From the Center for Human Genetics, Molecular
Oncology and
Neuronal Cell Biology Laboratories, Katholieke
Universiteit Leuven and Flemish Interuniversitary Institute for
Biotechnology, Herestraat 46, 3000 Leuven, Belgium and
ZMBH, University of Heidelberg,
Heidelberg, Germany
Received for publication, August 2, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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The amyloid peptide is the main constituent of
the amyloid plaques in brain of Alzheimer's disease patients. This
peptide is generated from the amyloid precursor protein by two
consecutive cleavages. Cleavage at the N terminus is performed by the
recently discovered The brain of patients suffering from Alzheimer's disease
(AD)1 is characterized by the
presence of amyloid plaques composed mainly of the 39-42 amino acid
amyloid Only recently the molecular identity of these enzymes has been
elucidated. Although this evidence is impressive, only limited information is
available on the cell biology of Bace. Bace is an
N-glycosylated transmembrane protein encoded in a 501-amino
acid open reading frame, from which the first 21 amino acids correspond
to the signal peptide. N-terminal sequencing of Bace purified from
human brain revealed that the mature protein starts at glutamic acid 46 (10), indicating that Bace is further processed after its translocation into the endoplasmic reticulum. Other proteases, e.g.
proprotein convertases (PCs) and members of the ADAM family, are also
synthesized as inactive proenzymes that require the removal of the
propeptide to become active (13). It has recently been shown that
pro-Bace is predominantly located in the endoplasmic reticulum and that constitutive propeptide cleavage takes place in the Golgi apparatus C-terminal to the Arg-Leu-Pro-Arg motif (14, 15), suggestive for the
involvement of members of the PC family in this process. PCs are
subtilisin-like serine proteases involved in the activation of many
neuropeptides, peptide hormones, growth and differentiation factors,
membrane-associated receptors, adhesion molecules, blood coagulation
factors, plasma proteins, and some pathogenic proteins like viral coat
proteins and bacterial toxins (13, 16, 17). Precursors are usually
cleaved C-terminal to basic motifs like Lys/Arg-(X)n-Lys/Arg, where n = 2, 4, or 6 and X is essentially any amino acid but Cys and
rarely Pro (13). Seven members have been thus far isolated as follows:
furin, PC1 (also called PC3), PC2, PC4, PC6 (also called PC5), PACE4,
and LPC (also called PC7 or PC8). All enzymes have a specific, albeit
partially overlapping, expression pattern and similar but not identical
substrate specificities.
Recently furin was implied in the production of amyloidogenic peptides
in familial British dementia (18, 19). This observation stimulated us
to ask whether proteases of the PC family could be involved in the
regulation of the activity of Bace. Although the answer to this
question is important from a cell biological point of view, we
obviously also speculated that new insights in the regulation of Bace
activity could foster new ideas for therapeutic intervention in AD.
We investigate here the posttranslational maturation of Bace in cells
in culture, and we demonstrate that mainly furin but, in addition,
although to a lesser extent, other PCs like PACE4, LPC, PC6A, and PC6B
could cleave the Bace propeptide in vivo, indicating some
redundancy in this controlling step. We find also that Bace activity on
APP is not significantly affected by the absence of furin or by PC
inhibitors, strongly suggesting that pro-Bace can process APP. We
conclude that it is unlikely that the proteolytic maturation of
pro-Bace is a valid therapeutic target.
