(Received for publication, September 13, 1995; and in revised form, December 11, 1995)
From the
Progressive cerebral deposition of the amyloid -peptide
(A
) is an early and constant feature of Alzheimer's disease.
A
is derived by proteolysis from the
-amyloid precursor
protein.
-Amyloid precursor protein processing and the generation
of A
have been extensively characterized, but little is known
about the mechanisms of degradation of this potentially neurotoxic
peptide. We identified and purified a proteolytic activity in culture
medium that can degrade secreted A
but not larger proteins in the
medium. Detection of the activity in conditioned medium required the
presence of fetal bovine serum and the passage of the cells with a
pancreatic trypsin preparation. Its inhibitor profile showed that the
activity was a serine protease other than trypsin or chymotrypsin. The
protease occurs as a stable
700-kDa complex with the inhibitor,
-macroglobulin (
M), that retains
activity against small substrates such as A
. Its
NH
-terminal sequence suggests that the protease is
previously unidentified. Our results indicate that the A
-degrading
protease we have detected is a non-trypsin component of a pancreatic
trypsin preparation or else derives from a zymogen in serum that is
activated by a protease in the latter preparation. Because
A
-bearing plaques in Alzheimer's disease brain contain both
M and receptors of
M-protease
complexes, the same or a similar
M-protease complex
could arise in vivo and play a role in A
clearance.
All patients with Alzheimer's disease (AD) ()develop extracellular amyloid deposits that are intimately
associated with dystrophic axons and dendrites, reactive astrocytes,
and activated microglia (neuritic plaques)(1) . The amyloid is
principally composed of the 40-42-residue amyloid
-protein
(A
)(2) , a proteolytic fragment of the
-amyloid
precursor protein (
APP)(3) .
APP is an integral
membrane glycoprotein that is encoded by a gene on human chromosome 21
and undergoes constitutive proteolytic processing to generate several
distinct secreted derivatives, including A
and a large, soluble
ectodomain fragment designated APP
(1) .
Several
lines of evidence support the hypothesis that A deposition is an
early and critical event in AD pathogenesis. First, missense mutations
in and around the A
region of
APP are genetically linked to
early onset AD in several families (reviewed in (4) ). The
biochemical effects of these mutations have been modeled in cultured
cells and shown to involve increased secretion of A
peptides(5, 6, 7, 8) . Second,
patients with trisomy 21 (Down's syndrome) have enhanced
expression of
APP due to increased gene dosage and gradually
develop the typical histopathological lesions of AD(9) . Third,
trisomy 21 patients develop immature, non-neuritic A
deposits
(diffuse plaques) prior to any other histopathological feature of
AD(9, 10) . Fourth, transgenic mice expressing a
mutant
APP protein develop A
deposits, surrounding neuritic
and glial dystrophy and synaptic loss resembling those of
AD(11) .
Based on these and other observations, it is likely
that A deposition plays a role in initiating AD lesions that
ultimately lead to neuronal dysfunction and hence dementia. As a
result, there is intense interest in finding compounds that
down-regulate A
production, retard its aggregation into
potentially cytotoxic amyloid fibrils, protect cells from
A
-mediated neurotoxicity, or enhance A
clearance from the
brain. Although recent research has focused almost entirely on the
biosynthetic processing of
APP and the production and
fibrillogenesis of A
, the degradation of the peptide is of
potentially equal importance in the process leading to progressive
amyloid deposition. Because A
is a normal product of
APP
metabolism that is present in cerebrospinal fluid and plasma at
10
to 10
M(12, 13, 14) , there must be
mechanisms for its clearance that prevent its accumulation in most
individuals until very late in life.
During the course of
experiments on A-secreting transfected cells, we observed a
time-dependent decrease in the amount of A
in the conditioned
medium. We have determined that the factor principally responsible for
the decrease in A
in our cultures is a serine protease that
degrades A
efficiently and is proteolytically active in a stable
700-kDa complex with the protease inhibitor,
-macroglobulin (
M). Detection of this
complex in culture medium required the presence of serum and a
pancreatic trypsin preparation used to passage the cells.
Amino-terminal sequencing of the purified enzyme suggests that it is a
previously unidentified endoprotease, not trypsin. Our results indicate
that serine protease-
M complexes can efficiently
degrade secreted A
peptides under physiological conditions,
raising the possibility that the regulation of A
levels in brain
could involve a similar mechanism.
During purification of the protease (below),
A-degrading activity was monitored by incubating 0.5-7
µl of the fractions obtained at each purification step with 7
µg of cold synthetic A
-(1-40) in 14 µl of reaction
buffer (187 mM NaCl, 0.02 M NaHPO
, 10
mM Tris-HCl, pH 7.5) at 37 °C for various times. After
quenching the reaction with 0.5 volume of glacial acetic acid, the
sample was analyzed by reverse phase HPLC, or 5 µl of 0.1% pyronin
Y was added before analysis on acid-urea-PAGE gels (see below). One
unit of enzyme activity is defined as the amount of protease required
to degrade 1 µg of A
-(1-40) substrate in 1 h at 37
°C under the above conditions. Protease inhibition assays were
conducted under the same conditions, except in the presence of 0.3
mM diisopropyl fluorophosphate (DFP). For some experiments,
excised gel bands or immunoprecipitates which contained the protease
were incubated in 50 µl of the same reaction buffer containing 25
µg of cold A
-(1-40) at 37 °C for 2 h. After a 10,000
g spin for 3 min, the supernatants were collected,
quenched, and analyzed by acid-urea-PAGE.
