From the Departments of Biological Chemistry and
Medicinal Chemistry, Merck Research Laboratories,
West Point, Pennsylvania 19486
Received for publication, October 9, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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The amyloid precursor protein
(APP)1 is cleaved
sequentially by two proteolytic activities, Pro domains are commonly found in protease precursors where they have
been shown to virtually abolish catalytic activity (7) and assist in
protein folding (8). The Pro domain is typically cleaved from the
protease precursor to generate the mature active protease. Edman
degradation of purified human brain BACE revealed a single N-terminal
protein sequence that began at the protease domain (3) (this species is
designated mature BACE). BACE isolated from human embryonic kidney 293T
cells that were transfected with the full-length BACE cDNA
exhibited an N-terminal sequence that began mostly at the protease
domain, whereas only ~10% began at the Pro sequence (3). In
addition, N-terminal sequencing of full-length Asp2 (i.e.
BACE), which was expressed in Chinese hamster ovary cells, also
revealed proteolytic processing at the junction between the putative
Pro and protease domains (4). These results establish that processing
of membrane-anchored BACE after Arg-45 to release the Pro segment and
generate mature BACE is prevalent in mammalian cells.
In the present study, we expressed in insect cells a soluble form of
the BACE precursor that is truncated at the junction between the
putative protease and transmembrane regions (PreProBACE460). A
BACE-related species, ProBACE460, that displays an N-terminal sequence
that corresponds exactly to mammalian cell-derived ProBACE was purified
from the conditioned medium (CM). The studies described herein show
that the Pro domain of ProBACE460, rather than abolishing catalytic
activity as in the case of true zymogens, appears to facilitate the
proper folding of the BACE protease domain.
Cloning of the BACE cDNA--
The BACE cDNA was cloned
by reverse transcription-polymerase chain reaction amplification of
human brain mRNA (CLONTECH; Palo Alto, CA)
using oligonucleotides that were designed from the published BACE
cDNA sequence (2-6). In particular, the 5'-oligonucleotide (5'-CGGGATGCGGCCGCATGGCCCAAGCCCTGCCCTGGCTCCTGCTG-3') contained a
NotI site upstream of the ATG start codon and the
3'-oligonucleotide (5'-GGATCGGTCGACTTAAATTTGAGGTGACCAAGA-3') spanned
the SalI site located in the 3'-untranslated region (262 base pairs downstream from the TGA stop codon). The 1771 base pair
polymerase chain reaction product was subcloned into pLHCX
(CLONTECH) to create the BACE retrovirus expression
construct, pRBR133. The integrity of the BACE cDNA was confirmed by
DNA sequencing (Midland Reagent Company, Midland TX).
Expression of BACE by Recombinant Baculovirus in Insect
Cells--
The cDNA encoding truncated PreProBACE (amino acids
1-460) was polymerase chain reaction amplified from pRBR133 using a
5'-oligonucleotide containing a BamHI site upstream
of the ATG start codon,
5'-GCTAGAGGATCCATGGCCCAAGCCCTGCCCTGGCTCCTGCTGTGG-3' and a
3'-oligonucleotide containing a stop codon after the Tyr460 residue
followed by an XhoI site,
5'-GTCGCACTCGAGTCAATAGGCTATGGTCATGAGGGTTGACTC-3'. The 1389-base
pair fragment was subcloned into the BamHI and
XhoI sites of a baculovirus expression vector, pBacPAC8
(CLONTECH), to generate pRBR136. The integrity of
the BACE cDNA clone was again confirmed by DNA sequencing (Midland
Reagent Company). pRBR136 was transfected into Spodoptera
frugiperda (Sf21) insect cells along with BacPAK6 viral DNA
to generate recombinant virus (BACE460 virus stock) according to the
manufacturer's instructions. Recombinant virus was harvested 5 days
post-transfection, plaque-purified and titered as described in the
CLONTECH manual. Sf21 cells (30 liters) were
grown in Grace's insect medium, 10% heat-inactivated fetal bovine
serum, 2 mM glutamine, 1× penicillin/streptomycin to
~9.0 × 105 cells/ml and were infected with BACE460
virus stock at a multiplicity of infection of 4 (Cell and Molecular
Technologies, Inc., Lavallette NJ). After 72 h, the CM was
harvested by filtration and immediately stored at 4 °C.
Production of Anti-BACE Antibodies--
A peptide corresponding
to the Pro segment of BACE (TQHGIRLPLRSGLGGAPLGLRLPR-C) was conjugated
to keyhole limpet hemocyanin and used as antigen for production of
rabbit polyclonal antibodies (Covance Inc., Denver PA). Another peptide
(RDLRKGVYVPC) that is situated within the protease domain of
BACE (amino acids 122-131) was similarly used as an antigen for
production of rabbit polyclonal antibodies (Covance Inc.). The anti-Pro
antibody was purified by passing the serum over an affinity column
containing the immobilized hapten.
