From Biochemical Neuroendocrinology, Clinical
Research Institute of Montréal, Montreal, Quebec H2W 1R7,
§ Diseases of Aging Unit, Loeb Health Research Institute,
Ottawa Hospital, Ottawa, Ontario K1Y 4K9, ¶ Neuropeptides
Structures and Metabolism, Clinical Research Institute of
Montréal, Montreal, Quebec H2W 1R7, Canada, the
Department of Pharmacological and Physiological Sciences,
University of Chicago, Chicago, Illinois 60637, and ** IPMC du CNRS,
UPR411, 660 Route des Lucioles, Sophia Antipolis,
06560 Valbonne, France
Received for publication, October 31, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Processing of the Alzheimer's disease is a progressive degenerative disorder of the
brain characterized by mental deterioration, memory loss, confusion,
and disorientation. Among the cellular mechanisms contributing to this
pathology are two types of fibrous protein deposition in the brain,
intracellular neurofibrillary tangles consisting of polymerized tau
protein, and abundant extracellular fibrils largely composed of
An alternative, anti-amyloidogenic cleavage carried out by
The amyloidogenic pathway of The second step in the amyloidogenic pathway of In the current study, we investigate whether PCs are responsible for
the cleavage of the prosegment of BACE, as well as the consequences of
blocking this maturation. In addition, we examine several
post-translational modifications of BACE and their possible influence
on the processing of Mouse BACE and Its Mutants--
Full-length mouse BACE
(mBACEF) was cloned from AtT20 cells by reverse
transcriptase-polymerase chain reaction (Titan One-Tube, Roche
Molecular Biochemicals) using the following nested sense (S) and
antisense (AS) oligonucleotides: S1 = AAGCCACCACCACCCAGACTTAGG; S2 = CTCGAGCTATGGCCCCGGCGCTGCGCTG
(XhoI site at 5') and AS1 = GAGGGTCCTGAGGTGCTCTGG;
AS2 = CCTCCTCACTTCAGCAGGGAGATG. The final product (1519 base
pairs) was completely sequenced, matched with the published structure
(11), and then subcloned into the expression vector pcDNA3.1/Zeo
(Invitrogen). To detect recombinant BACEF, we added, in
phase (by polymerase chain reaction), either a V5 (GKPIPNPLLGLDST;
[BACEF]V5) or FLAG (DYKDDDDK;
[BACEF]FG) epitope to the C-terminal amino
acid of the cytosolic tail of mouse BACE. We also prepared a
BACEF construct in pIRES2-EGFP (Invitrogen) in which the
FLAG epitope was introduced just after the signal peptide cleavage site
(giving the sequence ...
GMLPA Transfections and Biosynthetic Analyses--
All transfections
were done with 2-4 × 105 HK293 cells using Effectene
(Qiagen) and a total of 1-1.5 µg of BACE construct cDNAs subcloned into the vector pIRES2-EGFP. Two days post-transfection, the
cells were washed and then pulse-incubated for various times with
either 200 µCi/ml [35S]Met; 400 µCi/ml
Na2[35SO4],
[3H]Leu, [3H]Arg, [3H]Ser; or
1 mCi/ml [3H]palmitate (PerkinElmer Life Sciences) (20).
Pulse-chase experiments with [35S]Met were carried out as
described previously (21). The cells were lysed in immunoprecipitation
buffer (150 mM NaCl, 50 mM Tris-HCl, pH 6.8, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and a protease inhibitor
mixture (Roche Molecular Biochemicals)), after which the lysates and
media were prepared for immunoprecipitations (22). The monoclonal
antibodies used were directed against either the FL (FLAG-M2; 1:500
dilution; Stratagene) or V5 (1:1000 dilution; Invitrogen) epitopes.
Rabbit polyclonal antisera included those directed against aa 122-131
(PA-(1-756)) and 485-501 (PA-(1-757)) of human BACE (Affinity
Bioreagents Inc., Golden, CO), both used at a 1 µg/ml concentration;
aa 1-16 of human A Western Blot of PC-digested
BACE--
Pro-BACES/BACES preparations were
obtained by concentrating the media (5-10-fold) of HK293 cells
transiently transfected with the cDNAs of
[BACES]FG/V5 or
[BACES-R45A]FG/V5. Proprotein convertases were obtained from the media of BSC40 cells infected with vaccinia virus recombinants of either human furin, human PACE4, mouse PC5-A (29), or rat PC7 (30).2
PC-mediated digestions of pro-BACES were carried out by
preincubating identical
aliquots3 of
pro-BACES/BACES-containing HK293 media with the
appropriate volume of PC-containing BSC40 medium for 1-4 h at 37 °C
in a final volume of 200 µl (adjusted to 50 mM Tris
acetate, pH 7.0, 2 mM CaCl2 and 0.1% Triton
X-100 (v/v)). PC activity-inhibited controls comprised identical
incubations for 4 h along with 1 µM of the appropriate purified PC cognate prosegment (24, 25). Western blot
analyses of the reaction products were carried out following 10%
SDS-PAGE using either the FG (1:1000 dilution) or V5-HRP (1:5000 dilution) monoclonal antibodies (Stratagene). The secondary antibody for FG consisted of anti-mouse HRP-coupled IgGs (Roche Molecular Biochemicals). The disappearance of the N-terminal FLAG epitope on the
Western blot was taken to represent the degree of prosegment removal by
the PCs.
In Vitro Enzymatic Activity Assays--
Biosynthesis and Processing of BACE--
To characterize the
biosynthetic pathway of BACE and its post-translational modifications,
we first cloned the enzyme from the mouse corticotrophic cell line
AtT20. The resultant, fully sequenced 1519-base pair product
corresponded to the published mouse sequence (11). To detect
membrane-bound pro-BACE or BACE, we used the V5 epitope at the C
terminus of the cytosolic tail. Alternatively, we employed the
N-terminal FLAG epitope (FG) immediately following the signal peptidase
cleavage site to detect specifically pro-BACE. This double-tagged,
full-length protein ([BACEF]FG/V5) was
coexpressed in human kidney epithelial cells (HK293) either with a
control (CTL) (brain derived neurotrophic factor (BDNF)) or
N-terminal radiosequencing (26, 30) was carried out on
SDS-PAGE-purified immunoprecipitates. The C-terminally flagged 72-kDa
[pro-BACEF]FG, labeled with
[3H]Leu and produced in the presence of
To determine whether a proprotein convertase(s) could carry out the
processing of pro-BACE to BACE, we transiently coexpressed in HK293
cells the double-tagged [BACEF]FG/V5 with an
array of PC inhibitors including
To define better the region of the Golgi where pro-BACE processing
occurs, we coexpressed in HK293 cells
[BACEF]FG/V5 with either furin or
In the next set of experiments, we attempted to demonstrate directly if
PCs could process pro-BACE in vitro. In preliminary work, we
first tested which of the PCs expected to be active in the constitutive
secretory pathway could correctly cleave a peptide (pro-BACE-(38-54))
spanning the N-terminal furin consensus site. The best processing rates
were observed with furin and PC5 (not shown), followed distantly by
PACE4, whereas PC7 could barely cleave this sequence even when a
10-fold excess (as assessed by pERTKR-MCA hydrolysis) of activity was
employed. At the same time, we observed no detectable cleavage of this
peptide by either crude or partially purified soluble BACE
[BACES]V5 (not shown), lending further
support to the view that the BACE does not autocatalytically remove its
own propeptide. We next examined the PC-mediated processing of a
double- tagged soluble (S) form of pro-BACE
[pro-BACES]FG/V5 expressed in HK293 cells.
