Sequential proteolysis of the
amyloid precursor protein (APP) by
- and
-secretase activities
yields the amyloid
peptide that is widely deposited in the brains
of individuals with Alzheimer's disease. The membrane-anchored
aspartyl protease
-site
APP-cleaving enzyme (BACE) exhibits
all of the characteristics of a
-secretase and has been shown to
cleave APP at its
-site in vitro and in vivo. We found that BACE undergoes cleavage on a surface-exposed
-helix between amino acid residues Leu-228 and Ala-229, generating stable N- and C-terminal fragments that remain covalently associated via a disulfide bond. The efficiency of BACE endoproteolysis was observed to depend heavily on cell and tissue type. In contrast to
brain where holoprotein was predominant, BACE was found primarily as
endoproteolyzed fragments in pancreas, liver, and muscle. In addition,
we observed a marked up-regulation of BACE endoproteolysis in C2
myoblasts upon differentiation into multinucleated myotubes, a well
established model system of muscle tissue specification. As in liver,
BACE exists as endoproteolyzed fragments in the hepatic cell line,
HepG2. We found that HepG2 cells are capable of generating amyloid
peptide, suggesting that endoproteolyzed BACE retains measurable
-secretase activity. We also found that BACE endoproteolysis occurs
only after export from the endoplasmic reticulum, is enhanced in the
trans-Golgi network, and is sensitive to inhibitors of vesicular
acidification. The membrane-bound proteases tumor necrosis factor
-converting enzyme and furin were not found to be responsible for
this cleavage nor was BACE observed to mediate its own endoproteolysis by an autocatalytic mechanism. Thus, we characterize a specific processing event that may serve to regulate the enzymatic activity of
BACE on a post-translational level.
 |
INTRODUCTION |
Alzheimer's disease
(AD)1 is characterized
pathologically by the florid accumulation of insoluble amyloid
peptide (A
) in the central nervous system (1). A causative role for
A
in AD progression has been accepted by many for some time (2, 3),
and recent evidence from mouse models has underscored the importance of
this peptide in the process of cognitive decline (4-6). A
is
produced by the sequential processing of the amyloid precursor protein
(APP) by two proteolytic activities that have historically been
referred to as
- and
-secretase. Alternatively, cleavage of APP
by so-called
-secretase activity within the A
region of the
protein precludes the generation of the peptide (7). Numerous studies
have identified the membrane-anchored aspartyl protease BACE as the
-secretase (8-12), whereas evidence continues to mount that
-secretase activity is mediated at least in part by the presenilin
proteins (PS1 and PS2) (13-17). A group of metalloprotease
disintegrins, including tumor necrosis factor-
converting enzyme
(TACE), appears to be responsible for at least a significant fraction
of cellular
-secretase activity (18, 19).
The central role of A
in AD pathogenesis requires careful study of
the proteins involved in APP/A
processing and metabolism. The
-secretase activity of TACE, for instance, may depend on C-terminal
phosphorylation of the enzyme by protein kinase C (18, 19). The
presenilins have also been demonstrated to undergo phosphorylation
(20-22), although the functional impact of these modifications remains
unclear. More thoroughly characterized is the endoproteolysis of PS1
and PS2, an event that generates distinct presenilin N- and C-terminal
fragments that are thought to represent the biologically active forms
of the proteins. At endogenous levels the cleavage fragments of PS1 and
PS2 predominate, and under conditions of overexpression, the vast
majority of the presenilins exist as holoproteins, presumably due to
the saturation of the endoproteolytic pathway (23-27).
Despite its fairly recent discovery, BACE has already been the subject
of numerous investigations designed to elucidate its basic cell
biology. During maturation in the secretory pathway, BACE undergoes
glycosylation at 3-4 N-linked sites and is separated from
its propeptide domain (28-30). This latter processing step is mediated
by a member of the proprotein convertase family of proteases (31, 32).
In addition, BACE is palmitoylated at cysteine residues within its
cytoplasmic domain and C-terminally phosphorylated (33, 34). Mature
BACE has a t1/2 of 12-16 h and cycles between the
cell surface, the endosomal system, and the TGN multiple times through
the course of its life span (29, 30, 34). The cleavage site specificity
of BACE for its APP substrate has been shown to depend heavily on the intracellular localization of the protease (35).
In this study, we describe a specific endoproteolytic event that may
have a significant effect on BACE activity. Whereas the existence of
BACE breakdown products has been documented (10), the protein fragments
themselves have not been well characterized, and the process by which
they are generated remains unclear. We demonstrate BACE endoproteolysis
occurs between amino acid residues Leu-228 and Ala-229, generating
stable N- and C-terminal fragments that remain associated in a
heterodimeric complex stabilized in part by a disulfide bond. This
endoproteolytic process is regulated in a cell type- and tissue
type-dependent fashion. We show that BACE endoproteolysis
does not appear to take place in neurons. However, the processing event
occurs robustly in a variety of native tissues and may play an
important role in the differentiation of muscle cells. We also
demonstrate that endoproteolyzed BACE appears to retain at least some
-secretase activity. Finally, we investigate the cell biological
determinants and molecular mechanisms underlying the cleavage of
BACE. Our results indicate that BACE, like the presenilins,
undergoes an endoproteolytic cleavage event with intriguing functional implications.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction and Expression--
The generation of BACE,
BACE-KK, and BACE-TGN constructs has been described (30, 35). TACE and
m-furin-HA were expressed in pcDNA3.1 (19, 36). Expression of DNA
constructs in cultured cells was obtained using Geneporter transfection
reagent (Gene Therapy Systems, San Diego) with a DNA/Geneporter ratio
of 1 µg/10 µl. For some experiments BACE and APP were expressed
using a replication-defective Semliki Forest virus (SFV) vector
expressing wild-type human BACE and wild-type APP695. The
vectors were generated by cloning cDNA into the pSFV-1 expression
plasmid. Virions were produced and titered as described elsewhere (37).