BACE Activity Assay--
CM from Sf21 cells
infected by BACE460 virus stock (or control virus stock), purified
ProBACE460, or purified BACE460 were incubated with
acetyl-TTRPGSGLTNIK(6-(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVNLDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide (L-405,492), a modified 31-residue polypeptide that is designed from
the amino acid sequence of APP in the immediate vicinity of the
Purification of Recombinant Baculovirus Expressed BACE--
The
CM from BACE460 virus stock-infected Sf21 cells was spiked with
NaCl (350 mM), aprotinin (0.5 µM), and
leupeptin (1 µM) and applied to a concanavalin
A-Sepharose (Amersham Pharmacia Biotech, Piscataway NJ) column (1.5 liters of bed volume). The lectin affinity column was washed with
Buffer A (20 mM Tris-HCl, pH 7.4, and 0.5 M
NaCl) plus 0.5 µM aprotinin, 1 µM leupeptin and eluted with Buffer A plus 0.3 M
Generation of BACE460 by Treatment of ProBACE460 with
Furin--
ProBACE460 or BACE460 (500 nM) were incubated
with furin (20 units/ml; Affinity Bioreagents Inc, Golden, CO) at
30 °C in 0.1 M Hepes, 2.5 mM
CaCl2, and 0.5% Triton X-100, pH 7.5. At increasing times,
aliquots were removed and furin activity was quenched with EDTA (5 mM). The production of BACE460 by furin treatment of
ProBACE460 was assessed by SDS-PAGE/immunoblotting using antibodies
against the Pro or protease regions of BACE. Signals from the
immunoblots were quantified by scanning analysis using a densitometer
SI (Molecular Dynamics, Sunnyvale, CA). In addition, the level of BACE
activity after furin treatment was evaluated with L-405,492. For the
isolation of preparative amounts of BACE460, ProBACE460 (2 µM) was incubated with furin (100 units/ml) in 0.1 M Hepes, pH 7.6, 2.5 mM CaCl2, 0.5% Triton X-100 (final volume, 6.5 ml) for 14 h at 30 °C.
The sample was dialyzed versus 20 mM sodium
acetate, pH 5.0, 125 mM NaCl. In vitro generated
BACE460 was purified by affinity chromatography with immobilized
P10-P4' Stat(Val) as described above (except that pepstatin was
eliminated from the column wash step).
Immunoprecipitation of ProBACE460--
CM (100 µl) from the
BACE460 virus stock-infected Sf21 cells was spiked with control
antibody (normal rabbit IgG; 0.3 µM) or anti-Pro antibody
(0.3 µM; affinity purified) and immobilized protein G (10 µl of a 75% slurry) (Amersham Pharmacia Biotech). After overnight
storage at 4 °C with mixing, the samples were centrifuged, and the
supernatant fractions were evaluated by SDS-PAGE/immunoblotting (probing with either anti-Pro antibody or anti-protease antibody) and
assayed for BACE activity using L-405,492. Immunoprecipitation of the
purified ProBACE460 and BACE460 preparations (0.5 µM) was performed similarly except that 2.7 µM control and
anti-Pro antibody were used. The pellet fractions were extensively
washed with phosphate-buffered saline and assayed for BACE activity
using L-405,492.
Denaturation/Renaturation of ProBACE460 and
BACE460--
ProBACE460 and BACE460 (7 mg/ml) were denatured for
1 h at 23 °C with 6 M guanidine HCl in Buffer D in
the absence or presence of 50 µM BACE Pro peptide,
NH2-TQHGIRLPLRSGLGGAPLGLRLPR-COOH (Synpep Inc., Dublin CA).
The samples were renatured by rapid addition of Buffer D (60 volumes)
to yield a final concentration of 0.1 M guanidine HCl and
set at room temperature for 2 h. Control ProBACE460 and BACE460
samples to determine maximal BACE activity were adjusted to 7 mg/ml in
an equivalent volume of Buffer D and treated similarly. Samples were
assayed for BACE activity using L-405,492. Aliquots of the
mock-denatured/renatured or denatured/renatured ProBACE460 and BACE460
samples were analyzed by SDS-PAGE/immunoblotting using the
anti-protease antibody. In addition, aliquots of ProBACE460 with and
without treatment by guanidine denaturation/renaturation were loaded
onto a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) using an
AKTAexplorer chromatography system (Amersham Pharmacia Biotech). The
gel filtration column was eluted with 20 mM Tris-HCl, pH
7.2, 125 mM NaCl, and 0.1 M guanidine HCl.
Analytical Analyses--
The amino acid compositions of
protein/peptide samples were determined with a Beckman 6300 analyzer
after hydrolysis of the samples using 6 N HCl containing
0.2% phenol in sealed evacuated vials at 110 °C for 20 h.
N-terminal sequence analysis of purified protein samples was performed
by Edman degradation using an Applied Biosystems PROCISE Model 494 protein sequencer. Verification of product identity by liquid
chromatography/mass spectometry analysis was performed with an Agilent
Technology 1090 liquid chromatograph coupled to a PE Sciex API-3000
triple quadrupole mass spectrometer.
Expression of PreProBACE460--
A truncated BACE precursor
(encompassing amino acids 1-460) that contains the Pre, Pro, and
protease domains (PreProBACE460) was produced in Sf21 cells
using a baculovirus expression construct (Fig.
1). This expression system was designed
to direct the secretion of a soluble nonmembrane tethered form of BACE
from the cells. SDS-PAGE/immunoblot analysis of CM from cells that were
infected with BACE460 virus stock revealed the presence of a protein
(molecular mass, ~55,000) that reacted with anti-BACE antibodies
raised against the Pro domain (Fig.