Western blots of the secreted enzyme probed by the FG antibody revealed
that some of the enzyme was still in the form of pro-BACES.
We thus incubated identical aliquots of pro-BACES from
concentrated HK293 cell media with equivalent hydrolytic activities
(estimated using the fluorogenic substrate pERTKR-MCA) of partially
purified furin, PC5, PACE4, and PC7 for 1-4 h. The digestion products
were then run on SDS-PAGE and revealed by Western blotting using either
the FG or V5 antibodies. The results demonstrated that furin could
completely process pro-BACE into BACE within 2 h, whereas PC5 and
PACE4 had failed to complete this cleavage even after 4 h (Fig.
3). PC7 is barely, if at all, able to
perform this reaction. As confirmation of the identity of the enzyme(s)
carrying out this conversion, we treated the 4-h pro-BACE digestion
reaction with 1 µM of purified PC prosegments (pPCs)
produced in bacteria as reported previously (24). Correspondingly, the
pPCs of furin, PC5, and PACE4 inhibited pro-BACE processing. Analysis
of the R45A mutant (Fig. 3, right-hand side) of
pro-BACES with both the V5 and FG epitopes indicated that
none of the PCs tested could cleave this form, consistent with
processing occurring at Arg45 of the
42RXXR45 Post-translational Modifications of BACE and Their Effects on
Fig. 4C shows the results of SDS-PAGE analysis of
FG-immunoreactive BACE following a 2-h labeling with
[3H]palmitate of HK293 cells transiently overexpressing
either BACEF, its cytosolic tail Cys mutants, BACE-
The BACE Shedding--
We next tested whether BACEF could
be transformed into a soluble shed form. As shown in Fig.
6A, we could indeed detect a small amount of an ~6-kDa cellular form of [35S]Met
FG-immunoreactive BACEF but not FG-labeled
BACES. This suggested that at least a small amount of
shedding of membrane-bound BACEF could occur. Furthermore,
a similar [3H]palmitate Cys-labeled product was also
observed (not shown), supporting the notion that it represents a
C-terminal stump of BACE. To confirm this and to test the
importance of palmitoylation of the cytosolic tail Cys in the shedding
of BACE, we used three different antibodies. As shown in Fig.
6B, the ~6-kDa stump is detected by either the C-terminal
(CT) FLAG epitope antibody or a commercially purchased polyclonal
antiserum directed against the 485-501 CT sequence of BACE. The latter
antiserum also detected the ~6-kDa stump using wild-type
BACEF that does not carry a FLAG epitope (not shown). In
addition, we consistently observed that the cytosolic tail Cys-to-Ala
triple mutant C478A,C482A,C485A BACEF, which was minimally
palmitoylated, produced a significant increase (about 3-fold) in the
level of the ~6-kDa stump (Fig. 6B). This result suggests
that palmitoylation diminishes the shedding of BACE or enhances the
clearance of the ~6-kDa stump. Finally, to verify the presence of the
extracellular shedding product, we used a third antibody directed
against the N-terminal (NT) 122-131 sequence of BACEF.
This allowed us to detect the secreted shed form of BACE in the media
of HK293 transfectants (Fig. 6C), the level of which is also
enhanced in absence of Cys palmitoylation. Consistent with these data,
the apparent mass of the shed form of BACE (~58 kDa) is
smaller than that of its cellular counterpart.
Wild-type and Mutant BACE Processing of
We next analyzed the secreted
When we analyzed the levels of secreted APPS
To examine further the possibility that pro-BACE has The discovery of the unique type I membrane-bound BACE has
provided a new perspective in our understanding of In this work we focused on BACE, the more plausible Mutation of either of the arginines found to be critical for the
prosegment removal, i.e. R42A or R45A, did not result in significant alteration of the trafficking rate of pro-BACE to the TGN,
as estimated by pulse-chase (Fig. 2A) and sulfation rate analyses. While this article was in preparation, we became aware of two
reports (43, 44) on the biosynthesis of BACE that reported some
observations similar to ours. In the report by Capell et al.
(44) regarding the prosegment removal of human BACE, their data, like
ours, revealed that this processing occurs in the TGN and that
BACES traffics more rapidly than BACEF toward
the TGN. However, our data also differ from theirs, which suggests that the R45A mutant of human BACE does not exit the ER. Our triplicate pulse-chase data (Fig. 2A) clearly demonstrate that the exit
of both pro-BACEF and pro-BACEF-R45A (or R42A)
to the TGN is slow but does in fact occur to a similar extent for both forms.
An interesting observation occurred when we analyzed the rate of exit
of pro-BACE from the ER at 20 °C, a temperature that normally blocks
the budding of TGN vesicles but should not prevent movement from the ER
to the TGN (38). We found that under these conditions, pro-BACE cannot
exit the ER, as is the case with BFA and, much less so, bafilomycin
treatments (Fig. 5A). This is reminiscent of the observation
that Next, we showed that full-length BACEF is palmitoylated at
Cys residues 478, 482, and 485 within the cytosolic tail and that a
soluble form of BACES is not (Fig. 4C).
Interestingly, BACES seems to be secreted rapidly from and
does not accumulate within the cell, suggesting that the cytosolic
segment of BACEF must contain determinants that control
cellular trafficking rates and destination. One such element could be
Cys palmitoylation, since we found by pulse-chase experiments that the
triple mutation C478A,C482A,C485A slows the exit of pro-BACE from the
ER (not shown). However, immunocytochemical analysis of the
localization of [BACEF]FG and
[BACEF-C478A,C482A,C485A]FG failed to reveal
gross qualitative differences in their cellular distribution (not
shown). In contrast, our data show that preventing palmitoylation of
these Cys residues significantly enhances the shedding of a soluble
form of BACE into the medium (Fig. 6, B and C).
Thus, although the role of palmitoylation of BACE, which begins to
occur in the ER, remains to be fully elucidated, this modification most
likely provides a second anchor to the plasma membrane, possibly
directing the protein to discrete membrane microdomains and/or
resulting in a remodeling of the structure of its cytoplasmic region
(36).
