloid
-peptide (A
). N-terminal cleavage is
mediated by
-secretase (BACE) whereas C-terminal intramembraneous
cleavage is exerted by the presenilin (PS)
-secretase complex.
The A
-generating
-secretase cleavage principally occurs
after amino acid 40 or 42 and results in secretion of A
-(1-40) or
A
-(1-42). Upon overexpression of BACE in cultured cells we
unexpectedly noticed a reduction of secreted A
-(1-40/42). However,
mass spectrometry revealed a truncated A
species, which terminates
at amino acid 34 (A
-(1-34)) suggesting an alternative
-secretase cut. Indeed, expression of a loss-of-function variant of
PS1 inhibited not only the production of A
-(1-40) and A
-(1-42)
but also that of A
-(1-34). However, expression levels of BACE
correlate with the amount of A
-(1-34), and A
-(1-34) is produced
at the expense of A
-(1-40) and A
-(1-42). Since this suggested
that BACE is involved in a C-terminal truncation of A
, we incubated
purified BACE with A
-(1-40) in vitro. Under these
conditions A
-(1-34) was generated. Moreover, when conditioned media
containing A
-(1-40) and A
-(1-42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, A
-(1-34) was efficiently produced in vivo. These
data demonstrate that an apparently
-secretase-dependent
A
derivative is produced after the generation of the non-truncated
A
via an additional and unexpected activity of BACE.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-peptide (A
),
which is deposited in senile plaques and microvessels in the brain.
A
is generated by endoproteolytic processing of the
-amyloid
precursor protein (
APP), involving
- and
-secretase (1).
-secretase (also called BACE;
-site APP-cleaving
enzyme) was identified as a membrane-associated aspartyl protease
(2-6). BACE mediates the primary amyloidogenic cleavage of
APP and
generates a membrane-bound
APP C-terminal fragment (APP CTF
),
which is the immediate precursor for the intramembraneous
-secretase
cleavage (1). BACE also generates N-terminally truncated A
species starting with amino acid 11 of the A
domain (2, 7-9). A
close homologue (BACE-2) (6, 10-12) can also mediate the typical
-secretase cut although with much lower efficiency (13). BACE-2
rather exhibits an
-secretase-like activity, which cleaves in the
middle of the A
domain at amino acid 19 and 20 (9, 13, 14).
Apparently BACE-2 does not contribute to the amyloidogenic processing
of A
, since the deletion of BACE fully abrogates A
generation
(15-17).
-Secretase activity is associated with a protein complex, composed
of presenilins (PS1 or PS2), Nicastrin (Nct), PEN-2, APH-1a, and APH-1b
(18-25). The expression of these complex components is coordinately
regulated, and
-secretase activity is only detected in the presence
of all subunits (21, 23-25). Removing a single subunit results in the
destabilization or reduced maturation of the remaining components
(23-26). The catalytic activity is most likely contributed by the PSs
(1, 27). PSs are polytopic transmembrane proteins, which together with
the signal peptide peptidases and the type-4 prepilin peptidases may
belong to a novel family of aspartyl proteases of the GXGD
type (for review see Ref. 1). The cleavage of BACE-generated CTF
by
-secretase results in the secretion of A
into biological fluids
(1). This cleavage principally occurs after amino acid 40 and 42, the latter being enhanced by numerous familial AD-associated mutations in
the PS genes and
APP itself (28). Beside the predominant cleavage after amino acid 40 and 42 slightly shorter peptides have been
observed as well, suggesting that the
-secretase has loose sequence
specificity (29). This includes peptides terminating after amino acid
34, 37, 38, and 39 (29). In addition or in parallel to
these cleavages,
-secretase also cleaves within the transmembrane domain shortly before the cytoplasmic border after amino acid 49 to
liberate the
APP intracellular domain (AICD) (30-33), which may be
involved in nuclear signaling (34, 35). The biological function of
-secretase is related to the very similar intramembraneous processing of Notch. Indeed, a depletion of PS1 leads to a very severe
Notch phenotype (summarized in Ref. 36).