2A, lane 2) or
protease domain (data not shown). In contrast, no immunoreactivity was
evident with CM from Sf21 cells infected with control virus
stock when probed similarly using anti-BACE antibodies
versus the Pro domain (Fig. 2A, lane 1) or protease domain (data not shown).
Treatment of CM from BACE460 virus stock-infected Sf21 cells
with the antibody versus the Pro region of ProBACE in the
presence of immobilized protein G fully depleted the BACE-related
species that cross-reacts with the anti-Pro antibody (Fig. 2,
lanes 3 and 4). Nevertheless, the
ProBACE460-immunodepleted CM still possessed a less abundant species
that reacts with the anti-protease antibody and migrates in the
vicinity of ProBACE460 (which is indistinguishable from the mobility of
BACE460, see below) (Fig. 2A, lanes 5 and 6). These observations indicate that most but not all of the
BACE-related protein in the CM contains the Pro region.
BACE activity in the CM was measured using L-405,492, the modified
31-mer polypeptide derivative designed from the APP sequence (Swedish-type familial Alzheimer's disease variant) in the vicinity of
the
CM from cells infected with the BACE460 virus stock that was
extensively immunodepleted of proteins possessing the Pro region of
BACE (as described above) exhibits an approximate 60% reduction of
apparent BACE activity when compared with control antibody-treated CM
(Fig. 2C). This result provided the initial indication that a Pro region-containing form of BACE displayed activity. The decrease of BACE activity in CM caused by the immunocapture step with anti-Pro antibody was totally blocked when the hapten, Pro peptide, was also
present (Fig. 2C). The presence of both residual BACE
activity and anti-protease (but not anti-Pro) immunoreactivity after
exhaustive immunodepletion of ProBACE460 in the CM indicates that a
lesser non-Pro region-containing BACE species in the CM also displays BACE activity.
Isolation of ProBACE460 and BACE460--
The CM from BACE460 virus
stock-infected Sf21 cells was spiked with aprotinin/leupeptin
and fractionated by lectin affinity chromatography using immobilized
concanavalin A. The BACE activity was partially captured and eluted
with
The BACE preparation that was isolated from the CM of BACE460 virus
stock-infected Sf21 cells exhibited a single band when subjected
to SDS-PAGE and visualized by Coomassie Blue staining (Fig.
3A, lane 1).
SDS-PAGE/immunoblot analysis using the antibodies against the Pro or
protease regions of BACE both reacted with this species (Fig.
3B, lane 1, upper and lower
panels, respectively). The purified BACE-related species is a
glycoprotein as judged by its capture by immobilized concanavalin A and
its increased mobility during SDS-PAGE after treatment with
endoglycosidases (data not shown). Edman degradation of purified BACE,
which was isolated from CM revealed an N terminus that began
exclusively at the Pro region of BACE (Fig.
3C).2 Control
N-terminal sequencing runs with ProBACE460 spiked with varying amounts
of mature BACE460 (prepared as described below) established that a 5%
or greater contamination of ProBACE460 with BACE460 would be detected.
The apparent homogeneity of the ProBACE460 preparation was corroborated
by the absence of BACE immunoreactive material, using either the
anti-Pro (Fig. 3B, lane 3, upper
panel) or anti-protease (Fig. 3B, lane 3,
lower panel) antibodies, after the ProBACE460 preparation
was immunodepleted using antibody versus the Pro region.
The cellular protease that processes ProBACE to yield mature BACE was
recently shown to be furin (10). The minimal consensus sequence for
furin-mediated cleavage, Arg-Xaa-Xaa-Arg (11), is situated at the C
terminus of the Pro region of BACE. We thus treated purified ProBACE460
with furin and used SDS-PAGE/immunoblotting to monitor for
time-dependent processing. The reactivity of ProBACE460 with the anti-Pro antibody is markedly diminished and eventually eliminated as the incubation time with furin is lengthened (Fig. 4A, upper panel).
On the other hand, the reactivity with the anti-protease antibody was
largely unaffected (Fig. 4A, lower panel).
Importantly, N-terminal sequence analysis established that furin
cleaves ProBACE460 at the junction between the putative Pro and
protease domains to yield BACE460 (Fig. 3C).
A larger amount of BACE460 was obtained for further characterization by
treating ProBACE460 (625 µg) with furin and rechromatographing the
sample on the P10-P4' Stat(Val) affinity column. The protein recovered
from the affinity column (386 µg) was shown to be BACE460 from its
failure to react with anti-Pro antibody by immunoblot analysis (Fig.
3B, lane 4, upper panel) and
N-terminal sequencing analysis (data not shown). Moreover, in contrast
to the ProBACE460 preparation, treatment of BACE460 with immobilized
anti-Pro antibody failed to deplete the BACE-related species as
detected by immunoblotting using the anti-protease antibody (Fig.
3B, compare lanes 3 and 6, lower
panel).
ProBACE460 and BACE460 Both Display Catalytic Activity--
The
purified ProBACE460 preparation exhibits appreciable activity toward
L-405,492. Indeed, assaying the proteolytic activity of ProBACE460
enabled its isolation. Treatment of the purified ProBACE460 preparation
with immobilized anti-Pro antibody resulted in the loss of >90% of
the BACE activity (data not shown). Moreover, assay of the pellet
containing ProBACE460 tethered to the immobilized anti-Pro antibody
revealed appreciable BACE activity (data not shown). The results of
this immunocapture experiment establish that ProBACE460 itself, and not
a non-Pro region-containing contaminant, is responsible for the
observed activity in the ProBACE460 preparation.