In an effort to define the importance of cellular trafficking on the
production of C99 and A In conclusion, our data revealed that BACE can process
-amyloid precursor protein
(
APP) by
- and
-secretases generates the amyloidogenic peptide
A
, a major factor in the etiology of Alzheimer's disease. Following
the recent identification of the
-secretase
-amyloid-converting
enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the
proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network
of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant
APPsw
-secretase product C99. BACE is palmitoylated
at three Cys residues within its transmembrane/cytosolic tail and is
sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE
is shed into the medium and that the extent of ectodomain shedding is
palmitoylation-dependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a
decrease in the
-secretase product APPs
. Although there is little
increase in the generation of A
by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one
lacking the prosegment leads to enhanced A
levels. These findings
suggest that the shedding of BACE may play a role in the amyloidogenic
processing of
APP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid1 (for reviews see
Refs. 1-3).
-Amyloid, also known as A
, arises from proteolytic
processing of the
-amyloid precursor protein (
APP) at the
-
and
-secretase cleavage sites. The cellular toxicity and
amyloid-forming capacity of the two major forms of A
(A
40 and especially A
42) have been well
documented (1-3).
-secretase(s) is located within the A
peptide sequence of
APP, thus precluding the formation of intact insoluble A
. This cleavage by
-secretase within the (His-His-Gln-Lys
Leu-Val) sequence
of
APP is the major physiological route of APP maturation. The
products of this reaction are a soluble 100-120-kDa N-terminal
fragment (
APPs
) and a C-terminal membrane-bound ~9-kDa segment
(C83). In several recent reports, metalloproteinases such as ADAM9,
-10, and -17 were shown to be involved in the
-secretase cleavage of
APP (4-6). Enzymes within this family are typically synthesized as inactive zymogens that subsequently undergo prodomain cleavage and
activation in the trans-Golgi network (TGN). Evidence has been presented showing that several ADAMs are activated in a
nonautocatalytic manner by other enzymes such as the proprotein
convertases (PCs) (7). Thus, it is conceivable that such enzymes may
participate in a cascade leading to the activation of
-secretase. In
support of this proposal, we recently demonstrated that inhibition of PC-like enzymes in HK293 cells by the
1-antitrypsin
serpin variant
1-PDX (8) blocks the
-secretase
cleavage of
APPsw (9). Correspondingly, overexpression
of a PC (i.e. PC7) increased
-secretase activity. Of the
previously mentioned candidate
-secretases, our ontogeny and tissue
expression analyses suggest that, in adult human and/or mouse brain
neurons, ADAM10 is a more plausible
-secretase than ADAM17 (10).
APP processing begins with
-secretase(s). This enzyme generates the N terminus of A
by
cleaving
APP within the Glu-Val-Lys-Met-
-Asp-Ala
sequence, or by cleaving the Swedish mutant
APPsw within
the Glu-Val-Asn-Leu-
-Asp-Ala sequence. In addition,
cleavage has been reported to occur within the A
sequence
Asp-Ser-Gly-Tyr10-
-Glu11-Val, generating
A
11-40/42 (11). Very recently, five different groups
simultaneously reported the isolation and initial characterization of
two novel human aspartyl proteinases, BACE (11-15) and its closely
related homologue BACE2 (14, 15). BACE appears to fulfill all of the
criteria of a
-secretase. Whereas in vitro cleavage
specificity analyses of BACE and BACE2 did not reveal clear consensus
recognition sequences (11, 15), they did lead to the development of
novel, modified statine inhibitors (13). Comparative modeling of the
three-dimensional structure of BACE complexed with a substrate
suggested that BACE would preferentially cleave substrates having a
negatively charged residue at P1' and a hydrophobic residue at P1 (16).
In fact, this is the case for the
-secretase sites in
APP and
APPsw as well as for the site leading to the formation
of the A
11-40 peptide. Both BACE and BACE2 are type I
membrane-bound proteins with a prodomain that, at least for BACE (12),
is rapidly cleaved intracellularly. However, little else is known about
the mechanism of zymogen processing of these enzymes, including whether
their activation is autocatalytic or carried out by other enzymes.
Recent data derived from BACE overexpressed in bacteria (15) suggested
that the zymogen processing of the
Arg42-Leu-Pro-Arg45
site of the prosegment,
which is reminiscent of PC-cleavage sites (7), is not autocatalytic;
rather it is effected by another proteinase(s). Moreover, our
developmental analysis of the comparative tissue expression of mouse
BACE and BACE2 suggested that BACE, but not BACE2, is a good candidate
-secretase in the brain (10).
APP maturation
involves cleavages at the
-secretase sites
(Val-Val-
-Ile-Ala-
-Thr-Val) to generate either A
40
or A
42. Recently, in neuronal N2a cells, A
40 was shown to be produced within the TGN and
subsequently packaged into post-TGN secretory vesicles, suggesting that
the TGN is the major intracellular compartment within which the
A
40-specific
-secretase is active (17). Although
some insoluble, N-terminally truncated A
x-42
originates in the endoplasmic reticulum (ER), A
40 and
A
42 are formed primarily in the TGN. This compartment is
composed of the major source of the constitutively secreted pool of
A
that is deposited as extracellular amyloid plaques. Furthermore,
the generation of either peptide requires that
APP or its
membrane-bound,
-secretase cleavage product C99, passes at least
once through endosomal compartments (18). Thus,
APP trafficking to
or retention in particular cellular compartments may critically
influence its processing. Although the identification of the
-secretase(s) has not yet been conclusively established (18), some
reports have suggested presenilins as candidates (19).
APP and the generation of amyloidogenic A
peptides.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DYKDDDDK-QGTHL ... ) and the V5 epitope at
the C terminus of the molecule [BACEF]FG/V5. Other BACE constructs were also prepared as follows: 1) an active site
D93A mutant single- [BACEF-D93A]FG or
double-tagged [BACEF-D93A]FG/V5; 2) a
prosegment deletion mutant [BACEF-
p]FG in
which the signal peptide ending at Ala19 was fused directly
to the sequence ...
Met-Leu-Pro-Ala19-
-Glu46-Thr-Asp-Glu-Glu-;
3) a prosegment deletion mutant of the active site mutant
[BACEF-
p-D93A]FG; 4) PC cleavage site
(42RLPR45,
where boldface with underlines indicate sequence position of arginine) mutants [BACEF-R45A]FG as well as
the double-tagged [BACEF-R42A]FG/V5 and
[BACEF-R45A]FG/V5; and 5) cytosolic tail Cys
mutants, including single [BACEF-C478A]FG,
[BACEF-C482A]FG, [BACEF-C485A]FG, double
[BACEF-C482A,C485A]FG, and triple
[BACEF-C478A,C482A,C485A]FG Cys
substitutions. Soluble forms of BACE (BACES) were also
prepared by deleting the transmembrane domain and cytosolic tail (CT), leaving the sequence ... TDEST454 followed by a V5
epitope. These constructs included [BACES]V5, [BACES]FG/V5,
[BACES-R42A]FG/V5, and
[BACES-R45A]FG/V5.