Cloning and Mutagenesis of Bace--
Two primers were designed
based on the sequence of mouse Bace/Asp2 cDNA
(GenBankTM accession number AF200346 (11)), corresponding
to positions 1-23 (5'atggccccagcgctgcactggct3', sense primer) and
1483-1507 (5'tcacttgagcagggagatgtcatc3', antisense primer) and
used for amplification. The PCR product was cloned into pGEM-T
(Promega). This was subsequently used as template to introduce a
C-terminal Myc tag in Bace and sBace. The latter encompasses the entire
ectodomain of Bace but lacks the transmembrane domain and cytoplasmic
tail. The sense primer contained the start codon, preceded by a
BamHI restriction site (5'ctcggatccatggccccagcgctgcactgg3'),
the antisense primer contained the Myc tag, followed by a stop codon,
and an EcoRI restriction site (Bace,
5'ctcgaattctacaagtcctcttcagaaatgagcttttgctccttgagcagggagatgtcatc3'; sBace,
5'ctcgaattcctccaagtcctcttcagaaatgagcttttgctcataggctatggtcataagtg3'). The PCR products were digested with BamHI and
EcoRI and cloned in pcDNA3 (Invitrogen). Bace-ALPA and
sBace-ALPA, in which the propeptide cleavage site RLPR Antibodies--
A polyclonal antibody was raised in New Zealand
White rabbits against a synthetic polypeptide (ETDEEPEEPGRRGSFV)
corresponding to the region immediately C-terminal to the propeptide,
coupled to keyhole limpet hemocyanin. Generation of antibody GM 190, directed against the propeptide of Bace, has been described before
(15). Mouse anti-Myc monoclonal antibody clone 9E10 was used to detect the Myc-tagged Bace proteins. All antibodies used for immunodetection of the different members of the proprotein convertase family were obtained from Alexis Biochemicals. The polyclonal antibodies against the APP C terminus and against the ectodomain of APP have been described elsewhere (4). Monoclonal 4G8 and 6E10 antibodies were raised
against A Immunofluorescence--
Hippocampal neurons were cultured from
embryonic day 17 C57 black embryos and co-cultured with a glial
feeder layer. At day 15 post-plating, neurons were fixed using 4%
paraformaldehyde in 0.1 M phosphate buffer for 30 min at
room temperature followed by ice-cold methanol/acetone incubation to
permeabilize the cells (36). After blocking (4 °C, overnight),
neurons were incubated with primary antibodies anti-Bace polyclonal and
anti- Cell Lines and DNA Transfer--
Medium, serum, and supplements
used for the maintenance of cells were obtained from Life Technologies,
Inc. Chinese hamster ovary (CHO) and the furin-deficient derivative
RPE.40 cells (21), N2A, and COS cells were maintained in Dulbecco's
modified Eagle's medium/F12 (1:1) supplemented with 10% fetal calf
serum. 8-10 × 105 cells/10-cm2 culture
plate were transfected with 2 µg of DNA and 6 µl of Fugene (Roche
Molecular Biochemicals) and were used for experiments the next day (CHO
and RPE.40) or after 2 days (N2A and COS cells).
Radiolabeling and Immunoprecipitation--
Cells (8-10 × 105 cells/10 cm2) were starved for 1 h in
methionine-free RPMI 1640 medium and then labeled in the same medium containing 100 µCi/ml [35S]methionine and chased with
Dulbecco's modified Eagle's medium/F12 (1:1) for the times indicated
in the figure legends. In case of overnight labeling, 5% dialyzed
fetal calf serum was added to the labeling medium, and starvation was
omitted. For immunoprecipitation of Bace and PCs, cells were lysed in 1 ml of DIPA (50 mM Tris/HCl, pH 7.8, 150 mM
NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS).
Immunoprecipitations and endoglycosidase H and F digestions were
performed as described (22, 23). Immunoprecipitation of Bace and Bace
mutants with either anti-Myc 9E10 or anti-Bace antibody gave identical
results (data not shown). The figure legends indicated which antibody
was used.
To study APP processing, cells were cotransfected with plasmids
encoding APP, Bace constructs, furin, and PDX as indicated in the
figures. Twenty four (for CHO and RPE.40 cells) or 48 h (N2A and
COS cells) after transfection, cells were pulse-labeled for 4 h
and immediately lysed. APP full-length and C-terminal fragments (CTFs)
were immunoprecipitated from the cell extracts, whereas A
To discriminate between APPs originating from One-dimensional Isoelectric Focusing--
Separation of Bace
immunoreactive bands in isoelectric focusing was performed essentially
as described (25). Briefly, a 5% acrylamide (w/v) reducing gel
containing 2% Triton X-100, 9.1 M urea, 4% ampholytes, pH
5-7, and 1% ampholytes, pH 3.5-10, was run 13-16 h at a starting
voltage of ~20 V/cm and a limiting voltage of 50 V/cm. Prior to IEF
samples were deglycosylated with endoglycosidase F as described above.