Antibodies--
Several C-terminal antibodies were used in the
recognition of BACE and BACE-Ct. BACE C-terminal antisera 1 is a rabbit
polyclonal antisera directed against the C terminus of BACE (30).
Polyclonal rabbit antisera (88.6) recognizing the C terminus of BACE
was generated by immunizing rabbits with the 17-amino acid synthetic peptide
Cys-Leu-Arg-Gln-Gln-His-Asp-Asp-Phe-Ala-Asp-Asp-Ile-Ser-Leu-Leu-Lys conjugated to keyhole limpet hemocyanin. Antibody pre-adsorption experiments were carried out with non-conjugated synthetic peptides. The specific competing peptide was the BACE C-terminal sequence listed
above. The nonspecific competitor peptide was a 17-amino acid peptide
sequence in the BACE N terminus
(Glu-Thr-Asp-Glu-Glu-Pro-Glu-Glu-Pro-Gly-Arg-Arg-Gly-Ser-Phe-Val-Glu). Another affinity-purified rabbit polyclonal antibody recognizing the BACE C terminus was obtained from Affinity Bioreagents. HA-tagged BACE constructs were recognized with either a monoclonal antibody (mAb
HA11; Covance, Richmond, CA) or a rabbit polyclonal antisera (rb-HA11;
Covance) directed against the HA epitope. The N terminus of APP was
recognized with the goat polyclonal antisera Karen (38). APP C terminus
was detected with a rabbit polyclonal antibody (Zymed
Laboratories Inc.), and A
was detected with 4g8 and 6e10 (Signet). Immunoprecipitations and Western blots were performed using
standard methods. Radiolabeled gels were exposed to PhosphorImager screens which were then read 24-48 h later on a Storm 860 Scanner (Amersham Biosciences) and analyzed in ImageQuant version 1.2 (Amersham
Biosciences).
Cell Culture and Chemical Reagents--
Human embryonic kidney
293 cells, HepG2, and baby hamster kidney (BHK) cells were cultured in
Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented
with 10% fetal bovine serum (FBS). C2 cells (39) were cultured in DMEM
supplemented with 10% FBS. The C2 cells were induced to differentiate
by switching to DMEM, 1% FBS, and 1 µg/ml insulin (Sigma) for 4 days. All media were additionally supplemented with 1%
penicillin/streptomycin. In certain experimental situations, additional
chemical reagents were utilized at the following concentrations:
phorbol 12-myristate 13-acetate (Sigma), 10 µM;
bafilomycin A1 (Sigma), 1 µM; chloroquine (Sigma), 100 µM.
Metabolic Labeling and Pulse-Chase Experiments--
Cells
expressing BACE were preincubated in methionine/cysteine-free DMEM
supplemented with 10% dialyzed FBS (starve medium) for 30 min after
which they were incubated in the same medium containing 0.5 mCi of
[35S]cysteine/methionine per well for a predetermined
period. The cells were then washed once in phosphate-buffered saline
(PBS), lysed in radioimmunoprecipitation assay (RIPA) buffer (1%
sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 5 mM
EDTA, 50 mM Tris, pH 8, 150 mM NaCl), and
quantitatively immunoprecipitated with the desired antibody. For
pulse-chase experiments, cells were metabolically labeled as described
above for 30 min after which they were quenched with starve medium
containing a 100 molar excess of cold methionine/cysteine. The cells
were then chased in normal growth medium for times ranging from 1 to
24 h, washed in PBS, lysed in RIPA, and immunoprecipitated with
the desired antibody.
In some experiments wild-type human APP695 or wild-type
human BACE were expressed using recombinant, replication-defective SFV
vectors (37). In these experiments the cells were incubated with virus
using a multiplicity of infection of 2-10 for 1 h in DMEM without
serum. Cells were rinsed and incubated in normal growth medium for
~3-4 h prior to metabolic [35S]methionine labeling as
described above. For experiments expressing APP695wt in
HepG2 cells, SFV-infected cells were metabolically labeled for 6 h
following a 4-h incubation period.
N-terminal Radiosequencing--
Custom DMEM deficient in either
methionine or isoleucine was prepared using a minimum Eagle's
medium-
Selectamine kit (Invitrogen) and supplemented with 10%
dialyzed FBS. 293 cells expressing HA-tagged BACE were preincubated in
either the methionine-free or isoleucine-free medium for 30 min
followed by an 18-h incubation in the same media supplemented with
either [35S]methionine or [3H]isoleucine,
respectively. The cells were then washed in PBS, lysed in RIPA, and
immunoprecipitated with mAb HA11. Immunoprecipitates were eluted in 1×
Laemmli sample buffer plus 0.34 M
-mercaptoethanol, electrophoresed on 8% polyacrylamide gels, and transferred to ImmobilonTM-P polyvinylidene difluoride membrane
(Millipore). Bands corresponding to BACE-Ct were then cut from the
membrane and sequenced by automated Edman degradation collecting each
amino acid separately. The amino acids were assayed for radioactivity
in a Beckman LS 6500 scintillation counter.