Processing of ProBACE460 to BACE460 by furin is accompanied by a modest
increase in BACE activity toward L-405,492, which inversely mirrored
the loss in anti-Pro reactivity that was detected by immunoblotting
(Fig. 4B). There was an approximate 2-fold increase in BACE
activity after a 4-h incubation with furin. In an independent experiment, treatment of ProBACE460 with furin for 4 and 20 h increased BACE activity by 2.0- and 2.8-fold, respectively (Fig. 4C). As expected, treatment of BACE460 with furin did not
increase BACE activity (Fig. 4C). Incubation of L-405,492
with furin alone did not cause the generation of product (data not shown).
Cleavage of L-405,492 by purified ProBACE460 and BACE460 displayed a
linear dependence on substrate concentration up to 100 µM
of substrate (Fig. 5). The absence of
detectable saturation is in agreement with the results from a previous
BACE study that used a polypeptide substrate of similar length (3). The
estimated kcat/Km values of
ProBACE460 and BACE460 for L-405,492 are 3110 and 7300 M
ProBACE460 and BACE460 also cleave L-405,525, a substrate that is
identical to L-405,492, except that it contains KM at the P2 and P1
positions, respectively, which are the residues that are found in the
wild type APP sequence. There was no evidence for saturation of either
enzyme up to 30 µM of L-405,525 (Fig. 5B).
Mass spectrometric analysis confirmed that cleavage of L-405,525 occurs
at the
ProBACE460 and BACE460 display similar susceptibility to inhibition by
P10-P4' Stat(Val) (Fig. 6). The
IC50 values of P10-P4' Stat(Val) for ProBACE460 and BACE460
are 250 and 150 nM, respectively. The inability of P10-P4'
Sat(Val) to discriminate between ProBACE460 and BACE460 is concordant
with the virtually indistinguishable binding/elution behavior of these
BACE derivatives during affinity chromatography with this immobilized
ligand (data not shown). Both ProBACE460 and BACE460 are relatively
insensitive to inhibition by pepstatin. The residual activities of
ProBACE460 and BACE460 in the presence of 100 µM
pepstatin were 74 and 58%, respectively, of the uninhibited control
activities.
We considered that the apparent activity of ProBACE460 might arise from
its conversion to BACE460 during incubation with substrate under
standard assay conditions (pH 4.5). To explore this possibility, N-terminal sequencing was performed on ProBACE460 after its incubation with substrate (during which ~20% of the substrate was converted to
product). The observed amino acid sequence began exclusively at the N
terminus of Pro region; hence, conversion of ProBACE460 to BACE460
because of the presence of substrate or exposure to assay conditions
does not appear to occur. SDS-PAGE/immunoblot analysis using the
anti-Pro antibody also failed to show diminished immunoreactivity of
ProBACE460 after its incubation with substrate under standard assay
conditions (data not shown).
The Pro Region of BACE Assists in the Folding of the Protease
Domain--
To assess the potential role of the Pro domain in protein
folding, ProBACE460 and BACE460 were denatured with 6 M
guanidine HCl at neutral pH for 1 h and renatured by rapid
dilution into neutral pH buffer. After a 2-h renaturation period,
aliquots of the samples were added to BACE assay mixture containing the
substrate, L-405,492. The final guanidine HCl concentration during the
BACE assay, 0.1 M, has no effect on the activity of
ProBACE460 or BACE460. No BACE activity is observed when denatured
ProBACE460 or BACE460 is transferred directly to the assay mixture (at
pH 4.5) without an intervening renaturation step (at pH 7.2). However,
transfer of denatured ProBACE460 to the renaturing milieu leads to
recovery of BACE activity. Approximately 35% of the ProBACE460
activity is recovered after a denaturation/renaturation cycle (Fig.
7A, bar 1). In
contrast, the recovery of activity from the denatured BACE460
preparation is only ~5% (Fig. 7A, bar 3).
SDS-PAGE/immunoblot analysis of the denatured/renatured ProBACE460 and
BACE460 samples revealed similar levels of immunoreactivity when probed
with the anti-protease antibody (Fig. 7B). Hence, the lower
recovery of BACE activity after treatment of BACE460 with guanidine HCl
and subsequent renaturation is not due to greater susceptibility to protein degradation.
The protein species in the denatured/renatured ProBACE460 sample
that displays BACE activity elutes during gel filtration at the same
position as unperturbed ProBACE460 (data not shown). The refolded
active ProBACE460 species represents only partial recovery of total
protein (~37%) based on absorbance measurements and comparison with
the elution profile of unperturbed ProBACE460. Importantly, the
specific activity of the refolded ProBACE460 that elutes during gel
filtration as a monomeric species is indistinguishable from that of
ProBACE460 (data not shown). The inability to recover all of the BACE
activity after denaturation/renaturation of ProBACE460 probably
reflects the formation of misfolded ProBACE460 aggregates.
The presence of the BACE Pro peptide (75 µM) in
trans during the denaturation/renaturation of BACE460 serves
to increase the recovery of BACE activity to ~23% (Fig.
7A, bar 4), but the Pro peptide essentially has
no effect on the recovery of activity from denatured ProBACE460 (Fig.