-secretase are obvious targets for therapeutic strategies
aimed to inhibit A
generation. Unfortunately
-secretase inhibitors not only block A
generation but also interfere with Notch
signaling (37-39). Therefore treatment of patients with such inhibitors remains problematic. On the other side it has been shown
that the gene encoding BACE can be removed without any deleterious effects (15-17). Therefore inhibition of BACE with small chemical compounds seems to be a safer approach for long term treatment. However, a detailed understanding of the cleavage specificity and the
substrate specificity of BACE is required for the generation of
selective drugs. Surprisingly, overexpression of BACE in cell culture
models leads to reduced A
secretion (2). In order to investigate
this paradox we analyzed A
peptides secreted from cells stably
expressing various levels of BACE and made the surprising observation
that BACE can also cleave 34 amino acids C-terminal from its primary
cleavage site, thus mimicking a PS-like cleavage specificity.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APP695 (40) or
APP695 containing the
Swedish double mutation (
APPsw) (41) and cell lines co-expressing either PS1 wt or PS1 D385N have been described (42). To transfect BACE
into these cell lines, the BACE cDNA was cloned into the EcoRI/XhoI sites of pcDNA3.1 hygro (+)
expression vector (Invitrogen). Transfection was carried out using
FuGENE 6 reagent (Roche Molecular Biochemicals). Pooled stable cell
clones were selected in 150 µg/ml hygromycin (Invitrogen). The cell
lines expressing BACE-2 have been described previously (9).
domain of
APP, as well as the
antibodies 6687, against the C terminus of
APP, and 5313, against
the N terminus of
APP (46), have been described previously. The
monoclonal antibody 6E10 directed against amino acids 1-17 of the A
domain was obtained from Senetek Inc. For immunodetection of PS1 the polyclonal and monoclonal antibodies against the large hydrophilic loop
of PS1 (3027 and BI.3D7) were used (47, 48). Metabolic labeling,
immunoprecipitations, and Western blotting were carried out as
described previously (9).
as follows: HEK
293 cells expressing sBACE were incubated with Optimem 1 containing Glutamax (Invitrogen) for 24 h. 400 ml of the conditioned medium were purified using a mono Q-Sepharose column (Amersham Biosciences). 20 µl of the fraction derived from cells expressing sBACE or from control fractions not containing sBACE were incubated with 50 µg of
synthetic A
-(1-40) for MALDI-TOF MS and with 6 µg of synthetic A
-(1-40) for gel analysis at 37 °C for the indicated time
points. The pH was adjusted to 4.5 with acetic acid. Samples were dried in a Speed Vac and resuspended in acetic acid. Subsequently samples were purified by using a Zip-Tip column and were then subjected to
MALDI-TOF MS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
species secreted by BACE-expressing
cells we collected conditioned media from HEK 293 cells stably transfected with Swedish mutant
APP695 (
APPsw) and
BACE. Cells expressing either endogenous levels of BACE or moderate or
high levels of transfected BACE were investigated (Fig.
1A). To prove the catalytic
activity of BACE in these cell lines we performed in vitro
activity assays using solubilized membranes of the respective cell
lines (46). As expected we found substantially increased
-secretase
activity in the cell line expressing high levels of BACE as compared
with non-transfected cells or cells expressing low levels of BACE (data
not shown). These cell lines were labeled with
[35S]methionine and conditioned media were
immunoprecipitated with the anti-A
antibody 3926. Surprisingly,
increasing BACE expression negatively correlated with A
production
(Fig. 1B). This is consistent with previous findings by
Vassar et al. (2) who observed reduced A
production
despite increased BACE activity in cells transfected with
APPsw (2).
This paradoxical finding raised the possibility that A
species
produced under these conditions could either not be metabolically
labeled or not detected by conventional gel electrophoresis. We thus
used an independent method and attempted to identify secreted A
species by a combined immunoprecipitation/MALDI-TOF MS method. As
expected cells expressing endogenous BACE secreted predominantly A
-(1-40) (Fig. 1C). In addition we also obtained small
amounts of A
-(1-42) and A
-(1-37/38/39) (Fig. 1C).