Fluorescence Microscopy--
Antibody staining of HeLa cells was
performed as described previously (30). Briefly, cells were fixed and
permeabilized in ice-cold methanol followed by incubation in primary
antibody diluted in PBS supplemented with 2% FBS and 0.02% saponin.
BACE constructs were recognized using rb-HA11, and a monoclonal
antibody directed against GM-130 (Transduction Laboratories) was used
to detect Golgi. Cells were then incubated in fluorescent-conjugated secondary antibodies diluted in the same solution as the primaries. All
micrographs were taken with a Nikon E600 microscope utilizing UV illumination.
 |
RESULTS |
BACE Is Endoproteolytically Processed to Generate Stable N- and
C-terminal Fragments--
While characterizing the biochemical
properties of BACE under conditions of overexpression in cell culture,
we repeatedly noticed the presence of an ~37-kDa band by SDS-PAGE
whose immunoreactivity closely paralleled that of the BACE holoprotein.
This species could be readily demonstrated in cell lysates by Western
blot using a variety of antibodies directed against the C terminus of
BACE (Fig. 1, B and
C). Furthermore, peptide competition experiments confirmed
that the band was specific and not a background artifact (Fig.
1C). We interpreted these findings to suggest that a portion of the BACE holoprotein undergoes endoproteolysis generating a distinct
~37-kDa C-terminal fragment (BACE-Ct), a mechanism by which the
activity of the protein could be regulated (Fig. 1A). We
observed significant levels of BACE endoproteolysis in a variety of
cell types including human embryonic kidney 293 cells (Fig. 1B), baby hamster kidney (BHK) cells (Fig. 1C),
Chinese hamster ovary cells, HeLa cells, and N2a cells (data not
shown).

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Fig. 1.
A, schematic showing topological
models of BACE constructs used. Luminal and cytoplasmic domains are
indicated. White circles denote the positions of the
catalytic aspartic acid residues. The cytoplasmic domain of murine
furin used to design BACE-TGN is shown in blue. HA epitope
tag is shown in red. Endoproteolysis of BACE at Ala-229 is
also demonstrated, a process that generates the BACE cleavage products
BACE-Nt (green) and BACE-Ct (gray). B,
lysates from 293 cells expressing a C-terminally HA-tagged BACE
construct (lane B) or mock-transfected (lane M)
were subjected to Western blot with mAb HA11. Arrows
denote BACE-Ct. IP, immunoprecipitation. C, lysates from BHK
cells expressing untagged wild-type BACE were analyzed by Western blot
with 88.6 (rabbit polyclonal antisera recognizing BACE C terminus).
Blots were probed with antibody preadsorbed with specific
(S), nonspecific (NS), or no preadsorption
( ) (see "Experimental Procedures").
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By using metabolic pulse-chase analysis, we investigated the kinetics
of BACE endoproteolysis. 293 cells expressing BACE were radiolabeled
with [35S]methionine/cysteine for 30 min and chased in
non-radioactive media for various times up to 24 h, after which
the cells were lysed and subjected to immunoprecipitation. In these
experiments, BACE-Ct first emerged 4 h into the chase after which
the fragment accumulated slightly, reaching a maximum at the 12-h time
point, before being slowly turned over (Fig.
2A). The initial appearance of
BACE-Ct several hours after synthesis is characteristic of a
physiological breakdown product and is not consistent with BACE-Ct being a post-lysis artifact. In addition, our data indicate that endoproteolyzed BACE is stable and that the cleavage event itself does
not simply promote BACE degradation. Pulse-chase experiments were also
performed using an antibody directed against the N terminus of BACE
(Fig. 2B). These studies demonstrated the accumulation of an
~37-kDa N-terminal fragment (BACE-Nt) with kinetics parallel to those
of BACE-Ct. Thus, BACE endoproteolysis results in the production of
stable N- and C-terminal fragments.

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Fig. 2.
Pulse-chase analysis of BACE
endoproteolysis. A, 293 cells expressing BACE were
metabolically labeled for 30 min with
[35S]cysteine/methionine and chased in cold medium for
the indicated amount of time. Cells were then lysed and
immunoprecipitated with the C-terminal antibody BACE C-terminal
antisera 1. B, BHK cells infected with SFV-expressing
wild-type BACE were metabolically labeled for 45 min as in
A. Lysates for each time point were divided in half and
immunoprecipitated with BACE-Ct (88.6) or BACE-Nt antibodies.
Arrows denote BACE endoproteolytic fragments.
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BACE Endoproteolysis Occurs between Leu-228 and Ala-229, and the
Resulting Fragments Remain Associated after Cleavage--
The exact
site of BACE endoproteolysis was determined by N-terminal
radiosequencing of BACE-Ct after metabolic labeling with either
[35S]methionine or [3H]isoleucine (Fig.