7A, bar 2). Consistent with the predominant
effect of the Pro domain on protein folding and not suppression of
activity, the BACE Pro peptide (75 µM) has only a modest
effect (16% inhibition) on the activity of BACE460 (data not shown).
In stark contrast, Pro peptides for other aspartyl class proteases such
as chicken pepsin and human cathepsin D are relatively potent
inhibitors (Ki values < 10 nM and
30 nM, respectively) that completely block the activity of
their cognate proteases (12).
Comparison of the predicted amino acid sequence of full-length
BACE with members of the aspartyl protease family revealed the
existence of a 24-residue polypeptide (Pro domain) that is situated
between the putative Pre and protease domains (2-5). The peptide bond
at the junction of the Pro and protease domains in the BACE precursor
is efficiently cleaved in mammalian cells to generate mature BACE (2,
4). A typical function of the Pro region in aspartyl proteases is to
confer zymogen-like properties on the unprocessed precursor species
(7). In this report, we show that the Pro segment of ProBACE has a
relatively minor inhibitory effect on BACE activity, but it does appear
to play a role in the proper folding of the protease domain.
The activity of ProBACE460 was not markedly different from that of
BACE460 when assayed with modified 31-mer polypeptide substrates corresponding to the wild type and Swedish-variant-type APP sequences (74 and 43% of the BACE460 activity, respectively). The apparent homogeneity of the ProBACE460 preparation that was evident by SDS-PAGE
and N-terminal sequencing points to ProBACE460 as being responsible for
the observed BACE activity. Consistent with this conclusion, greater
than 90% of the BACE activity was removed from the ProBACE460
preparation by immunoprecipitation using the anti-Pro antibody.
Moreover, the pellet fraction from the anti-Pro immunocapture step
displayed BACE catalytic activity. Conversion of ProBACE460 to BACE460
during incubation with substrate under standard assay conditions was
excluded by N-terminal sequencing and immunoblot analysis using the
anti-Pro antibody.
The binding of full-length ProBACE (3) and ProBACE460 (shown herein) to
the P10-P4' Stat(Val) affinity column is also consistent with the
conclusion that ProBACE is catalytically active because only active
forms of aspartyl proteases are expected to bind tightly to immobilized
transition state analogs. Although some true zymogens of aspartyl
proteases bind to immobilized transition state analogs (13-15), in
these instances, the precursors invariably display markedly lower
affinity for the immobilized ligand, which is not the case with ProBACE460.
Our observation that furin converts purified ProBACE460 to authentic
BACE460 is concordant with a recent claim that furin or a furin-like
proprotein convertase in mammalian cells is responsible for the
processing of ProBACE (10). In addition to using cell-based assays,
these investigators showed that furin correctly processed a recombinant
soluble BACE precursor in vitro to release the Pro domain
(10). Furin-like activity exists in insect cells; in particular, it is
present in cells derived from Drosophila melanogaster (16).
However, proteins that are expressed recombinantly in insect cells are
typically not cleaved at putative furin processing sites unless furin
is coexpressed in the cells (17, 18). These results are consistent with
the inefficient processing of ProBACE460 to BACE460 by the Sf21 cells.
The finding that ProBACE460 displays activity bears on the prediction
as to where Whereas the putative Pro region in ProBACE does not confer strict
zymogen-like properties on BACE, it does appear to play a role in the
proper folding of the protease domain. The remarkable ability of the
BACE Pro peptide to assist folding of its cognate mature protease
domain in trans is reminiscent of results observed with
During the preparation of this manuscript, a study appeared showing
that a purified, soluble (C-terminal truncation 6 residues upstream of
the putative transmembrane domain) derivative of ProBACE (pro-memapsin
2) expressed in Escherichia coli displays catalytic activity
(27). Surprisingly, pro-memapsin 2 was not cleaved by furin, a finding
that differs from that of our study and Vassar and colleagues (10). The
inability of furin to process pro-memapsin 2 may reflect differences in
the structure of this BACE derivative. Pro-memapsin 2 contains a
8amino acid extension (i.e. part of the putative signal
peptide) upstream of the established N terminus for ProBACE in
mammalian cells (2). In addition, the absence of protein glycosylation
on the E. coli-derived pro-memapsin 2 might influence
processing by converting enzymes. These investigators also studied two
"mature" BACE analogs of memapsin 2, Leu28p-memapsin 2 and
Gly45p-memapsin 2, which possess a 5-residue N-terminal extension and
13-residue N-terminal deletion, respectively, as compared with
mammalian-cell derived BACE (27). The potential impact on activity
because of these deviations from the structure of the bona
fide mammalian cell-derived mature BACE remains to be determined.
Our current investigation provides novel insight into molecular
mechanisms that impact the catalytic activity of BACE, a protease that
purportedly plays a pivotal role in the etiology of Alzheimer's disease. The observation that ProBACE460 displays substantial activity
relative to BACE460 distinguishes this aspartyl protease from typical
members of this general protease class. However, from a teleological
perspective, the anticipated role of the Pro segment in blocking
premature exposure of BACE activity within the cell may be superfluous
because of overriding pH-mediated control of enzymatic activity.