(produced in our laboratory, used at a 1:200
dilution); anti-
-amyloid, recognizing mostly the C-terminal part of
A
40 (Sigma A8326, used at a 1:200 dilution); FCA18, recognizing all
A
peptides beginning with the N-terminal Asp; FCA3340, specifically
recognizing the C terminus of A
40; and FCA3542,
recognizing the C terminus of A
42 (23). Immunoprecipitates were resolved by SDS-PAGE (either 8 or 14% Tricine
gels) and autoradiographed (21). All PC inhibitor proteins were cloned
in pcDNA3 (Invitrogen), including those of
1-PDX (8); the preprosegments of furin, PC7 (24), PC5 (25), and SKI-1 (26,
27); and wild-type (
2M) and furin site-mutated (
2MG-F)
2-macroglubulin (28).
-Secretase activity
was evaluated using a 20-aa synthetic peptide (SW20) that spans the
cleavage site (KTEEISEVNL
DAEFRHDSGY) of
APPsw.
Reactions were carried out for 4-18 h at 37 °C in 100 µl of 50 mM NaOAc (pH 4.5), 10-30 µM of SW20, and 10 µg/ml of leupeptin (to inhibit low levels of a non-
-secretase
proteolytic activity). The digestion products were separated via
reverse transcriptase-high pressure liquid chromatography using a
1%/min trifluoroacetic acid/acetonitrile gradient (15-45%) on a C-18
column (Vydac). Peaks were quantitated according to their absorption
values at 210 nm and were definitively identified using matrix-assisted laser desorption ionization/time of flight mass spectroscopy
(Voyager/PerkinElmer Life Sciences). Digestions by PCs of the BACE
maturation site-spanning peptide (LGLRLPR
ETDEESEEPGRRG;
pro-BACE-(39-58)) were carried out as described above for the
pro-BACES/BACES Western blot preincubations (except in 100 µl), whereas digestions of this peptide by BACE were
as for
-secretase activity determinations (either at pH 4.5 or 6.5).
Incubations with the peptide comprising the entire prosegment of mBACE
(THLGIRLPLRSGLAGPPLGLRLPR; pro-BACE-(22-45)) were carried out as
described for the
-secretase activity measurements, first
preincubating at either pH 4.5 or 6.5 with 10-30 µM of
this peptide. Again, the digestion products were quantitated by reverse transcriptase-high pressure liquid chromatography and matrix-assisted laser desorption ionization/time of flight-mass spectroscopic analyses.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-PDX cDNA. Two days after transfection, the cells
were pulse-labeled with [35S]Met for 15 min (P15). They
were then chased for 1 or 2 h in the presence or absence of the
fungal metabolite brefeldin A (BFA), which promotes fusion of the
cis-, medial, and trans-Golgi (but not the TGN)
with the ER (31). Cell extracts were immunoprecipitated with either FG
or V5 monoclonal antibodies and analyzed by SDS-PAGE (Fig.
1). In the absence of BFA and
1-PDX at P15 (Fig. 1A), the FG epitope
reveals a 66-kDa pro-BACE form that is gradually transformed first into
a 64-kDa (C1 h) and then into a minor 72-kDa (C2 h) pro-BACE form. The
72-kDa form is not visible in the presence of BFA, the major band
appears at 63 kDa. In contrast, the 72-kDa form is greatly enriched in
the presence of
1-PDX (Fig. 1B). Treatment
with endoglycosidases revealed that the 63- and 64-kDa pro-BACE forms
are sensitive to both endoH and endoF, whereas the 72-kDa form is
sensitive only to endoF (not shown). These data suggest that the 63- and 64-kDa bands represent immature (likely ER-resident),
N-glycosylated pro-BACE, whereas the 72-kDa form represents
maturely glycosylated pro-BACE. Only in the presence of
1-PDX does pro-BACE immunoreactivity accumulate in the
Golgi apparatus. In immunoprecipitation experiments employing the V5 epitope, the 2-h chase period revealed mainly a 68-kDa band (Fig. 1C). In the presence of
1-PDX (Fig.
1D), we observed an accumulation of a 72-kDa protein
reminiscent of pro-BACE (Fig. 1C). An additional control,
using wild-type
1-antitrypsin to replace of
1-PDX, gave the same results as the BDNF controls
presented here (not shown).
View larger version (38K):
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Fig. 1.
HK293 cells were transiently cotransfected
with either ([BACEF]FG/V5 + BDNF](control
(CTL)) (A and C) or
([BACEF]FG/V5 + 1-PDX) (B and
D) cDNAs. Two days post-transfections the
cells were pulse-labeled in the absence or presence of 5 mM
BFA for 15 min with [35S]Met and then chased for 1 or
2 h. Cell lysates were immunoprecipitated with either the FG or V5
mAbs and analyzed by SDS-PAGE on 8% Tricine gels. The migration
position of the 53-kDa molecular mass standard and those of pro-BACE
(pBACE) and BACE as well as their molecular masses are
emphasized.
1-PDX, had a Leu at positions 3, 7, 9, and 13 (not
shown). This is consistent with the protein starting at
Thr22
(Ala-Gln-Gly21-
-Thr22-His-L-Gly-Ile-Arg-Leu-Pro-Leu-Arg-Ser-Gly-Leu) just after the signal peptidase cleavage site (8, 9). The corresponding
68-kDa protein, labeled with [3H]Ser, revealed a Ser at
position 6 (not shown), compatible with the protein being mature BACE
obtained following removal of the prosegment (aa 22-45) at the
Arg-Leu-Pro-Arg45-
-Glu46-Thr-Asp-Glu-Glu-Ser-Glu-Glu
sequence (12).
1-PDX (8, 21); the
pre-prosegments of furin, PC7 (24), PC5 (25), and SKI-1 (27); and the
wild-type (
2M) and furin-inhibiting mutant
(
2M-F) forms of
2-macroglubulin (28). In
addition, we prepared mutant forms of BACE in which the PC consensus
cleavage site Arg residues in the prosegment were replaced by Ala at
positions 42 or 45 (R42A or R45A, respectively). The transfected cells
were pulse-labeled for 20 min with [35S]Met and then
chased for 90 min without label. Following immunoprecipitation of the
cell lysates with a FG antibody, the material was analyzed by SDS-PAGE.