3A), which demonstrated that
cleavage occurs between amino acid residues Leu-228 and Ala-229.
Analysis of the position of these residues in the published BACE
crystal structure (40) revealed that both lie in a solvent-exposed
-helix connecting the two extracellular lobes of the protein (Fig.
3, B and C). Cleavage at this site would split
apart the BACE catalytic domains and could potentially inactivate the
enzyme. However, structural studies have indicated the existence of a
disulfide bond between residues Cys-216 and Cys-420 (Fig.
3C) (29, 40). Such a connection could serve to maintain the
association of BACE-Ct and BACE-Nt even after endoproteolysis.

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Fig. 3.
A, graph showing the results of
N-terminal radiosequencing performed on BACE-Ct (see "Experimental
Procedures"). The positioning of radiolabeled methionine and
isoleucine residues identified the BACE endoproteolysis site at
Ala-229. B and C, the BACE endoproteolysis site
shown from two different views in the context of the published crystal
structure (40). The N-terminal and C-terminal extracellular lobes of
BACE are shown in green and gray, respectively,
and the catalytic aspartic acid residues of the protein are indicated
in red. The endoproteolysis site (white asterisk)
lies in a solvent-exposed -helix connecting the two extracellular
lobes of the protein (blue). Also shown is a disulfide bond
between residues Cys-216 and Cys-420 (yellow) covalently
linking the two extracellular domains of BACE. D, lysates
from 293 cells expressing HA-tagged BACE were treated with an
appropriate amount of Laemmli sample buffer with or without 2 M -mercaptoethanol (BME) and incubated at
either 37 or 55 °C for 10 min. Lysates from mock-transfected 293 cells were also included as negative controls ( ). The
samples were then subjected to SDS-PAGE and Western blot with mAb HA11.
In the presence of -mercaptoethanol, BACE-Ct could be readily
visualized (arrow), whereas in the absence of reducing agent
the fragment was undetectable.
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In light of this possibility, we asked whether a distinct BACE
C-terminal fragment could be visualized under non-reducing conditions
that should preserve the integrity of disulfide bonds. Triton X-100
lysates from 293 cells expressing BACE were analyzed by Western blot
after incubation at either 37 or 55 °C in the presence or the
absence of
-mercaptoethanol. Under non-reducing conditions, the BACE
holoprotein migrated at a lower apparent molecular weight than under
reducing conditions, suggestive of intramolecular disulfide bonding and
consistent with previous results (Fig. 3D) (30). Whereas
BACE-Ct was readily apparent in the presence of
-mercaptoethanol, it
could not be detected in the absence of reducing agent, consistent with
the notion that the BACE N- and C-terminal fragments remain covalently
associated by a disulfide bond even after endoproteolysis (Fig.
3D).
BACE Endoproteolysis Occurs Readily in Several Different
Non-neuronal Tissues and May Play a Role in Cellular
Differentiation--
To determine whether BACE endoproteolysis takes
place at significant levels in vivo, we assayed for the
presence of BACE-Ct in a variety of native tissue samples derived from
rat. In lysates from pancreas, liver, and muscle, Western blot
demonstrated high levels of a C-terminal BACE fragment that co-migrated
with BACE-Ct derived from BHK cells (Fig.
4A). BACE holoprotein was not
readily apparent in these tissues. By contrast, samples from cerebral cortex, cerebellum, diencephalon, and brain stem exhibited negligible amounts of BACE-Ct, whereas the holoprotein was clearly evident (Fig.
4A). Analysis of human tissue yielded similar results.
Samples from cerebral cortex and cerebellum revealed only BACE
holoprotein, whereas lysates from muscle tissue exhibited a large band
co-migrating with BACE-Ct. The specificity of the antibody was
confirmed by peptide blocking (Fig. 4B). To determine
whether BACE-Nt is also present at significant levels in native tissue,
we performed Western blots on rat muscle lysates using a BACE
N-terminal antibody (Fig. 4C). Although the N-terminal BACE
antibody (left panel) was less sensitive than the C-terminal
BACE antibody (right panel), the N-terminal antibody still
detected an ~35-37-kDa band co-migrating with BACE-Ct. The similar
size of the Nt- and Ct-BACE fragments in muscle is consistent with data
showing that immunoprecipitations using both Nt- and Ct-BACE antibodies
detected apparently identical, closely spaced doublet bands at ~37
kDa in size (Fig. 2B). This would be expected under
immunoprecipitating conditions because BACE-Nt and -Ct fragments remain
attached via a common disulfide bond. These findings are also in
keeping with the expected size of the fragments based upon the position
of the endoproteolytic cleavage. Thus these results indicate that N-
and C-terminal cleavage products of BACE endoproteolysis selectively
accumulate in cell lines and in vivo. The existence of
highly stable, BACE cleavage fragments suggests that this form of BACE
may have a specific physiological function in certain tissue and cell
types.

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Fig. 4.
A, Western blot with affinity-purified
BACE-Ct antibody shows relative amounts of BACE holoprotein and BACE-Ct
in lysates with equal protein content derived from rat frontal cortex,
cerebellum (cereb.), diencephalon (dienc.), brain
stem (b.stem), pancreas (pancr.), muscle, and
liver (two lanes for each tissue correspond to 25 and 12 µg of
protein). Transfected BHK cell lysate serves as positive control.