Bace Subcellular Localization in Hippocampal Neurons--
We used
confocal microscopy to study the intracellular localization of Bace in
primary cultures of hippocampal neurons (Fig. 1). Mouse neurons indeed express Bace,
and some overlap in the distribution of Bace and the Golgi marker
Biosynthesis of Bace--
We next cloned and sequenced the
cDNA encoding Bace from a brain-specific mouse cDNA library
(Stratagene) and confirmed its identity to the sequence published by
Yan et al. (11). To characterize the biosynthesis and
maturation of Bace, pulse-chase experiments were performed. Transiently
transfected CHO cells were radiolabeled and chased for various times
(Fig. 2A). Immediately after
the pulse labeling, three specific protein bands were observed as follows: a major one migrating with an approximate mass of 65 kDa and two minor ones migrating at 50 and 75 kDa, consistent with
previous reports (14, 15). The 50-kDa protein disappeared during the
chase, whereas the amount of 65-kDa protein decreased but remained
prominent and the 75-kDa increased during the first 2 h of chase
and remained constant afterward. Deglycosylation experiments with
endoglycosidase H (Fig. 2B) demonstrated that the 75-kDa
protein but not the 65-kDa protein carries complex-type oligosaccharides. Removing all N-linked sugars using
endoglycosidase F resulted in a single band of 50 kDa. This indicates
that the three protein species contain the same polypeptide backbone.
The 50-kDa protein is therefore the unglycosylated Bace precursor; the
65-kDa protein is Bace containing simple N-linked
oligosaccharides; and the 75-kDa species finally is the fully complex
glycosylated protein. The large shifts in molecular weight upon
glycosylation indicate that the four potential glycosylation sites of
Bace are probably all utilized, although some heterogeneity is possible (14).
Analysis of the N terminus of Bace reveals a potential propeptide of 24 amino acids, ending in the sequence Arg-Leu-Pro-Arg45.
Basic motifs are often recognized and cleaved by PCs (13, 16, 17).
Since Bace purified from human brain or transfected cells starts at
Glu46 (9-11), it is therefore likely that Bace is
processed by a member of this PC family. Since the basic residues are
essential for recognition by PCs (13), we substituted the arginine
residues at the P4 and P1 positions in the basic motif (Arg-Leu-Pro-Arg
We also generated the soluble forms of Bace-WT and Bace-ALPA (sBace-WT
and sBace-ALPA, respectively) lacking the transmembrane and cytoplasmic
domains. Upon transfection with both constructs, soluble Bace is
synthesized and secreted and can be recovered from the conditioned
medium (Fig. 2D). The soluble secreted sBace-WT and
sBace-ALPA are complex glycosylated and an endoglycosidase H
glycosidase-sensitive precursor can be detected in the cells (data not
shown). This indicates that sBace matures during its traffic through
the secretory pathway in a way similar to the wild-type full-length
protein, validating it as a tool for our further investigations.
Cleavage of the propeptide should decrease the molecular mass of
pro-Bace by 2,560 Da. From the experiments described above it is clear
that this small difference between pro-Bace and processed Bace is too
small to allow discrimination in the gel electrophoresis system used.
We noticed, however, that the propeptide of Bace contains 4 arginines
and no acidic residues, whereas the mature Bace protein is acidic in
nature. We therefore decided to use isoelectric focusing (IEF) to
separate processed from unprocessed Bace.
We used the furin-deficient CHO cell strain RPE.40 (21) to determine
the possible role of furin in the processing of both Bace and sBace. We
studied first whether sBace became processed by the PCs, because the
pattern of protein bands to be analyzed is much less complex than that
for wild type Bace (see Fig. 2). Cotransfection of sBace with furin
cDNA resulted in further migration of the immunoprecipitated
protein toward the anode, at the bottom of the gel (Fig.
3A), suggesting that the
propeptide had indeed been removed by furin. This was confirmed using
the propeptide-specific antibody APP Processing by Wild-type and Mutant Bace--
To determine
whether the prodomain cleavage and membrane anchorage of Bace affect
The Bace-ALPA mutant is, as expected, as active as wild-type Bace in
cleaving APP (Fig. 6, A and B). Expression of
sBace, finally, induces cleavage of APP mainly at position
Asp1 (Fig. 6, A and B). The fact that
sBace does not cleave at Glu11 when overexpressed in
vivo suggests that sBace is less efficient in reaching the APP
substrate or at least the Glu11 site, which is closer to
the cell membrane than the Asp1 site (9) and that the
transmembrane domain of Bace is needed to allow for efficient cleavage
at the Glu11 site.