We first note that these treatments do not affect the lower
ER-associated form of pro-BACE (66 kDa) but only modulate the relative
level of the pro-BACE (72 kDa) associated with the Golgi, where
processing to BACE takes place. When BACE was coexpressed with either
1-PDX, pro-Fur, pro-PC5 or
2M-F, the
quantity of the 72-kDa pro-BACE (pBACEG, Golgi form) was
elevated (Fig. 2A). Similar
results were seen for the both the R42A or R45A prosegment cleavage
site mutants. In contrast, the 72-kDa pro-BACE was barely detectable in
the control, pro-PC7, pro-SKI-1, or
2M coexpressions. Parallel control experiments (not shown) verified that the prosegments of PC7 (24) and SKI-1 (27) were able to inhibit processing of
appropriate substrates by their cognate enzymes. These data strongly
support the hypothesis that a PC-like enzyme may be involved in the
processing of pro-BACE into BACE. The prosegment results implicate
furin and PC5 as likely PC candidates, whereas PC7 and SKI-1 appear
unlikely to mediate this process. The finding that the Arg residues at
the predicted
42RXXR45
site are essential for pro-BACE processing is also consistent with the
reported cleavage specificities of furin and PC5 (7).
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Fig. 2.
A, HK293 cells were transiently
cotransfected with cDNAs coding for either
([BACEF]FG/V5 + BDNF) (control,
CTL),
([BACEF-R45A]FG/V5 + BDNF) or ([BACEF-R42A]FG/V5
+ BDNF) or ([BACEF]FG/V5
+ either 1-PDX, the prosegments of furin,
PC5, PC7, SKI-1, furin-mutated (
2M-F) or wild-type
(
2M)
2-macroglobulin. The cells were
pulse-labeled for 20 min with [35S]Met and then chased
for 90 min. Cell lysates were immunoprecipitated with the FG mAb and
analyzed by SDS-PAGE on 8% Tricine gels. B, HK293 cells
were transiently cotransfected with cDNAs coding for either
([BACEF]FG/V5 + BDNF) (CTL),
([BACEF]FG/V5 + furin), or
([BACEF]FG/V5 +
1-PDX). The
cells were then pulse-labeled for 2 h with
Na2[35SO4]. Cell lysates were
immunoprecipitated with the FG or V5 mAbs and analyzed by SDS-PAGE on
8% Tricine gels. (Note that the higher apparent size of
BACEG in the CTL lane compared with the
furin lane is due to end-lane distortion.) The migration
positions of those pro-BACE in the ER (66 kDa; pBACEER)
or Golgi (72 kDa; pBACEG) and (68 kDa; BACEG)
are emphasized.
1-PDX and then labeled the cells for 2 h with Na2[35SO4]. SDS-PAGE analyses of
the FG or V5 immunoprecipitates are shown in Fig. 2B. By
using the FG antibody, we observed that pro-BACE is weakly sulfated
(CTL, better seen on overexposed gels). In the presence of
1-PDX, the intensity of the 72-kDa
35SO4-labeledpro-BACE (pBACEG) was
greatly enhanced. The V5 immunoprecipitates clearly demonstrated that
BACE is sulfated and further revealed that furin digestion appears to
lower the average apparent mass of sulfated BACE from 72 (pBACEG) to 68 kDa (BACEG). Finally, the data
suggest that processing of pro-BACE by a PC-like enzyme into BACE
occurs at the TGN or in a subsequent compartment. Not only are
sulfotransferases located in this region of the secretory pathway (32,
33), but, with the exception of PC5-B (34), all other PCs become active
only at or beyond the TGN (7), which is also a major site where
1-PDX acts (21).
PC consensus site. Similar results were obtained using the R42A mutant
(not shown). Finally, coexpression of
[BACEF]FG in furin-deficient LoVo cells (35)
with each of the above PCs or with the yeast PC homologue kexin
revealed that furin, kexin, and, to a lesser extent, PC5 could best
mediate efficient intracellular processing of pro-BACE into BACE (not
shown).
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Fig. 3.
Western blot analysis of 1-4 h in
vitro processing of wild-type (WT)
[pro-BACES]FG/V5 or the (R45A) mutant
[pro-BACES-R45A]FG/V5 by either furin, PC5-A,
PACE4, or PC7 in the absence or presence of 1 µM of PC prosegments (pPCs).
FLAG-M2 (FG) or V5-HRP monoclonal antibodies were
used.
-Secretase Activity--
To investigate the functions of the
prosegment and the transmembrane/cytosolic tail of BACE, we prepared a
series of mutants singly tagged at the C terminus with a FG or V5
epitope. The first construct was a truncated form of full-length BACE
in which the prosegment was removed (BACE-
p). We also created Ala
mutants of three Cys residues located within the cytosolic tail of
BACEF that are potential Cys-linked palmitoylation sites
(36). Accordingly, we made three single (Cys478,
Cys482, and Cys485), as well as double
(C482A,C485A) and triple (C478A,C482A,C485A) mutants. As described
previously, transiently transfected HK293 cells were pulse-labeled for
20 min with [35S]Met followed by a chase of either 1 or
2 h. SDS-PAGE analysis of the FG-immunoprecipitated products (Fig.
4A) revealed that, in contrast
to the wild-type [BACEF]FG, the truncated
[BACE-
p]FG remains mostly in the ER, with only small
amounts reaching the TGN (seen on overexposed
autoradiograms). This mutant also demonstrated a high level of
endoH sensitivity and a very low level of sulfation (not shown).
Furthermore, microsequencing of the [3H]Arg
[BACE-
p]FG revealed an Arg11,12 sequence,
clearly showing that the signal peptide was removed (not shown). These
data suggest that the majority of BACE-
p remains in the ER, with
only a small fraction reaching the TGN and being sulfated. This was
further corroborated by immunocytochemical evidence showing that the
majority of BACE-
p immunoreactivity was concentrated in the ER (not
shown). Hence, the prodomain appears to be critical for the efficient
exit of pro-BACE from the ER. On the other hand, BACES
passes rapidly through the secretory pathway, as evidenced by its
accumulation in the medium after 1 h of chase (Fig. 4A)
and the relatively low amounts of pro-BACES in the ER
(endoH-sensitive, lower band in cells; not shown) after either 1 or
2 h of chase. By transfecting [BACES]FG
into HK293 cells and then labeling for 2 h with
Na2[35SO4], we were able to
examine the intramolecular site(s) at which sulfation of BACE occurs.
Equal aliquots of the FG-immunoprecipitated media were digested with
endoH, endoF, or arylsulfatase. Only endoF removed the
35SO4 label (Fig. 4B), demonstrating
that sulfation occurred on one or more mature
N-glycosylation sites (32), although not on tyrosine
residues (33).
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Fig. 4.