B, Western blot carried out as in A shows BACE
expression in lysates with equal protein content from human tissue
(cortex (CRX), cerebellum (CRB), and muscle
(MUS)). For peptide block, antibody pre-adsorbed with
BACE-Ct peptide was used on an equivalent companion blot (right
panel). C, Western blot of endogenous BACE-Nt
(left panel) and BACE-Ct (right panel) fragments
from rat muscle (duplicate lanes). B lane indicates
recombinant BACE overexpressed in BHK cells. D, Western blot
with affinity-purified BACE-Ct antibody shows differentiated mouse C2
cells (C2 Diff) endogenously express large amounts of
BACE-Ct in comparison to undifferentiated C2 myoblasts (C2).
In order to make low levels of BACE holoprotein visible, 8 × 105 undifferentiated cells were loaded per lane as compared
with 2 × 105 differentiated cells per lane.
N indicates endogenous BACE from primary rat cortical
neurons used as a positive control (note lack of BACE-Ct fragments).
Peptide block control was done with an equivalent companion blot.
E, differentiated C2 cells expressing SFV-derived wt BACE
were metabolically labeled for 30 min with
[35S]cysteine/methionine and chased in cold medium for
the indicated time. Cells were then lysed and immunoprecipitated with
the C-terminal antibody, 88.6. Lower panel is darker
exposure of the portion of the upper panel showing BACE-Ct.
Arrowheads indicate BACE-Ct. NI, non-infected
controls.
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We further investigated the endoproteolytic processing of BACE in
muscle tissue using C2 murine myoblasts that can be induced to
differentiate into multinucleated myotubes (39). We found that
differentiation dramatically altered the processing of endogenously expressed BACE. Undifferentiated C2 myoblasts exhibited relatively low
levels of BACE holoprotein but not BACE-Ct (Fig. 4D).
However, upon differentiation into myotubes, this expression pattern
was reversed. Whereas BACE holoprotein was down-regulated (note that four times the number of cells were loaded in the C2 lane
compared with the C2(Diff)
lane in order to visualize the holoprotein), BACE-Ct
expression was markedly up-regulated (Fig. 4D). The
specificity of the BACE antibody was confirmed by BACE-Ct peptide
antibody preadsorption (Fig. 4D). Pulse-chase analysis of
BACE metabolism in differentiated C2 myotube cultures (Fig.
4E) confirmed the existence of a precursor-product
relationship between BACE and BACE-Ct in muscle tissue with kinetics
similar to those observed in other non-neuronal cell types (Fig. 2,
A and B). These findings demonstrate that
endoproteolytic conversion of BACE is a marked feature attending muscle
differentiation, suggesting a physiological role in the specification
of muscle cells and perhaps, more broadly, in tissue development.
Cell Types Containing Endoproteolyzed BACE Retain
-Secretase
Activity--
We next asked if
-secretase activity and A
production can occur in cells where endogenous BACE is expressed in the
endoproteolyzed form. Because BACE-Ct fragments are highly expressed in
liver, we examined BACE expression in the human hepatic cell line,
HepG2. Western blot analysis of HepG2 cells revealed high levels of
BACE-Ct with virtually no detectable BACE holoprotein (Fig.
5A). The specificity of the
antibody for BACE was confirmed by peptide blocking (Fig. 5A). These findings indicate that stable levels of
endogenous BACE exist almost exclusively as endoproteolyzed fragments
in HepG2 cells. APP processing was examined by metabolically labeling HepG2 cells expressing SFV-induced wild-type human APP695.
Cells expressing SFV-
-galactosidase (lacZ) served as
controls. Immunoprecipitation with antibodies directed against the
C-terminal domain of APP revealed the presence of APP C-terminal
fragments consistent with
- and
-secretase-mediated APP cleavage
(Fig. 5B). Immunoprecipitation of the conditioned medium
with 6e10, a monoclonal antibody recognizing amino acid residues 1-10
of A
, readily detected A
(Fig. 5B). The same
experimental approach also detected the production of A
in
differentiated C2 myotube cultures (data not shown). These results
strongly suggest that endoproteolyzed BACE retains at least some APP
-site cleaving enzyme activity.

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Fig. 5.
A, Western blot shows expression of
BACE-Ct, but not holoprotein, in human hepatic HepG2 cells.
Equivalent Western blot was treated with antibodies preadsorbed with
BACE-Ct peptides to show specificity of antibody for BACE.
BHK indicates BACE overexpressed in BHK cells. B,
HepG2 cells generate APP C-terminal fragments consistent with - and
-secretase activity and also generate A . HepG2 cells expressing
SFV-mediated APP695 or SFV-lacZ were metabolically labeled
with [35S]methionine/cysteine, lysed, and
subjected to immunoprecipitation with an antibody recognizing APP
C-terminal fragments. Conditioned medium was immunoprecipitated with
6E10 to reveal secreted A .
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Cell Biological Characteristics of BACE Endoproteolysis--
To
determine more accurately the cellular compartment(s) where the BACE
endoproteolytic event takes place, we used BACE constructs targeted to
specific organelles. Placing a dilysine motif on the C terminus of BACE
(BACE-KK) effectively retains the protein in the ER (30), whereas
replacing the cytoplasmic tail of BACE with the intracellular domain of
the proprotein convertase furin targets the protease to the TGN
(BACE-TGN) (Fig. 1A) (35). The cellular distributions of
BACE-KK and BACE-TGN were confirmed by immunofluorescence (Fig.