In conclusion, we confirm and extend previous work that demonstrated
the glycosylation and maturation of Bace in the secretory pathway (14,
15). We demonstrate in particular that pro-Bace is processed by furin
to its mature form. There is redundancy in the proteolytic maturation
of Bace, since other members of the PC family can compensate for loss
of furin activity. We present evidence suggesting that this maturation
step is not essential for the
Finally, our finding that pro-Bace is able to cleave APP implies that
it could be active as a -secretase (Bace). This aspartyl protease
contains a propeptide that has to be removed to obtain mature Bace.
Furin and other members of the furin family of prohormone convertases are involved in this process. Surprisingly,
-secretase activity, neither at the classical Asp1 position nor at the
Glu11 position of amyloid precursor protein, seems to be
controlled by this maturation step. Furthermore, we show that
Glu11 cleavage is a function of the expression level of
Bace, that it depends on the membrane anchorage of Bace, and that
Asp1 cleavage can be followed by Glu11
cleavage. Our data suggest that pro-Bace could be active as a
-secretase in the early biosynthetic compartments of the cell and
could be involved in the generation of the intracellular pool of the
amyloid peptide. We conclude that modulation of the conversion of
pro-Bace to mature Bace is not a relevant drug target to treat Alzheimer's disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(A
) peptide (1, 2). A
derives from a type I single
membrane-spanning protein termed amyloid precursor protein (APP) by
post-translational proteolytic cleavage (3). Two cleavages by
-and
-secretases, respectively, are required to release A
from APP.
-Secretase is apparently a large complex, with presenilin being an essential component of it (4-7).
-Secretase has
been identified independently by 5 groups and was named Bace (beta-site APP cleaving
enzyme), Asp-2, or memapsin 2 (membrane-anchored aspartic
protease of the pepsin family) (8-12). Bace is a type I integral
membrane protein, with a typical aspartyl protease motif in its luminal
domain. Bace fulfills most of the requirements expected for a candidate
-secretase. It has broad tissue distribution with higher expression
in the brain (8-10). It localizes mainly in Golgi and endosomes (8,
9). Bace overexpression increases, and treatment of cells with
antisense oligonucleotides complementary to Bace mRNA decreases
-secretase cleavage of APP (8-12). Bace is a transmembrane protein
whose predicted topology is correct with respect to the
-secretase
cleavage site in APP. It cleaves more efficiently APP carrying the
Swedish mutation than wild-type APP (9-12). The purified enzyme
cleaves synthetic APP substrates encompassing the
-secretase site
(9-12). Finally, Bace has an acidic pH optimum and is resistant to the
aspartic protease inhibitor pepstatin A (9, 10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was mutated
into ALPA, were made using QuikChange Site-directed Mutagenesis Kit
(Stratagene), according to the suppliers guidelines and using Bace-Myc
as template. Note that these constructs also contain the Myc tag. All
constructs were verified by sequencing.
-(17-24) and A
-(1-16), respectively, and were
obtained from Senetek. Polyclonal rabbit 53/4 specifically recognizes
the
-secretase-generated neoepitope and was kindly provided by Dr.
Savage, Cephalon (20).
-COP monoclonal antibody (clone MAD, Sigma) for 2 h at
room temperature. For detection, Alexa488 and Alexa546 dyes coupled to
secondary antibodies (Molecular Probes) were used (1 h at room
temperature). Analysis was done on a NIKON inverted microscope DIAPHOT
300 (PlanApo 60/1.40 oil) connected to a Bio-Rad MRC1024 confocal
microscope, and images were captured by Lasersharp (version 3.2) and
processed using Adobe Photoshop 5.0 (Adobe, CA).
and total
secreted APP (APPs) were immunoprecipitated from the conditioned medium
as described (4, 24).
- versus
-cleavage, samples from the conditional medium were resolved by 10% PAGE. Western blotting was subsequently performed with either 6E10 or
53/4 antibodies.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-COP is observed. Bace is, however, also present in
-COP negative
vesicles, most likely endosomes. Previous studies have addressed the
issue of Bace subcellular distribution in non-neuronal cells using Bace
overexpressed from transfected cDNA (8, 9, 12, 14, 15). Our data
confirm these previous findings at the endogenous levels of expression and indicate that transfected Bace localizes to the relevant
subcellular compartments. Since the levels of endogenous expression of
Bace are very low (results not shown), further biochemical analysis to
characterize the maturation and the activity of Bace was performed in
transfected cells.