A, HK293 cells were transiently
transfected with cDNAs coding for either
[BACEF]FG,
[BACEF- p]FG, or
[BACES]V5. The cells were
pulse-labeled for 20 min (
) with [35S]Met and then
chased for 1 or 2 h. Cell lysates and media (for
BACES) were immunoprecipitated with the FG or V5 mAbs and
analyzed by SDS-PAGE on 8% Tricine gels. B, HK293 cells
were transiently transfected with [BACES]V5
cDNA. The cells were then pulse-labeled for 2 h with
Na2[35SO4]. Cell lysates were
immunoprecipitated with the V5 mAb. Equal aliquots of SDS-PAGE-purified
proteins were then digested overnight at 37 °C with 5 milliunits of
either endoH or endoF (Glyco Inc.) or 80 milliunits of arylsulfatase
(Sigma). The products were analyzed by SDS-PAGE on 8% Tricine gels.
C, HK293 cells were transiently transfected with cDNAs
coding for either [BACEF]FG,
[BACEF-C482,485A]FG,
[BACEF-C478,482,485A]FG,
[BACEF-
p]FG, or
[BACES]V5. The cells were pulse-labeled for
2 h with [3H]palmitic acid. Cell lysates were
immunoprecipitated with FG or V5 (for BACES) mAbs and
analyzed by SDS-PAGE on 8% Tricine gels.
p or
BACES. Both BACEF (68 kDa) and the
ER-concentrated BACE-
p (64 kDa) were palmitoylated. When each of the
three Cys residues was individually mutated, we observed a significant
decrease in the degree of palmitoylation (not shown). The double
(C482A,C485A) mutant had
30% as much palmitoylation as the wild-type
BACEF, whereas the triple mutant C478A,C482A,C485A was
barely palmitoylated. We verified that each of the mutants was
expressed to similar degrees based on their FG-immunoprecipitated
reactivities following a 2-h pulse labeling with [35S]Met
(not shown). These data demonstrate that palmitoylation can occur at
all three of the Cys (478, 482, and 485) residues within the cytosolic
tail of BACEF. Predictably, soluble BACES was
not palmitoylated. The fact that the 64-kDa BACE-
p was
palmitoylated, as opposed to the mature 68-kDa BACEF,
suggests that this type of post-translational modification can begin at
the level of the ER (36).
secretase activity of [BACEF]FG was
first tested in HK293 cells transfected with
APPsw
cDNA. Following a 3-h pulse labeling with [35S]Met
(Fig. 5), the cells were exposed to
either BFA, bafilomycin (an inhibitor of vesicular acidification) (37),
or a 20 °C incubation (which prevents most secretory proteins from
leaving the TGN) (38). Fig. 5A shows that BFA and the
20 °C incubation prevented FG-immunoprecipitated 66-kDa pro-BACE
from escaping the ER and becoming either the 72-kDa pro-BACE or mature,
endoH-resistant BACE (not shown), whereas bafilomycin exerted a
retarding effect in the ER (compared with untreated cells). As shown in
Fig. 5B, coexpression of wild-type BACEF and
APPsw leads to the production of a membrane-bound
~10-kDa intracellular product (C99) that was detected by a polyclonal
antiserum raised against the N-terminal 16 aa of A
. This band was
also observed using the A
N-terminal-specific antibody FCA18 (23),
confirming that this cleavage product began with the correct N terminus
of A
(starting at the
-secretase cleavage site sequence
653DAEFRHDS ... ) and likely ended at the C terminus
of
APP, as reported previously (11, 12). Unexpectedly, regardless of
the relative levels of BACE and pro-BACE,
APPsw was well
processed in the ER. However, C99 accumulation in the presence of
bafilomycin may also result from a decreased turnover due to lysosomal
alkalinization (37). In other pulse and pulse-chase experiments we
observed that the maximal amount of C99 product was generated by
BACEF after a 20-min pulse, consistent with production of
C99 in an early secretory compartment, most likely the ER.
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Fig. 5.
HK293 cells were transiently transfected with
cDNAs coding for either (A and B)
(BDNF + APPsw)
(CTL) or ([BACEF]FG +
APPsw). The cells were
pulse-labeled for 3 h with [35S]Met at either
37 °C in the absence or presence of 90 µM BFA or 250 nM bafilomycin or at 20 °C. Cell lysates were
immunoprecipitated with either the FG mAb (A) or the 1-16
A
antibody (B) and analyzed by SDS-PAGE on 8% Tricine
gels.
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Fig. 6.
HK293 cells were transiently transfected with
cDNAs coding for either [BACEF]FG or
[BACES]FG (A)
[BACEF]FG or
[BACEF-C478A,C482,C485A]FG (B
and C). The cells were pulse-labeled for
either 3 h (A) or 2 h (B and
C) with [35S]Met at 37 °C. Cell lysates
were immunoprecipitated with either the FG (A and
B), CT-(485-501) (B), or NT-(122-131)
(C) antibodies and analyzed by SDS-PAGE on 8% Tricine gels.
The arrowheads in A and B point to an
~6-kDa intracellular stub of BACEF, whereas in
C the arrow shows the position of secreted
~58-kDa shed BACE. Notice that the exposure times for all cellular
products are for 1 day, whereas the autoradiogram of the media proteins
in C was obtained after 10 days of exposure.
APPsw--
In the next set of experiments (Fig.
7), wild-type BACE and selected BACE
mutants were coexpressed with
APPsw. As shown in Fig.
7A, C99 production was evident in cells coexpressing
wild-type BACEF and
APPsw following pulse
labeling for 4 h with [35S]Met. Unexpectedly, the
same band, although less intense, was also obtained with the mutants
[BACEF-R45A] and BACEF-
p (Fig. 7A), as well as with the [BACEF-R42A],
[BACEF-C482A,C485A], and [BACEF-C478A,C482A,C485A] mutants (not shown), indicating
that all of these isoforms have at least some
-secretase activity. The absence of C99 production by the active site mutant
[BACEF-D93A] confirms that these activities actually
correspond to BACE and its mutant forms (Fig. 7A). Notably,
the soluble form of BACES produced much less C99 compared
with any of the other active forms analyzed, even though similar
amounts of immunoreactive BACE were expressed (not shown).
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Fig. 7.
HK293 cells were transiently cotransfected
with cDNAs coding for ( APPsw + BDNF) (
) or
APPsw together with
either [BACES]V5,
[BACEF]FG,
[BACEF-D93A]FG,
[BACEF-R45A]FG, or
[BACEF-
p]FG.
The cells were pulse-labeled for 3 h with [35S]Met.
The cell lysates (A) or media (B and
C) were immunoprecipitated (A and C)
with the 1-16 A
antibody, and in B with the 1-40 A
antibody (A8326), and analyzed by SDS-PAGE on 8% (A and
C) or 14% (B) Tricine gels. The migration
positions of C99, A
, A
x-40 APPS,
and A
17-40 known as p3 (generated by
- and
-secretases) are shown.