Upon expression of moderate levels of BACE we observed an
additional A
species (A
-(1-34); Fig. 1C). In order to
analyze if the production of this truncated species is related to BACE
expression levels, we next investigated A
species secreted from
cells expressing higher levels of BACE (Fig. 1A). This
revealed robust amounts of A
-(1-34), which was accompanied by
reduced levels of A
-(1
40), A
-(1
42), and A
-(1-37/38/39) (Fig. 1C). Similar results were obtained using cell lines
co-expressing wtAPP and BACE (data not shown). The detection of robust
levels of A
-(1-34) upon expression of BACE explains the lack of its detection upon metabolic labeling, since the single radioactively labeled Met residue at position 35 of the A
peptide has been removed
by the additional cleavage.
View larger version (28K):
[in a new window]
Fig. 1.
Production of
A -(1-34) correlates with BACE expression.
A, HEK 293 cells stably overexpressing
APP695
sw (control; lane 1) or
APP695 sw and BACE
(lanes 2 and 3) were labeled with
[35S]methionine. BACE was immunoprecipitated from cell
lysates with antibody 7520. B, conditioned media were
analyzed for A
accumulation by immunoprecipitation with antibody
3926. C, MALDI-TOF MS of A
peptides immunoprecipitated
from conditioned media of the three cell lines with antibody 3926. Arbitrary intensities are given on the y-axis
(a.i.). The tables below the spectra indicate the
peak masses obtained by mass spectrometry (mass) and the
respective calculated masses (mass calc.). Note that the
relative levels of A
-(1-34) correlate with increasing amounts of
BACE expression while other A
species are reduced.
Although the above described results suggest that BACE is directly
involved in the enhanced production of A-(1-34), previous observations indicated that C-terminally truncated A
species including A
-(1-34) are generated by the
-secretase complex in a
PS-dependent manner (52, 53). In order to analyze if a
PS-dependent
-secretase activity is required for
A
-(1-34) generation, we co-expressed BACE with either PS1 wt or the
non-functional PS1 D385N mutant (Fig.
2A). As shown previously (42),
PS1 wt undergoes endoproteolysis whereas no endoproteolysis was
obtained in cells expressing PS1 D385N (Fig. 2A,
right panel; Ref. 27). The non-functional PS1 D385N fully
replaced biologically active endogenous PS (Fig. 2A,
right panel). Whereas robust levels of A
-(1-34) and
A
-(1-40) (and all other minor A
species) were produced from
cells co-expressing PS1 wt and BACE, A
-(1-34) generation as well as
generation of all other A
species was almost completely inhibited in
the presence of the non-functional PS1 D385N (Fig. 2B,
right panel). This clearly demonstrates that a
PS-dependent
-secretase activity is involved directly or
indirectly in the production of A
-(1-34). However, the results
described in Fig. 1, demonstrated that upon BACE expression A
-(1-34) generation occurs to the expense of the production of all
other A
variants and thus suggests a direct involvement of BACE in
the cleavage of A
at position 34. This apparent paradox may indicate
that
-secretase activity is required first to produce secreted A
species, which are then trimmed at their C termini by a so far unknown
BACE activity.
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In order to prove this hypothesis, synthetic A-(1-40)
was incubated with purified BACE isolated from conditioned media of cells secreting a soluble version of BACE lacking the transmembrane domain and the C terminus (43). To prove the catalytic activity of
secreted BACE we carried out in vitro assays (46). Soluble BACE was fully active in the in vitro assay, whereas no
activity was obtained in control media (Fig.