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Fig. 1.
Double immunofluorescent staining of
fully polarized hippocampal neurons for endogenous Bace
(red) and the Golgi marker protein
-COP (green). A partial
overlap is observed in the Golgi region. Notice abundant vesicular
stainings at the periphery of the Golgi apparatus which are likely
endosomal structures. Bar, 5 µm.
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Fig. 2.
Biosynthesis of Bace. CHO cells were
transfected with empty vector ( ), Bace cDNA (B),
Bace-ALPA cDNA (BALPA), or the soluble variants (sB and
sBALPA), radiolabeled for 1 h, and either chased for
the times indicated (A and B) or for 4 h
(C and D). Bace from cell lysates
(A-C) or medium (D) was immunoprecipitated with
monoclonal antibody 9E10 and analyzed on SDS-PAGE. A,
pulse-chase experiment indicating the conversion of a 50-kDa protein
toward a 65- and 75-kDa protein. The mobility of endoglycosidase
H-resistant (endoHR) and endoglycosidase H-sensitive
(endoHS) Bace, as well as the deglycosylated Bace
are indicated by arrows. B, immunoprecipitated
Bace was either untreated (
) or treated with endoglycosidase H
(H) or N-glycosidase F (F).
C and D, no major differences in glycosylation
(C) or secretion (D) of the Bace-ALPA mutants are
observed. The positions of the molecular weight markers are
indicated.
Ala-Leu-Pro-Ala). We called this mutant protein accordingly "Bace-ALPA." Upon expression in CHO cells, Bace-ALPA is apparently synthesized and N-glycan maturated in a way
indistinguishable from Bace-WT (Fig. 2C).
-pro-Bace which should not react
with mature sBace and which, in fact, failed to recognize the bands
generated in the presence of furin (Fig. 3B). The
heterogeneity of the bands corresponding to mature sBace is commonly
observed with other proteins in IEF (25). This can be explained by the
heterogeneity in the glycosylation pattern of sBace together with the
asparagine to aspartic residue conversion that occurs during the
deglycosylation prior to IEF. Similar results were obtained with
full-length Bace. Upon furin cotransfection part of pro-Bace was
converted to the mature Bace form (Fig. 3, C and
D; see also Fig. 5, A and C). The
fastest migrating band in Fig. 3C could be
immunoprecipitated by antibodies directed against mature Bace but not
by antibodies directed against the propeptide (D). This
indicates that this band represents processed Bace. We observed,
however, that a fraction of pro-Bace was resistant to furin treatment
(indicated as pro-BacePRE). Based on the pulse-chase
experiments shown in Fig. 2, from which it is clear that a large part
of wild-type Bace remains endoglycosidase-sensitive after 4 h of
chase, we conclude that this fraction represents newly synthesized,
immature glycosylated Bace. Since this pool is localized in the early
compartments of the secretory pathway, it is not accessible for furin,
which is only active in the late Golgi apparatus. The fraction of
protein that is labeled Pro-Bace in Fig. 3, C and
D, on the other hand, represents the fully glycosylated
protein that has reached the Golgi compartment and is therefore
sensitive to furin cleavage. To our surprise, both sBace-ALPA and
Bace-ALPA are efficiently processed in RPE.40, furin-deficient cells
(Fig. 3), and their processing is not inhibited by the PC inhibitor
1-PDX (not shown) in contrast to their wild-type Bace
counterparts. Furthermore, preliminary results using protease
inhibitors indicate that this cleavage is performed by a trypsin-like
protease (data not shown). It is therefore likely that the double ALPA
mutation creates a novel site that becomes artificially cleaved by a
non-PC, trypsin-like protease. Regardless of the identity of this
protease and the precise site of cleavage, it is clear that this
processing event is physiologically
irrelevant.2 In any event,
the fact that wild-type Bace (Bace-WT) and "wild-type" sBace are
poorly cleaved in furin-deficient cells, together with the rescue of
the cleavage process after furin expression, indicates strongly that
furin is involved in pro-Bace maturation in vivo. That other
members of the PC-family could rescue the cleavage of Bace-WT cannot,
however, be excluded. Therefore, all other PCs that have broad tissue
distribution, i.e. PACE 4, PC6 (isoforms A and B), and LPC,
were tested for their activity in sBace (Fig. 4) and Bace (Fig.