Nevertheless, the Pro domain in ProBACE is not completely anachronistic
because it continues to operate to promote the proper folding of the
protease domain. Finally, the activity displayed by ProBACE460 suggests
that blocking the conversion of ProBACE to BACE might have little or no
impact on the production of the amyloid -Secretase (BACE) is a membrane-bound aspartyl
protease that cleaves the amyloid precursor protein to generate the N
terminus of the amyloid
peptide. BACE is expressed as a precursor
protein containing Pre, Pro, protease, transmembrane, and cytosolic
domains. A soluble BACE derivative (PreProBACE460) that is
truncated between the protease and transmembrane
domains was produced by baculovirus-mediated expression.
ProBACE460 was purified from conditioned media of infected
insect cells using immobilized concanavalin A and immobilized BACE
inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro
and protease regions to generate mature BACE460. The
kcat/Km of ProBACE460 when
assayed with a polypeptide substrate is only 2.3-fold less than that of
BACE460. This finding and the similar inhibitory potency of P10-P4'
Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has
little effect on the BACE active site. Exposure of ProBACE460 to
guanidine denaturation/renaturation results in a 7-fold higher recovery
of BACE activity than when BACE460 is similarly treated. The presence
of free BACE Pro peptide during renaturation of BACE460 but not
ProBACE460 increases recovery of activity. These findings show that the
Pro domain in ProBACE460 does not suppress activity as in a strict
zymogen but does appear to facilitate proper folding of an active
protease domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and
-secretase, to
generate the N and C termini, respectively, of the amyloid
peptides. Aberrant production or compromised clearance of the amyloid
peptides, which give rise to neuritic plaques in brain parenchyma, may be a root cause of Alzheimer's disease (1).
-Secretase (BACE;
Asp2; memapsin 2) was recently cloned (2-6) and shown to be a novel
type I transmembrane aspartyl protease. The cDNA encoding BACE
predicts a precursor protein with a modular structure containing Pre,
Pro, protease, transmembrane, and cytosolic tail domains (see Fig.
1).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-secretase cleavage site (the residues that flank the scissile bond
are underlined). L-405,492 contains the mutations NL in the P2 and P1
positions, respectively, that are observed in the Swedish-type familial
Alzheimer's disease kindreds (9). An alternative substrate,
L-405,525, that was used to assay purified BACE460 and
ProBACE460 is identical to L-405,492 except that it has KM at the P2
and P1 positions, respectively, which are the residues that are present
in the wild type APP sequence. All assays were performed in the
presence of 50 mM sodium acetate, 0.15 M NaCl,
pH 4.5, 0.2%
3-[(3cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid 0.1 mg/ml bovine serum albumin. Where indicated, BACE assays were also performed in the presence of the BACE inhibitor (3), P10-P4'Stat(Val) (Midwest Biotechnology, Fishers IN and Enzyme Systems
Products, Livermore, CA). Reactions were incubated at 37 °C for
2 h unless otherwise indicated and terminated by heating the
samples at 75 °C for 5 min. Samples were analyzed by reverse-phase HPLC using a Zorbax Extend C18 column (Waters, Milford, MA).
Coumarin-tagged product and substrate were monitored by fluorescence
detection with excitation and emission wavelengths of 340 and 440 nm, respectively.
-D-glucopyranoside, 0.3 M methyl-
-D-mannopyranoside, 0.5 µM
aprotinin, and 1 µM leupeptin. EDTA (1 mM)
was added to the eluate from the lectin affinity column. Diafiltration
with a hollow fiber 10,000 molecular weight cut-off column (A/G
Technologies, Needham MA) was used to concentrate the lectin affinity
column eluate ~15-fold and buffer exchange into 50 mM
sodium acetate, pH 5.0, 0.5 M NaCl, 0.5 µM
aprotinin, 1 µM leupeptin, and 1 mM EDTA. The
diafiltration retentate was applied to an affinity column (HiTrap
N-hydroxysuccinimide-activated; Amersham Pharmacia Biotech)
containing immobilized P10-P4' Stat(Val) (3). The affinity column was
washed sequentially with (a) Buffer B (20 mM
sodium acetate, pH 5.0, 125 mM NaCl) plus 0.5 µM aprotinin, 1 µM leupeptin, 1 mM EDTA, 100 µM pepstatin, (b)
Buffer B, (c) Buffer C (20 mM sodium acetate, pH
5.0, and 750 mM NaCl), and (d) Buffer B. The
P10-P4' Stat(Val) affinity column was eluted with Buffer D (20 mM Tris-HCl, pH 7.2, and 125 mM NaCl).
Fractions were collected and evaluated for BACE activity with
L-405,492. Peak activity fractions were pooled and stored as aliquots
at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the protein
structure of the BACE precursor protein as deduced from its cDNA
sequence. Full-length BACE is composed of several domains that
include the Pre (residues 1-21), Pro (residues 22-45), protease
(residues 46-460), transmembrane (TM; residues 461-477)
and cytosolic tail (residues 478-501) regions. The amino acid sequence
of the putative Pro region is shown. Also shown is the schematic
structure of the BACE derivative, PreProBACE460, that was produced in
insect cells using baculovirus-mediated expression.
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Fig. 2.