APP cleavage products using a
polyclonal antibody developed against A
40 as well as the
antibodies FCA3340 (not shown) recognizing the C terminus of
A
40 (23). Both antisera recognize A
40
(generated by the
- and
-secretases) and A
11-40
generated by overexpressed
-secretase (11). Amazingly,
BACES and, to a lesser extent, BACE-
p were by far the
forms of
-secretase that ultimately lead to the highest formation of
A
40 (Fig. 7B). In addition, BACES
did not lead to the production of A
11-40, suggesting
that the latter reaction requires a membrane-bound form of BACE.
Indeed, overexpression of either BACEF or
BACER45A (as well as the Cys mutants
[BACEF-C482A,C485A] and
[BACEF-C478A,C482A,C485A], not shown) resulted in an
elevation of the level of A
11-40 product with no
significant change in that of A
40. Again, as expected,
[BACEF-D93A] was inactive.
generated
by
-secretase using the same 1-16 A
antibody, we noticed an inverse relationship between the levels of C99 and those of secreted APPS. The constructs BACEF,
[BACEF-R45A], BACEF-
p generated higher amounts of C99 and A
x-40 along with lower levels
of secreted APPS, whereas control cells or cells
overexpressing the inactive [BACEF-D93A] mutant
secreted much more pronounced levels of APPS (Fig.
7C). These data argue that the APPS measured
with the 1-16 A
antibody is probably APPS
resulting
from cleavage of
APP by
-secretase either at the TGN or at the
cell surface (5, 39). In comparison, some of our other data (Fig. 5)
showed that overexpressed BACE or its mutants process
APPsw in an earlier compartment such as the ER and thus
precede the action of
-secretase.
-secretase
activity, digestion analyses of a synthetic peptide substrate (KTEEISEVNL[/underln]
DAEFRHDSGY) encompassing the
APPsw
-secretase cleavage site were carried out
in vitro using concentrated media of HK293 cells that
overexpressed BACES. Our preliminary findings (not shown)
indicate that preincubation of wild-type BACES (but not the
[BACES-R42A] or [BACES-R45A] mutants) with
furin appears to increase the cleavage of the SW20 synthetic peptide.
This is consistent with our Western blot (Fig. 3) that confirmed that furin had removed the FG epitope from the prosegment of the wild-type but not from either the [BACES-R42A] or
[BACES-R45A] mutants. Although these data imply that
removal of the prosegment from pro-BACE enhances the activity of this
enzyme, a definitive conclusion regarding the extent of activation
should await the findings of more detailed quantitative studies using
purified pro-BACE. In keeping with the hypothesis that the prosegment
of BACE could act as an autocatalytic inhibitor of its cognate enzyme,
we tested whether a synthetic peptide representing the full-length
prosegment (pro-BACE-(22-45)) could function as an inhibitor. When
preincubated with active BACE, 20 µM of this propeptide
resulted in only an ~20% inhibition of the Swedish peptide substrate
(at 10 µM) cleavage. Since this was a weak inhibition in
the presence of twice the amount of substrate, we concluded that this
peptide is, at best, a poor inhibitor of BACE and did not pursue this
point any further.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase (11-15). Our recent data on the tissue expression of BACE in mouse and
human brain (10) indicate that it colocalizes with
APP and ADAM10 in
the cortex and hippocampus of adult mice and in the cortex of human
presenile patients. Furthermore, the distributions of either BACE2 or
ADAM17 were not compatible with them being candidate brain
- or
-secretases, respectively.
-secretase, and
we sought to define some of its molecular and cellular trafficking
properties. We first showed that in HK293 cells BACE is synthesized as
pro-BACE in the ER and then moves to the TGN where it rapidly loses its
prosegment due to cleavage by an
1-PDX-inhibitable convertase(s). We next went on to show that, aside from
1-PDX and the furin-site mutated
2-macroglobulin, other inhibitors such as the
preprosegments of furin and PC5 can also inhibit pro-BACE processing.
N-terminal sequencing confirmed that this cleavage occurs at the site
42RLPR45
of pro-BACE sulfated at one or more of its carbohydrate moieties. The
observations that sulfation of sugars occurs in the TGN (32) and that
PCs, except perhaps PC5-B (34), are active only in this compartment or
beyond indicated that processing of pro-BACE to BACE occurs either in
the TGN or in post-TGN vesicles. This conclusion is consistent with
those of Huse et al. (40) who recently reported that
BACEF is rapidly and efficiently transported to the Golgi
apparatus and to a distal secretory pathway. Similarly, we are in
agreement with Bennett et al. (41) who showed that furin is
a potential processing enzyme of pro-BACEF. However, our
ex vivo and in vitro data complement and extend
these findings by more clearly defining the candidate PCs likely to be
responsible for BACE maturation. Thus, in vitro digestions
of pro-BACE (Fig. 3) and ex vivo coexpression of pro-BACE
with the PCs in furin-negative LoVo cells (not shown) demonstrated that
zymogen processing was best performed by furin and less so by PC5.
During revision of this article, we also became aware of an article by
Creemers et al. (42) who reached similar conclusions
regarding the involvement of several PCs in the processing of BACE,
although, in our hands, PC7 is not a candidate BACE convertase.
integrins do not exit the ER at 20 °C because of their
inability to form heterodimers (45). Whether this means that BACE is
part of a larger complex, such as the one involving
presenilins/
-secretase (46), is not yet clear. It was previously
reported that the production of A
40 and
A
42 was abrogated at 20 °C (17). Our data show that
pro-BACE can process
APPsw into C99 in the ER (Fig.
5B), suggesting that
-secretase activity could be the
limiting factor at 20 °C. Even though the holoenzymes BACE and
pro-BACE (not shown) exhibit an in vitro pH optimum of 4.5 for cleavage of synthetic peptides mimicking the
-site (11, 12, 15),
our data argue in favor of active BACE within the neutral pH
environment of the ER (Fig. 5B). The combined observations
that the active site mutant [BACEF-D93A] can lose its
prosegment (not shown), that BACE did not cleave the PC cleavage site
spanning peptide (aa 39-58 of BACE), and that PCs such as furin and
PC5 can remove the prosegment of BACE in vitro and ex
vivo support the notion that BACE does not auto-activate, but
likely requires a furin-like enzyme for zymogen activation. Alternatively, we cannot rule out the possibility that there are other
enzymes or proteins that can interact with pro-BACE and activate it by
cleavage or dislocation of its prosegment. Our finding that the BACE
zymogen is activated by a PC is similar to processing of the
relatively inactive prorenin to renin by PC5 (47). Modeling of mouse
pro-BACE, based on the structure of the closely related human
progastricsin, suggested that the full-length prosegment acts as a flap
covering the active site of BACE and that the furin-processing site
42RXXR45
is
quite accessible to cleavage (not shown).