5) maturation. Expression of the enzymes
was confirmed (Fig. 5B). From Fig. 4, it is obvious that
only furin was able to process pro-sBace. Furthermore, endogenous
processing activity on pro-sBace was observed in CHO cells (right
panel) but not in RPE.40 cells (left panel), and
activity could be stimulated by expression of furin and inhibited by
expression of the PC inhibitor
1-PDX.
1-PDX is a genetically engineered serine protease
inhibitor derived from the trypsin inhibitor
1-antitrypsin and has been shown to inhibit efficiently
furin and to a lesser extent PACE4, PC6A, and PC6B (26, 27). The same
experiment was performed with wild-type Bace (Fig. 5). Although
overexpression of furin resulted in efficient processing of pro-Bace,
other PCs were capable of cleaving pro-Bace to a various extent as
well. This is unlikely to be a cell type-specific effect, since similar
results were obtained in the neuron-based cell line N2A (Fig.
5C). On the other hand, the data obtained with sBace suggest
that furin has a preponderant role in pro-Bace processing in
vivo.
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Fig. 3.
Processing of sBace, Bace, and cleavage site
mutants. Furin-deficient CHO cell strain RPE.40 was transfected
with empty vector ( ), sBace (sB), sBace and furin
(sB+Fur), sBace-ALPA (sBALPA), Bace and
furin (B+Fur), or Bace-ALPA (BALPA), and
radiolabeled overnight (A and B) or for 1 h
followed by 4 h of chase (C and D). Medium
(A and B) or cell lysates (C and
D) were incubated with the anti-Bace or anti-pro-Bace
antisera as indicated at the bottom of the IEF slab gels. The mobility
of the different proteins is indicated. Pro-BacePRE and
pro-Bace refer to the furin-resistant and -sensitive bands,
respectively (see text). The asterisk indicates an
unspecific protein band. Heterogeneous migration of proteins in IEF is
not uncommon. Notice that Bace containing the ALPA mutation is
processed in a furin-independent way. (see text).
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Fig. 4.
Processing of sBace by PCs. CHO and
RPE.40 cells were transfected with empty vector ( ), sBace alone, or
together with furin (Fur), PACE4, PC6A, PC6B, LPC, or
1-PDX and radiolabeled overnight. Cell culture medium
was incubated with anti-Bace antibody and analyzed on IEF slab gels.
The position of sBace and pro-sBace is indicated.
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Fig. 5.
Processing of Bace by PCs. CHO
(A), RPE.40 (A-C), and N2A (C) cells
were transfected with empty vector ( ), Bace alone, or together with
furin (Fur), PACE4, PC6A, PC6B, LPC, or
1-PDX. Cells were radiolabeled overnight (B)
or for 1 h followed by 4 h of chase (A-C). Cell
lysates were incubated with anti-Bace antibody (A-C) or
antibodies directed against specific PCs (B) and analyzed on
IEF slab gels (A-C) or SDS-PAGE (B).
Pro-BacePRE and pro-Bace refer to the furin-resistant and
-sensitive bands, respectively (see text). The asterisk
indicates an unspecific protein band.
-secretase activity, RPE.40 cells were cotransfected with plasmids
encoding APP, wild-type Bace, Bace-ALPA, sBace, sBace-ALPA, and furin
or
1-PDX as indicated in Fig.
6 (A-C). Processing of APP
was analyzed by in vivo labeling and immunoprecipitation of
cell-associated C-terminal fragments (CTFs, Fig. 6A), total
secreted APP (APPs, Fig. 6B, bottom), and A
peptides (Fig. 6B, top) or by Western blotting to discriminate
between APPs
and APPs
generated by
and
secretase,
respectively (Fig. 6C). Expression in RPE.40 cells of APP
alone resulted in cleavage of APP mainly at the
-secretase site, as
shown by the accumulation of CTFs starting at the
-cleavage site
(Fig. 6A) and by the fact that most of the secreted APP
corresponded to APPs
(Fig. 6C, lane 1).
-Stubs (Fig.