3A). This activity was fully blocked by the BACE-specific inhibitor GL189 (Fig. 3A). Upon
incubation of synthetic A
-(1-40) with soluble BACE an additional
peptide, which co-migrated with synthetic A
-(1-34) was detected on
a gel system capable to separate low molecular weight peptides (Fig. 3B; Ref. 54). In vitro generation of A
-(1-34)
was completely inhibited upon addition of the specific BACE inhibitor
GL189 (Fig. 3B). Using MALDI-TOF MS we confirmed a
time-dependent generation of A
-(1-34) during incubation
of soluble BACE with A
-(1-40) (Fig. 3C). In contrast
incubation of synthetic A
-(1-40) with conditioned media, not
containing soluble BACE does not reveal truncated A
species (Fig.
3C). This finding demonstrates that BACE has the ability to
cleave A
after amino acid 34 and, together with the results shown in
Fig. 1, excludes artificial trimming by exopeptidases.
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Because in vivo, only very minor amounts of BACE are
secreted (data not shown and Ref. 55) we next investigated if
membrane-bound BACE can convert secreted A-(1-40/42) to
A
-(1-34) in living cells. To do so we collected conditioned media
(Fig. 4A) from cells
expressing
APPsw and endogenous BACE. These media were then added
either to cells expressing PS1 D385N alone or to cells co-expressing
PS1 D385N and BACE. Because of the lack of
-secretase activity in
the latter cell line almost no de novo synthesis of any A
species occurs (Ref. 42; compare also Fig. 2B). Therefore any truncation of A
should occur independent of
-secretase activity. In conditioned media incubated with cells
expressing PS1 D385N no detectable conversion of A
-(1-40) to
A
-(1-34) was observed (Fig. 4B). However, upon addition
of the conditioned media to cells expressing both PS1 D385N and BACE,
A
-(1-34) was readily produced (Fig. 4C). Taken together
these results demonstrate that BACE can proteolytically modify A
species, which were originally produced by a
-secretase-dependent pathway.
|
While BACE is the protease with the major -secretase activity
(15-17), the homologous BACE-2 can also proteolytically process
APP
to some extend (9, 13, 14, 56). To investigate if the BACE homologue
BACE-2 may also be able to convert A
-(1-40/42) into A
-(1-34) we
co-expressed wild type
APP together with either moderate or high
levels of BACE-2 (Fig. 5A).
Low level expression of BACE-2 allowed the recovery of very small
amounts of A
-(1-34), which were not detectable in cells expressing
no exogenous BACE-2 (Fig. 5B). However, high level
expression of BACE-2 allowed the generation of increased amounts of
A
-(1-34) thus demonstrating a BACE-2-dependent
generation of A
-(1-34). These data demonstrate that both BACE and
BACE-2 have the unexpected ability to generate C-terminally truncated
A
species.
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DISCUSSION |
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BACE plays a central role in the pathogenesis of AD and appears to
be the sole -secretase, since its knock-out in mice fully abolishes
A
generation (15-17). In addition and in contrast to the loss of
-secretase activity the knock out of BACE has no obvious phenotype
(15-17). Thus BACE became a primary target for the development of
therapeutic strategies. However, very little is known about the
biological function of BACE. In addition we do not yet know precisely
where the major proteolytic acitivity of BACE is localized within the
cell. BACE is co-translated into the endoplasmic reticulum (ER) as a
pro-enzyme (43, 57). During its trafficking through the secretory
pathway the pro-domain is removed, and complex glycosylation occurs
(43, 57-61). Upon reaching the plasma membrane BACE is reinternalized
and targeted to endosomes (44, 62). From endosomes BACE is retrieved in
a phosphorylation-dependent manner and transported back to
the trans-Golgi network (44). Although BACE has an acidic pH
optimum it is apparently active in early compartments such as the ER,
since small amounts of A
can accumulate in pre-Golgi compartments
(45). Work on the Swedish
APP mutation also demonstrated
-secretase activity within the trans-Golgi network (64).
In contrast wild type
APP is apparently processed by BACE within
early endosomes after reinternalization from the plasma membrane (65).
So far no proteolytic activity of BACE was demonstrated on the cell
surface. Here we show that A
can be truncated by BACE at its C
terminus after its generation by
-secretase (Fig.