Expression of PreProBACE460 by recombinant
baculovirus-infected insect cells. A,
SDS-PAGE/immunoblot analysis of CM. CM (5 µl) from Sf21 cells
infected with control virus stock (lane 1) or BACE460 virus
stock (lane 2) was probed by immunoblotting (IB)
with the anti-Pro antibody. CM from Sf21 cells infected with
BACE460 virus stock was immunoprecipitated (IP) with control
antibody (lanes 3 and 5) or anti-Pro antibody
(lanes 4 and 6) and treated CM was probed by
immunoblotting using anti-Pro antibody (lanes 3 and
4) or anti-protease antibody (lanes 5 and 6). B,
assay of BACE activity in CM from baculovirus-infected Sf21
cells. BACE substrate (L-405,492; 0.3 µM) was incubated
with increasing amounts of CM from cells infected with control virus
stock ( ) or BACE460 virus stock (
). The BACE460 virus stock was
also assayed in the presence of 10 µM P10-P4' Stat(Val)
BACE inhibitor (+). Product formation as detected by HPLC is expressed
as a percentage of the initial substrate concentration. Each sample was
assayed in triplicate; the mean ± S.D. is shown. C, CM
from BACE460 virus stock-infected Sf21 cells was
immunoprecipitated (IP) with control antibody (lane
1), anti-Pro antibody (lane 2), or anti-Pro antibody in
the presence of Pro-peptide (lane 3) and the "cleared"
CM samples were assayed for BACE activity using L-405,492. Data show
the means of duplicate values, which are normalized to the activity
observed after treatment with the control antibody (assigned a value of
100%).
-secretase scissile bond. The CM from Sf21 cells infected with BACE460 virus stock catalyzed the cleavage of L-405,492 (Fig. 2B). A linear dependence of product formation on the volume
of CM was observed. In contrast, BACE activity was not evident with CM
from Sf21 cells that were infected with control virus stock (Fig. 2B). Mass spectrometric analysis confirmed that
cleavage of L-405,492 was occurring at the
-secretase scissile bond
(data not shown). The addition of the protease inhibitors, EDTA,
aprotinin, and leupeptin did not block BACE activity in the CM from
BACE460 virus stock-infected cells. However, the BACE activity in the CM was completely suppressed by the BACE inhibitor, P10-P4' Stat(Val) (Fig. 2B).
-D-glucopyranoside and methyl-
-D-mannopyranoside (approximate 60% recovery).
The fractions containing the BACE activity were pooled, concentrated,
and subjected to affinity chromatography using immobilized P10-P4'
Stat(Val) (3). The BACE activity was bound and subsequently eluted at pH 7.2. The final recovery of BACE activity was ~15%.
View larger version (45K):
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Fig. 3.
Characterization of purified ProBACE460 and
BACE460. A, Coomassie Blue staining of purified
ProBACE460 (1 µg; lane 1) and BACE460 (1 µg; lane
2). The protein molecular weight markers and their corresponding
apparent molecular mass values are shown. B,
Immunoprecipitation (IP) of isolated ProBACE460 and BACE460
with anti-Pro antibody. Lane 1, ProBACE460; lane
2, ProBACE460 + control antibody; lane 3, ProBace460 + anti-Pro antibody; lane 4, BACE460; lane 5,
BACE460 + control antibody; lane 6, BACE460 + anti-Pro
antibody. The unbound protein was analyzed by SDS-PAGE/immunoblot
(IB) analysis using the anti-Pro antibody (top
panel) or anti-protease antibody (bottom panel).
C, N-terminal analysis of the purified ProBACE460 and
BACE460 preparations. The depicted residues are those obtained at each
cycle of the Edmund degradation and correspond to the residues expected
from the amino acid sequence (2-6).
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Fig. 4.
In vitro activation of ProBACE460
to yield BACE460 by treatment with furin. A,
SDS-PAGE/immunoblot (IB) analysis showing
time-dependent, furin-mediated conversion of ProBACE460 to
BACE460. Protein blots were probed with the anti-Pro (top
panel) and anti-protease (bottom panel) antibodies. The
incubation times with furin are indicated. Lane B contains
BACE460. B, impact of furin treatment of ProBACE460 on BACE
activity ( ) as measured with the BACE substrate (L-405,492) and HPLC
analysis. Also shown is the residual amount of ProBACE460 using the
data in A and gel scanning densitometric analysis (
).
C, assay of BACE activity of the ProBACE460 or BACE460
preparations using L-405,492 after incubation with and without furin
for the indicated times.
1 s
1, respectively. Hence,
ProBACE460 appears to display ~43% of the activity of BACE460 for
this particular substrate. Mass spectrometric analysis confirmed that
L-405,492 is cleaved at the
-secretase scissile bond by both
purified ProBACE460 and BACE460 (data not shown).
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Fig. 5.
Activity displayed by purified ProBACE460 and
BACE460. A, ProBACE460 ( ) or BACE460 (
) (each 10 nM) were incubated with varying concentrations of L-405,492
for 30 min at 37 °C. B, ProBACE460 (
) or BACE460 (
)
(each 500 nM) were incubated with varying concentrations of
L-405,525 for 120 min at 37 °C. The amount of product was measured
by HPLC. Reactions were performed in triplicate, and the data are
reported as the means ± S.D.
-secretase scissile bond (data not shown). The kcat/Km value of ProBACE460
for L-405,525, 46 M
1 s
1, is
~74% of the corresponding value displayed by BACE460, 62 M
1 s
1. Hence, the activities of
ProBACE460 and BACE460 with L-405,525 are not markedly different just
as was observed with the other substrate, L-405,492. However, the
kcat/Km values of ProBACE460
and BACE460 for L-405,525 are 1.5 and 0.9%, respectively, of the
corresponding values for L-405,492. This large difference is consistent
with earlier observations that BACE displays a striking preference for
the Swedish-type mutations at the P2 and P1 positions of the substrate
(2-4).