6A) and APPs
(Fig. 6C), on the other hand,
were almost undetectable, which is entirely consistent with data
obtained in many other cell lines showing that endogenous
-secretase
activity is very low, and APP processing is mainly by the
nonamyloidogenic pathway in non-neuronal cells (28). Coexpression of
Bace induced cleavage of APP at the 2
-secretase sites
(Asp1 and Glu11 (9)), with the expected
concomitant decrease in
-processing (Fig. 6, A and
C). Competition between
- and
-secretase for the
substrate APP has been reported previously (9-11, 29-31). It is
remarkable that CTFs (Fig. 6A) and secreted peptides (Fig. 6B) starting at position Glu11 are far more
abundant than those starting at Asp1 under the experimental
conditions used. The phenomenon is not specific for RPE.40 cells, since
similar results were obtained with N2A and COS cells (Fig.
6D). We speculated that Asp1 is still the
preferred
-secretase site under those conditions but that the 99 amino acids CTF that is generated by Asp1 cleavage
(CTF
1) can be further processed by overexpressed Bace to yield the
Glu11 C-terminal APP fragment (CTF
11). That this
interpretation is correct was proven by two different experiments.
First, if Bace cleaves preferentially at Asp1, then APPs
secreted into the medium should react with an antibody that
specifically recognizes the neoepitope generated after Asp1
cleavage. As shown in Fig. 6C, Bace transfection indeed
resulted in the substantial accumulation of secreted APP that contains this neoepitope (APPs
). Second, if the relative abundance of Glu11 cleavage in our experiments is a consequence of the
high levels of Bace expression, then decreasing Bace expression should
result in a switch toward Asp1 cleavage. This is, in fact,
what we observed (Fig. 7). Transfection of N2A cells with decreasing amounts of Bace cDNA resulted in decreasing levels of Bace protein expression (Fig. 7A, 2nd
panel). Immunoprecipitation of peptides secreted into the medium
showed that at high levels of Bace protein (1 µg of transfected
cDNA) most of the secreted A
peptides starts at
Glu11, whereas with decreasing Bace expression levels
(until they are undetectable in our assay) the majority of the secreted
peptide starts at Asp1 (Fig. 7A and
quantification in B). As expected, there was a direct correlation between the levels of Bace expression and the amount of
APPs
recovered in the medium, and decreasing levels of APPs
were
accompanied by increases in the amount of secreted APPs
(Fig.
7A, two lower panels). Altogether, these results show that the cleavage at Glu11 is a function of the expression level
of Bace. The Glu11 position is known to be a normal
cleavage site of Bace (see for example Vassar et al. (9)).
Moreover, peptides starting at this position are produced by primary
cultures of neurons and are also present in plaques of AD patients
(Ref. 28 and references therein). We therefore consider the
Glu11 cleavage as a reliable reflection of the Bace
activity in our experimental system. In addition we analyzed also the
production of APPs
, which reflects cleavage of APP at
Asp1. Our approach therefore allows us to evaluate the
proteolytic capacity of Bace at position Glu11 as well as
at position Asp1 and therefore to determine whether
propeptide processing is needed for Bace activity or not. In this
regard, processing of APP was observed even in the absence of furin,
suggesting that pro-Bace is an active enzyme (Fig. 6, A-C).
RPE.40 cells lack furin, and little or no mature Bace is detected after
overexpression (Fig. 5A, lane 2). Since other PCs, at least
when overexpressed, can cleave pro-Bace (Fig. 5), one could argue that
enough mature Bace is synthesized in RPE.40 cells that would explain
the observed processing of APP. Although we cannot definitively rule
out this possibility, we consider this as very unlikely, since
coexpression of
1-PDX, that inhibits several other PCs
in addition to furin, resulted in no detectable decrease in
-secretase cleavage as compared with Bace or Bace plus furin
(5th lane versus 3rd and 4th in Fig. 6A). Moreover, in preliminary
experiments, we found that when Bace is retained in the endoplasmic
reticulum by means of a KK motif, it is still capable of cleaving APP
(not shown). This Bace-KK is, as expected, not complex glycosylated.
Since propeptide cleavage occurs in the Golgi and trans-Golgi network, this mutant Bace-KK protein should still contain its propeptide.
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Fig. 6.
Processing of APP by Bace and Bace
mutants. RPE.40 (A-C), N2A (D), and COS
(D) cells were transfected with plasmids encoding APP, Bace,
furin or 1-PDX as indicated at the top of the
figures. Twenty four (for RPE.40 cells) or 48 h (for N2A and COS)
after transfection, cells were labeled for 4 h and lysed.