6). Thus it appears likely that secreted A
is further processed by BACE at or close to the plasma membrane although we cannot exclude uptake of A
prior to its processing by
BACE.
|
Our data demonstrate a novel cleavage site at position 34 of the A
domain. This was unexpected since full-length
APP is cleaved by BACE
in a highly sequence-specific manner (66). Moreover, previous work
demonstrated that in vivo a membrane bound substrate is
required for recognition by BACE (66). Obviously, soluble A
escapes
these requirements and is cleaved by BACE after amino acid 34 of the
A
domain. This suggests that the initial cleavage of BACE at the
Met-Asp bond of the A
domain occurs in a different structural
context than the secondary cut at position 34. This latter cleavage
occurs only after A
-(1
40/42) generation, whereas the first
cleavage requires the membrane bound precursor with a specific
recognition sequence at the Met-Asp bond. Furthermore, BACE-2, which
differs in its cleavage specificity of
APP (Fig. 6) by predominantly
generating an
-secretase-like cleavage after amino acid 19 of the
A
domain (9, 13, 14) is also able to generate A
-(1
34) from
A
-(1
40/42). Both enzymes apparently share similar sequence
requirements for recognition and cleavage of soluble A
but different
preferences for cleavage of full-length
APP. This may also have
implications for the search for physiological substrates of BACE. So
far only a sialyltransferase (ST6 Gal 1) has been identified as a
putative BACE substrate beside
APP (67). Our results suggest that
substrate-mimicking peptides have to be considered as natural BACE
substrates in vivo.
A-(1-34) has been shown to exist in vivo (29). The novel
cleavage activity of BACE and BACE-2 removes the most hydrophobic sequences of the A
domain. This may inhibit aggregation and thus facilitate proteolytic clearance by the insulin-degrading enzyme (68)
or neprilysin (63, 69). Thus BACE may play an unexpected role in A
clearance.
Finally, our data also demonstrate that an apparently PS- and
-secretase-dependent cut is in fact mediated by BACE.
Since the cleavage after amino acid 34 is fully dependent on the
previous PS/
-secretase cleavage, the involvement of proteases in the
generation of C-terminally truncated A
peptides may have been
misinterpreted (52, 53). Moreover, our results also solve the apparent
paradox that increased BACE expression results in reduced A
production (2), since the truncated A
-(1-34) species cannot be
detected by autoradiography due to the loss of the Met residue at
position 35 of the A
domain. Furthermore, under the electrophoretic
conditions used (54), the peptide aberrantly migrates at a higher
molecular weight as expected, again confusing the analysis of A
produced in BACE-expressing cells. Finally our findings may also
suggest that substrate-mimetic peptides could represent a novel
therapeutic approach in vivo.
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ACKNOWLEDGEMENTS |
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We thank H. Steiner, P. Kahle, and A. Capell for critical discussion and Klaus Maskos and Luis Moroder for the generation of the BACE inhibitor GL189.
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FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 596-Project B1 (to C. H.) and the Priority Program (to J. W., C. H., and G. M.)) and the DIADEM Project (to C. H.).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.
b These authors contributed equally to this work.
d Present address: Dept. of Chemistry/Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
g Present address: Dept. of Neurology, University of Bonn; Sigmund-Freud-Str. 25, 53105 Bonn, Germany.
h These authors contributed equally to this work.
i To whom correspondence may be addressed. Tel.: 49-89-5996-471/472; Fax: 49-89-5996-415; E-mail: chaass@pbm.med.uni-muenchen.de.
j To whom correspondence may be addressed. Tel.: 49-228-2879782; Fax: 49-228-2874387; E-mail: jochen.walter@ukb.uni-bonn.de.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M211485200
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ABBREVIATIONS |
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The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid
-peptide;
APP,
-amyloid precursor
protein;
PS, presenilin;
BACE,
-site APP-cleaving enzyme;
sBACE, soluble BACE;
wt, wild type;
HEK, human embryonic kidney;
MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.
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