, we compared the ability of various
engineered forms of BACE to process
APPsw and ultimately to generate amyloidogenic peptides following a 3-h pulse labeling. Surprisingly, overexpression of the soluble form of BACES
resulted in a very significant increase in the levels of secreted
A
40, but not A
11-40, as measured by an
A
40-specific antiserum A8326 (Fig. 6B). These
data were also confirmed with the FCA18, FCA3340, and FCA3542
antibodies (see "Experimental Procedures"), which revealed that
most of the processed material was indeed A
40 and not
A
42 (not shown). This experiment, which was repeated four times, revealed that, in the presence of BACEF,
intracellular C99 production is enhanced, and secreted
A
11-40 becomes clearly visible. In contrast, in the
presence of BACES, we note a small increase in
intracellular C99 accompanied by a large enhancement in the level of
secreted A
40 (Fig. 6B). Pulse-chase analyses demonstrated that C99 is formed early, since we could observe its
maximal production within the first 20 min of radiolabeling (not
shown). The only difference is that at the 20-min pulse, the level of
intracellular C99 is much higher with BACEF as compared with BACES, whereas following a 90-min chase, secreted
A
40 was only observed with BACES. These data
suggest that BACEF and BACES may process
APPsw in different intracellular compartments,
ultimately leading to the accumulation of either C99 or its
-secretase product A
40. In view of the rapid exit of
BACES from the ER and its fast trafficking through the TGN
and past the cell surface (Fig. 4A), the production of C99
by BACES may be favored in a micro-compartment close to
where
-secretase is active. An exciting extension of this model
(which will require extensive verification) is that shedding may
enhance the amyloidogenic potential of BACE. Indeed, a small amount of
an ~6-kDa C-terminal membrane-bound stub and a soluble 58-kDa form
resulting from BACEF shedding were observed in HK293
cells (Fig. 6). Moreover, palmitoylation of BACE cytosolic tail Cys
residues appears to suppress shedding (Fig. 6, B and C), perhaps by controlling the cellular localization of
BACEF as has been reported for other proteins (48). Whether
this processing reaction is accomplished by ADAMs (4-6) or other
sheddases is currently under investigation. Finally, overexpression of
the active site mutant [BACEF-D93A] in murine N2a cells
stably overexpressing
APPsw (17) did not affect the
generation of either C99 or A
by endogenous secretases (not shown),
suggesting that this mutant does not act as a dominant negative, as was
the case for the active site mutant of the candidate
-secretase
ADAM10 (5).
APPsw in the ER and that furin or PC5 process the
zymogen in the TGN, possibly to optimize its activity in acidic
cellular compartments. BACE undergoes a number of other
post-translational modifications such as carbohydrate sulfation and
cytosolic tail Cys palmitoylation that may finely regulate its rate of
trafficking and cellular destination(s). The latter modification seems
to reduce the level of BACE ectodomain shedding, which may provide a
safety mechanism to reduce the amyloidogenic potential of BACE. Since
expression of soluble BACE (as opposed to full-length BACE) ultimately
leads to a much higher production of the amyloidogenic peptide A
,
inhibiting the shedding of BACE may have value as an alternative
strategy in the treatment of Alzheimer's disease. Finally, the
in vivo physiological function of BACE remains to be
elucidated, along with the possibility that this enzyme may be part of
a larger complex with other proteins, including the other secretases
involved in the processing of
APP.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. Lemieux for her technical help throughout this study. The secretarial assistance of S. Emond is appreciated.
![]() |
FOOTNOTES |
---|
* This work was supported by Canadian Institutes of Health Research (CIHR) Group Grant MGC-11474, by CIHR Operating Grants MOP-14466 (to N. G. S. and M. C.) and MT 14766 (to C. L.), and by the Protein Engineering Network of Centres of Excellence Program supported by the Government of Canada.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 and reprint requests should be
addressed: Biochemical Neuroendocrinology, Clinical Research Institute of Montréal, 110 Pine Ave. West, Montreal, Quebec H2W 1R7,
Canada. Tel.: 514-987-5609; Fax: 514-987-5542; E-mail:
seidahn@ircm.qc.ca.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M009899200
2 The activities of the different PC preparations were established according to the initial hydrolysis rates of the fluorogenic synthetic peptidyl substrate pERTKR-MCA (29, 30). Thus, in each incubation, we used the necessary volume of enzyme preparation required to generate a hydrolysis activity of 180 pmol of AMC released per h at 100 µM pERTKR-MCA.
3 In lieu of a precise analytical means of quantitating our pro-BACES/BACES preparations, we used identical aliquots of these HK293 preparations to ensure that the tubes within each incubation series received exactly the same starting quantity of pro-BACES.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A,
-amyloid;
PC, proprotein convertase;
BDNF, brain-derived neurotrophic factor;
SKI-1, subtilisin-kexin-isozyme-1;
1-PDX,
1-antitrypsin Portland;
HRP, horseradish peroxidase;
BACE,
-amyloid-converting enzyme;
BFA, brefeldin A;
TGN, trans-Golgi network;
ER, endoplasmic reticulum;
FG, FLAG-M2
epitope;
2M,
2-macroglobulin;
BACEF, full-length BACE;
BACE-
P, prosegment-deleted
BACE;
BACES, soluble BACE;
[pro-BACEF]FG, full-length BACE with a
FLAG-epitope at the C terminus;
[BACEF]V5
full-length BACE with a V5-epitope at the C terminus, [BACEF]FG/V5, full-length BACE with a
FLAG-epitope at the N terminus and a V5-epitope at the C terminus;
[BACEF-D93A]FG, full-length BACE mutated at
the active site Asp93 into Ala and containing a
FLAG-epitope at the C terminus;
[BACEF-D93A]FG/V5, full-length BACE mutated
at the active site Asp93 into Ala and containing a FLAG
epitope at the N terminus and a V5-epitope at the C terminus;
[BACEF-
p]FG, full-length BACE lacking the
prodomain and containing a FLAG epitope at the C terminus;
[BACEF-R45A]FG, full-length BACE mutated at
Arg45 into Ala and containing a FLAG epitope at the C
terminus;
[BACEF-R42A]FG/V5, full-length BACE
mutated at Arg42 into Ala and containing V5 and FLAG
epitopes at the N and C termini, respectively;
[BACEF-R45A]FG/V5 full-length BACE mutated at
Arg45 into Ala and containing V5 and FLAG epitopes at the N
and C termini, respectively;
cytosolic tail Cys-mutants included single
[BACEF-C478A]FG, [BACEF-C482A]FG,
[BACEF-C485A]FG, double
[BACEF-C482A,C485A]FG, and triple
[BACEF-C478A,C482A,C485A]FG Cys to Ala
mutants flagged at the C terminus;
CT, cytosolic tail;
endoH, endoglycosidase H;
endoF, endoglycosidase F;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
PAGE, polyacrylamide gel electrophoresis;
aa, amino acids;
APP,
-amyloid precursor protein;
mAb, monoclonal antibody;
CTL, control;
pPC, PC prosegment;
ADAMs, a disintegrin and metalloprotease.
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