6A). When expressed in HeLa
cells, BACE-KK exhibited a fine reticular staining pattern, consistent with ER localization and identical to previous results (30), whereas
BACE-TGN displayed positive immunoreactivity in a perinuclear distribution that colocalized well with the Golgi marker GM-130. The
BACE targeting mutants were expressed in 293 cells, and
immunoprecipitations of radiolabeled protein were performed to assay
for endoproteolysis. Whereas BACE-Ct was undetectable in cells
expressing BACE-KK, the fragment accumulated at roughly twice the
wild-type level in cells expressing BACE-TGN (Fig. 6B).
These findings indicated that endoproteolysis occurs only after BACE is
exported from the ER and that the efficiency of the cleavage event
itself is enhanced in the TGN. The larger size of BACE-Ct in cells
expressing BACE-TGN simply reflects the increased length of the furin
cytoplasmic tail relative to its wild-type BACE counterpart.

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Fig. 6.
A, immunofluorescence micrographs of
HeLa cells expressing BACE, BACE-KK, or BACE-TGN. All cells were
fixed and permeabilized with methanol prior to BACE staining (see
"Experimental Procedures"). The lower row of
panels indicates the extent of co-localization
(yellow) between BACE-TGN (green) and the Golgi
resident protein GM-130 (red). B, 293 cells
expressing either BACE, BACE-KK, or BACE-TGN were radiolabeled with
[35S]cysteine/methionine for 18 h, lysed, and
immunoprecipitated. Arrows denote BACE-Ct derived from
either BACE-HA or BACE-TGN. C, 293 cells expressing BACE or
BACE-TGN were pretreated with either normal growth medium
( ), growth medium containing 100 µM
chloroquine (Chl), or growth medium containing 1 µM bafilomycin A1 (Baf) for 3 h. The
cells were then radiolabeled with
[35S]cysteine/methionine for 18 h with chloroquine
or bafilomycin A1 included as before, lysed, and immunoprecipitated.
Arrows indicate BACE-Ct. D, bafilomycin
(Baf) blocked the endoproteolysis of BACE in differentiated
C2 cells expressing SFV-mediated wt BACE. NI, non-infected
controls. Cells were metabolically labeled and then chased in cold
medium for the indicated time (hours). Lysates were immunoprecipitated
with BACE-Ct antibodies. Lower panel is darker exposure of a
portion of the upper panel. Arrows indicate BACE-Ct.
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To define further the cell biology of BACE endoproteolysis, we asked
whether the generation of BACE-Ct requires an acidic microenvironment.
A variety of proteases, including BACE, are optimally active at acidic
pH values (10, 12). We addressed this issue using two pharmacological
reagents. Chloroquine, a weak base, accumulates in acidic vesicles
where it counteracts the established pH gradient (41, 42), whereas
bafilomycin A1 selectively blocks vacuolar-type H+-ATPases
(43), protein complexes that pump protons into a variety of cellular
compartments including endosomes and the TGN (44). 293 cells expressing
BACE were labeled with [35S]cysteine/methionine for
16 h in the presence of either chloroquine or bafilomycin A1. This
treatment eliminated the production of BACE-Ct, as visualized by
immunoprecipitation of radiolabeled protein (Fig. 6C).
Furthermore, metabolic pulse-chase analysis revealed that bafilomycin
A1 also blocks BACE-Ct production in C2 myotubes, a cell type where
endoproteolyzed BACE is highly expressed (Fig. 6D). These
experiments indicate that BACE endoproteolysis most likely occurs in an
acidic domain of the cell, such as the TGN or the endosomal/lysosomal system.
BACE Is Not Cleaved by Either Furin or TACE and Does Not Appear to
Autocatalyze Its Own Endoproteolysis--
We next sought to determine
what enzyme(s) mediate the endoproteolytic processing of BACE. In light
of our results implicating the TGN as a likely site for BACE
endoproteolysis, we considered two proteases as good candidates. The
previously mentioned
-secretase TACE was originally found to be
involved with the cleavage of cell surface proteins (45, 46). However,
more recent evidence has implicated TACE in protein kinase C-regulated
-secretase activity within the TGN, where the protein competes with
-secretase for APP substrate (19). In addition to TACE, furin is
known to be active primarily in the TGN where it mediates the
processing of several different precursor proteins (47). Furin or
a furin-like protease has also been demonstrated to cleave BACE at its
propeptide site (31, 32), demonstrating that these two proteins
interact in cells. Arguing against a role for furin, however, is the
fact that the BACE endoproteolysis site is not marked by the consensus sequence, Arg-Xaa-(Lys/Arg)-Arg, typically associated with furin cleavage (47).