A, CTFs and full-length APP immunoprecipitated from cell
lysates. B, immunoprecipitation of APPs and A
from the
conditioned medium. C, Western blotting using antibodies
6E10 (top) and 53/4 (bottom) specific for
-secretase-cleaved APPs (APPs
) or
-secretase-cleaved APPs (APPs
) at the Asp1
site on samples from the conditioned medium. D,
immunoprecipitation of CTFs (upper panel), APP (middle
panel), and Bace (lower panel) from N2A
(left) and COS (right) cells that have been
cotransfected with APP and different Bace constructs, as indicated at
the top. B, Bace; BALPA,
Bace-ALPA; sB, sBace; sBALPA, sBace-ALPA;
1, CTF or A
starting at Asp1;
11, CTF or A
starting at Glu11;
, CTF starting at the
-cleavage site.
View larger version (31K):
[in a new window]
Fig. 7.
Effect of Bace expression level on
Asp1 and Glu11 cleavage of APP. N2A cells
were cotransfected with 1 µg of APP-encoding plasmid and the
indicated amounts of Bace-encoding plasmid (each transfection was with
2 µg of total DNA; empty vector was used to complete this amount).
A, cells were metabolically labeled, and the secreted
peptides (upper panel) and Bace (second panel)
were immunoprecipitated. A fraction of the conditioned medium was
analyzed by Western blotting to detect APPs and
forms
(lower two panels). B, PhosphorImager
quantification of the bands shown in the upper panel of
A. 1 and 11 refer to the start
position of the A
peptide.
-secretase activity of Bace on APP. We
conclude that the maturation of pro-Bace has little relevance as a
therapeutic target for Alzheimer's disease. Several other functions,
apart from inhibiting proteolytic activity of the proenzymes, have been found for propeptides, including roles in folding and intracellular transport. Possibly the propeptide of Bace is important for folding or
intracellular transport of pro-Bace. Alternatively, it is possible that
APP is not the only physiological substrate of Bace and that cleavage
of other yet unidentified Bace substrates is dependent on appropriate
removal of the propeptide.
-secretase in early biosynthetic cell
compartments, i.e. in endoplasmic reticulum and early Golgi. Therefore, it is likely that pro-Bace is involved in the generation of
the intracellular amyloid peptide pool as well (32-35). This pool is
considered by some investigators as the real culprit in Alzheimer's disease.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Gary Thomas (Portland, OR) for
providing us with 1-PDX cDNA and Dr. Mary Savage
(Cephalon) for the antibody 53/4 specific for the free C terminus of
-secretase-cleaved APPs.
![]() |
Addendum |
---|
While this work was under revision, a paper by Benett et al. (37) suggested that furin is responsible for the proteolytic removal of the propeptide from Bace.
![]() |
FOOTNOTES |
---|
* This work was supported by the European Union EU-TMR network ERBFMRXCT960023, the Fonds voor Wetenschappelijk Onderzoek, the Flanders Interuniversity Institute for Biotechnology, and the K. U. Leuven.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.
§ Both authors contributed equally to this work.
¶ Postdoctoral fellows of the "Fonds voor Wetenschappelijk Onderzoek, Vlaanderen."
** Postdoctoral fellow of EMBO.
§§ To whom correspondence should be addressed. Tel.: 32-16346227; Fax: 32-16347181, E-mail: Bart.Destrooper@med.kuleuven.ac.be.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M006947200
2 We have recently made a single amino acid substitution into the PC consensus cleavage site RLPR of Bace to generate a GLPR site. This mutant is not processed, in agreement with our hypothesis that the wild-type Bace is processed by a member of the PC family, whereas the ALPA mutant creates an artifactual cleavage site.
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ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
APP, amyloid precursor protein;
IEF, isoelectric focusing;
Bace, beta-site APP-cleaving
enzyme;
sBace, soluble Bace;
Bace-ALPA, Bace in which the
propeptide cleavage site RLPR is mutated into ALPA (single letter
amino acid code);
PCs, proprotein convertases;
PAGE, polyacrylamide gel
electrophoresis;
1-PDX,
1-antitrypsin
Portland;
A
, amyloid
;
CTFs, C-terminal fragments;
WT, wild type;
CHO, Chinese hamster ovary.
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