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Fig. 6.
Inhibition of ProBACE460 and BACE460 with
P10-P4' Stat(Val). ProBACE460 (20 nM; ) or BACE460
(20 nM;
) was incubated with BACE substrate (L-405,492)
in the presence of varying concentrations of P10-P4'Stat(Val). Product
formation was measured by HPLC. Data are expressed as the percentages
of BACE activity that is observed in the absence of P10-P4' Stat(Val)
with either ProBACE460 or BACE460. Reactions were performed in
triplicate, and the data are reported as the means ± S.D.
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Fig. 7.
Recovery of BACE activity after
denaturation/renaturation of ProBACE460 and BACE460. A,
aliquots of ProBACE460 (bars 1 and 2) and BACE460
(bars 3 and 4) were incubated in 6 M
guanidine for 60 min in the absence (bars 1 and
3) or presence (bars 2 and 4) of BACE
Pro peptide (50 µM), diluted in renaturation buffer for
2 h, added to assay buffer containing BACE substrate (L-405,492)
and incubated at 37 °C for 60 min. Product formation was measured by
HPLC. Activity is expressed as the percentage of the mock
denatured/renatured ProBACE460 or BACE460. B, immunoblot
analysis using the anti-protease antibody of ProBACE460 (lanes
1 and 2) and BACE460 (lanes 3 and
4) before (lanes 1 and 3) and after
the denaturation/renaturation (D/R) cycle (lanes
2 and 4). The mobilities of the molecular weight
markers are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-secretase mediated cleavage is expected to take place
in the cell. However, attempts to pinpoint the subcellular site of
-secretase activity must also take into account the pH optimum of
BACE (2, 3) or ProBACE,3 both
approximately pH 4.5. Hence, although it is tempting to attribute the
purported
-secretase-mediated cleavage in the endoplasmic reticulum
(19) to colocalized ProBACE (20), the neutral pH value in the
endoplasmic reticulum (21) does not appear to be conducive to ProBACE
(or BACE) catalysis. Others claim that
-secretase cleavage is absent
in the endoplasmic reticulum but is instead colocalized with markers
later in the secretory pathway (22), which is seemingly more compatible
with the aforementioned pH dependence of BACE activity. Another recent
investigation showed that BACE is actively targeted and localized to
endosomes (23), which is an acidic site (pH 5.0) (20) that should be
quite favorable for BACE-mediated catalysis (21). It is important to
note that furin-mediated processing of ProBACE probably occurs in the
secretory pathway upstream of subcellular sites with pH conditions that promote BACE activity (20); hence, the activity displayed by ProBACE
in vitro may not be realized within the cell. Further studies are required to establish the significance of cellular ProBACE activity.
-lytic protease (24) and subtilisin (25). Our observations indicate
that the Pro peptide interacts with BACE460 during refolding and may
well serve as a chaperone that thwarts aggregation of unfolded protein.
The poor ability of the Pro peptide to block BACE460 activity makes it
difficult to attribute the lower activity of ProBACE relative to
BACE460 to a direct interaction between the Pro domain and the active
site in ProBACE460. The fact that the BACE Pro region promotes folding
but is not a potent inhibitor of the protease is unusual but not
without precedent. A recent study showed that the inhibitory and
chaperone-like functions of the subtilisin Pro peptide are not
obligatorily linked (26). It should be noted, however, that additional
studies are required to exclude the possibility that membrane-tethered,
mammalian-cell derived, full-length ProBACE is a strict zymogen with
respect to its ability to process its natural membrane-bound substrate, APP.
peptide.
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FOOTNOTES |
---|
* 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.
§ To whom correspondence should be addressed: Merck Research Laboratories, WP42-213, Dept. of Biological Chemistry, West Point, PA 19486. Tel.: 215-652-3237; Fax: 215-652-3082; E-mail: steve_gardell@merck.com.
Published, JBC Papers in Press, December 28, 2000, DOI 10.1074/jbc.M009200200
2 Attempts to produce ProBACE460 in Sf21 cells by baculovirus-mediated expression using serum-free CM (Kemp Biotechnologies Inc., Frederick, MD) resulted in an approximate 50:50 mixture of ProBACE460 and mature BACE460 after purification by sequential concanavalin A and P10-P4'Stat(Val) affinity chromatography. ProBACE460 was further purified to apparent homogeneity from this mixture by immunoaffinity chromatography with anti-Pro antibody that is cross-linked to protein G-agarose (using the Immunopure Protein G IgG Plus Orientation kit; Pierce). The ProBACE460 was eluted from the antibody column using IgG elution buffer (Pierce). The ProBACE460 that was purified in this manner was homogenous as judged by N-terminal sequencing. Moreover, it displayed activity that was similar to ProBACE460, which was purified and characterized as described elsewhere in the manuscript (unpublished data).
3 X.-P. Shi, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
APP, amyloid
precursor protein;
CM, conditioned medium;
BACE, -site APP cleaving
enzyme;
L-405, 492,
acetyl-TTRPGSGLTNIK(6-(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVNLDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
HPLC, high pressure liquid chromatography;
PAGE, polyacrylamide gel
electrophoresis L-405, 525, acetyl
TTRPGSGLTNIK(6(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVKMDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide.
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REFERENCES |
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