The impact of TACE and furin on the endoproteolysis of BACE was
determined by co-expression studies. 293 cells were transfected with
either BACE alone, BACE and TACE, or BACE and mfurin-HA (an HA-tagged
murine furin construct). The cells were then labeled with
[35S]cysteine/methionine for 8 h, lysed, and
immunoprecipitated with mAb HA11. Analogous experiments using BACE-TGN
were also performed. We found that co-expression of TACE or mfurin-HA
with either BACE-HA or BACE-TGN did not lead to an appreciable increase
in the accumulation of BACE-Ct (Fig. 7,
A and C). We also attempted to up-regulate TACE
activity by treating the cells with the protein kinase C activator
phorbol 12-myristate 13-acetate (PMA) 4 h into the
[35S]cysteine/methionine labeling period. No effect on
BACE-Ct levels was observed (Fig. 7A). TACE activity was
confirmed by co-transfection with APP in 293 cells. A 30-min
[35S]cysteine/methionine label followed by PMA
stimulation led to an increase in secreted APP
derived from both
endogenous APP751 and overexpressed APP695
(Fig. 7B), consistent with reported results (19). We
confirmed the expression of furin-HA by Western blot (Fig.
7D). Thus, neither TACE nor furin was found to mediate the cleavage of BACE.

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|
Fig. 7.
A, 293 cells in 6-well plates expressing
either BACE alone or BACE and TACE were radiolabeled with
[35S]cysteine/methionine for 8 h, lysed, and
immunoprecipitated (2nd to 5th lanes). In half of
the wells, TACE/ADAM activity was stimulated with 10 µM
PMA 4 h into the labeling period (3rd,
5th, 7th, and 9th lanes). Analogous
experiments were also performed using BACE-TGN (6th to
9th lanes), and mock-transfected cells were employed as a
negative control (1st lane). Arrows indicate
BACE-Ct. B, as a control for TACE activity; 293 cells
expressing APP and TACE were radiolabeled with
[35S]cysteine/methionine for 30 min after which they were
either left untreated or incubated with 10 µM PMA for
another 30 min. Conditioned media were then collected and
immunoprecipitated for secreted APP (see "Experimental
Procedures"). APP species derived from APP751 and
APP695 are indicated. C, BACE and BACE-TGN were
either expressed alone or with mfurin-HA in 293 cells (2nd
to 5th lanes). Mock-transfected cells provided a negative
control (1st lane). Radiolabeling for 18 h followed by
cell lysis and immunoprecipitation did not reveal any increase in the
production of BACE-Ct (arrows) resulting from furin
co-expression. D, the presence of mfurin-HA was verified by
Western blot. E, 293 cells expressing either
BACE-D289N, BACE, or mock-transfected were radiolabeled for 18 h,
lysed, and immunoprecipitated. BACE-Ct was effectively generated from
both BACE-HA and BACE-D289N (arrow).
|
|
We also considered whether an autocatalytic mechanism might be
responsible for BACE endoproteolysis. Several enzymes, including furin
and possibly the presenilins, undergo autocatalytic processing events
that significantly impact the activity of the proteases themselves (13,
15, 47). In addition, the dependence of BACE endoproteolysis on an
acidic microenvironment correlates well with the documented acidic pH
optimum for BACE. To address this possibility, we generated a BACE
construct whose second active site aspartate residue (Asp-289) mutated
to asparagine (BACE-D289N). This mutation has been demonstrated to
eliminate completely the protease activity of BACE (8), and in our
hands, co-expression of BACE-D289N with APP in 293 cells did not lead
to the typically observed increase in A
secretion (data not shown).
If BACE does autocatalyze its own endoproteolysis, one would expect
BACE-Ct not to be produced in cells expressing BACE-D289N or to be
produced only at very low levels due to the presence of endogenous
BACE. We transfected BACE-D289N and BACE into 293 cells and assayed for
endoproteolysis by immunoprecipitation of radiolabeled protein. No
significant differences were found between the BACE-Ct levels of cells
expressing either BACE-D289N or BACE (Fig. 7E). Our
findings, therefore, are not consistent with BACE directly mediating
its own endoproteolysis. Nevertheless, an autocatalytic mechanism cannot be completely excluded (see "Discussion").
 |
DISCUSSION |
The proteolysis of APP by BACE is a crucial step in the generation
of A
in brain and represents an important potential target of
therapeutic strategies aimed at slowing or even halting the progression
of AD. Consequently, a better understanding of the cellular mechanisms
regulating
-secretase activity is of obvious importance. We and
others (29, 30) have found that BACE is a stable protein with a
t1/2 of 12-16 h. This long life span strongly
implies that, in order for cells to effectively exert tight control
over
-secretase activity, regulatory mechanisms must exist at a
post-translational level. We have described an endoproteolytic
processing pathway that cleaves BACE into distinct N- and C-terminal
fragments, each ~37-kDa in size. BACE endoproteolysis appears to be a
normal cellular metabolic process resulting in the generation of a
highly stable form of the protein. In this regard BACE may be similar
to a wide variety of proteins that are known to be regulated by
endoproteolysis including zymogens, clotting factors, complement
proteins, and hormones (48, 49). In addition, several membrane-bound
proteases such as the A Disintegrin
And Metalloprotease (ADAM) and proprotein convertase families require specific cleavage events during the maturation process to obtain biological activity (47, 50, 51).
Structural analysis of BACE endoproteolysis revealed that cleavage
occurs on a solvent-exposed
-helix bridging the two extracellular lobes of the protein. Analogous helices are absent from soluble aspartyl proteases like pepsin (40, 52), consistent with the fact that
these enzymes have not been shown to undergo endoproteolysis at similar
positions in their amino acid sequences (53, 54). Typically, cleavage
at this site would be expected to inactivate BACE by dissociating the
two catalytic aspartic acid residues. However, we found that the
BACE-Nt and -Ct remain covalently associated after proteolysis via a
common disulfide bridge. This finding suggests the possibility that
such post-translational regulation may alter and/or attenuate, rather
than abolish, the enzymatic activity of BACE.
Although BACE endoproteolysis occurs readily in a variety of cultured
cell types, only a relatively small proportion of the holoprotein
appears to undergo cleavage when overexpressed. By contrast,
endoproteolyzed BACE accumulates at high levels endogenously in muscle,
liver, and pancreas, coupled with undetectable or very low levels of
holoprotein. This dichotomy is reminiscent of the regulated
endoproteolysis of the presenilins, which exist primarily as
holoproteins when overexpressed, and yet are found almost entirely as
N- and C-terminal cleavage fragments endogenously (23, 24, 55, 56).
Whereas a specific physiological purpose cannot be assigned to BACE
endoproteolysis at this time, this post-translational event appears to
be a normal, regulated metabolic process yielding stable cleavage
products that accumulate in certain native tissues. Moreover, the
pattern of endoproteolyzed BACE expression is generally consistent with
BACE mRNA levels in different tissue types. For instance, the
pancreas exhibits robust BACE mRNA expression (10) and is among the
tissues harboring the highest levels of BACE cleavage fragments. In
addition, we have shown that an up-regulation of BACE endoproteolysis
is associated with myogenic differentiation in C2 cells, a well
established model of muscle development. Together, these findings
strongly suggest that endoproteolyzed BACE has a biologically
significant, but as yet undefined, function(s) in a number of different
cell types, particularly in cells of non-neuronal lineage.
We attempted to localize BACE endoproteolysis by targeting the protein
to different sites within the secretory pathway. These studies revealed
that the cleavage event takes place only after the protein is exported
from the ER and appears to be enhanced in the TGN. We also found BACE
endoproteolysis to be sensitive to inhibitors of vesicular
acidification, a result consistent with a TGN and/or endosomal
localization for BACE cleavage. Multiple lines of evidence implicate
the TGN in BACE endoproteolysis. Thus, we considered two membrane-bound
proteases, TACE and furin, both of which are active in the TGN, to be
excellent candidate enzymes. However, neither enzyme up-regulated the
production of BACE-Ct when co-expressed with BACE in 293 cells. We also
demonstrated that BACE-Ct could be generated from a catalytically
inactive BACE (BACE-D289N), suggesting that BACE does not cleave
itself. However, this finding does not exclude autocatalysis as a
mechanism for BACE endoproteolysis. If BACE cleavage occurs via an
intermolecular (trans) mechanism, as opposed to an
intramolecular (cys) mechanism, the endoproteolysis of the
recombinant BACE-D289N could have been mediated in our studies by
endogenous BACE.
The potential impact of BACE endoproteolysis on APP processing is
intriguing because
-secretase activity is known to be quite low in
non-neuronal tissues, such as the pancreas, that express high levels of
BACE mRNA (9, 10). Some have argued that a predominance of
alternatively spliced BACE variants with reduced APP processing
activity may account for this discrepancy (57, 58). However, more
recent data indicate that the most abundant BACE mRNA species in
pancreas correspond to full-length protein, implying that
down-regulation of
-secretase activity most likely occurs at a
post-translational level (59). We have shown that BACE exists primarily
as endoproteolytic fragments in pancreas, muscle, and liver, whereas
holoprotein predominates in neuronal tissue. This would imply that BACE
endoproteolysis significantly reduces
-secretase activity in these
tissue types. We observed definitive
-secretase activity and
subsequent A
production in HEPG2 cells, where virtually all
detectable BACE protein exists as endoproteolytic fragments. Although
we cannot completely exclude the possibility that trace amounts of
full-length BACE mediated
-secretase activity in these studies, it
appears far more likely that the readily measurable levels of A
were
generated by the highly abundant endoproteolyzed BACE species.
Therefore, these findings in conjunction with previous work (9, 10)
suggest that endoproteolysis may significantly attenuate, but most
likely does not abolish, the
-secretase activity of BACE in certain non-neuronal cell types. An improved understanding of this
post-translational event could aid considerably in the crafting of
methods to reduce cerebral amyloid deposition by specifically
activating BACE endoproteolysis in neural tissue.
Finally, the effects of BACE endoproteolysis may extend beyond APP
cleavage. For instance, BACE processing could modulate the ability of
the protease to act on other substrates, a possibility made all the
more feasible by recent findings implicating the protease in the
cleavage of the sialyltransferase, ST6Gal-1 (60). Much work remains to
be done to better understand what will likely emerge as numerous
functions for BACE, an enzyme that is widely expressed in a diverse
array of cell and tissue types.
We thank Dr. David W. McCourt at Midwest
Analytical, Inc., for help with the radiosequencing of BACE-Ct. We also
thank Mark Biscone for assistance in the design of structural figures.
As always, we appreciate the thoughtful comments and insights provided by the members of the Doms, Lee, and Schellenberg laboratories.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M213303200
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