Figure 1:
Degradation of A by COS
conditioned medium. A, CHO cells transfected with
APP695
were pulse-labeled with [
S]methionine for 2 h,
washed with isotope-free medium, and chased for 4 h in either
unconditioned medium (DMEM, 10% FBS; lane 1) or conditioned
medium of COS cells (lane 2). The chase media were
immunoprecipitated with the A
antibody, R1280, and electrophoresed
on Tris-Tricine SDS-PAGE gels, followed by autoradiography. Arrows indicate APP
, A
, and p3. Protein size markers are
shown. B,
I-labeled synthetic
A
-(1-40) peptide was added to either unconditioned medium
(DMEM; lane 1) or COS CM (lane 2) and incubated at 37
°C for 4 h. A
remaining after this in vitro reaction
was detected by Tris-Tricine SDS-PAGE and autoradiography. C,
proteolytic products of the A
-(1-40) peptide after
incubation in medium (DMEM, 10% FBS; lane 1) or COS CM (lane 2) at 37 °C for 4 h were resolved by 15%
acid-urea-PAGE and stained with Coomassie Blue. The position of intact
A
-(1-40) is shown. * indicates the A
cleavage
products.
To characterize the proteolytic activity, we
examined the effects of several different protease inhibitors. Using
concentrations known to cause maximal inhibition of other relevant
proteases(17) , the inhibitor profile demonstrated that an
Adegrading serine protease was present in the medium (Table 1). Both DFP and 4-(2-aminoethyl)-benzenesulfonyl fluoride
(Pefabloc) virtually abolished the degradation of secreted A
and
partially inhibited that of secreted p3 (Fig. 2). Because
Pefabloc actually decreased the levels of secreted A
in cells
cultured in plain DMEM, 10% FCS (Fig. 2, compare lanes 1 and 3), this compound may have an inhibitory effect,
either directly or indirectly, on the unidentified proteases that
generate A
from
APP, in addition to its inhibition of the
A
-degrading protease. Two other serine protease inhibitors,
elastatin and aprotinin, produced only 10-15% inhibition of
A
degradation by the COS medium.
Figure 2:
Degradation of A mediated by COS
conditioned medium is blocked by certain serine protease inhibitors.
A
secreted by
APP
-transfected CHO cells was
analyzed in pulse-chase experiments as described in Fig. 1,
using either unconditioned medium (DMEM, 10% FBS; lanes 1, 3, and 5) or COS CM (lanes 2, 4,
and 6) for the chase. The serine protease inhibitors Pefabloc (lanes 3 and 4) and DFP (lanes 5 and 6) were added to the chase media. Since Pefabloc decreased the
amount of A
in DMEM, 10% FBS, it may have additional effects on
APP processing (see ``Results''). A
and p3 (arrows) were precipitated by
R1280.
In further experiments, we
observed that the A-degrading activity in CM was highest
immediately after the passage of COS cells and decreased with time
after passage. Because trypsin, a serine protease, was routinely used
to passage the COS cells, we examined the possibility that A
degradation by the COS CM was mediated at least in part by the trypsin,
although trypsin inhibitors are present in the FBS that was used in all
experiments, and only very small amounts of a Life Technologies
pancreatic trypsin preparation were applied to detach the cells for
passage (see ``Experimental Procedures''). A
-degrading
activity in CM was compared in COS cultures that were detached by
trypsin purchased from Life Technologies, Inc., sequence-grade trypsin
purchased from Boehringer Mannheim or EDTA. As additional controls, the
same amount of Life Technologies trypsin as used for cell passage was
incubated in plain DMEM, 10% FBS in the absence of COS cells. These
various media were assayed for A
-degrading activity by the
pulse-chase/immunoprecipitation paradigm described above. The CM from
COS cells passed using sequence-grade trypsin from Boehringer Mannheim
or EDTA showed little or no A
-degrading activity in this paradigm,
while DMEM, 10% FBS incubated with the trypsin from Life Technologies
without the COS cells degraded A
(Fig. 3A),
indicating that the major A
-degrading activity observed under
these conditions did not require the presence of COS cells. To
determine whether the trypsin itself in the Life Technologies
preparation was responsible for the A
degradation, TLCK, an
inhibitor of trypsin, was included during the chase period. A
degradation was not prevented (Fig. 3B). In view of
this result and the fact that the more highly purified trypsin
preparation from Boehringer Mannheim did not result in A
degradation, it was not trypsin per se that degraded A
in
our assay. To determine whether the degradation was mediated by
chymotrypsin, which often copurifies with trypsin, we included TPCK, an
inhibitor of chymotrypsin, in the COS chase medium, but again A
degradation was not inhibited (Fig. 3B). In addition,
purified chymotrypsin was incubated with synthetic A
-(1-40)
in DMEM, 10% FBS, but a different pattern of A
fragmentation was
observed on acid-PAGE, and this was inhibitable by TPCK (data not
shown). Thus, chymotrypsin is not responsible for the A
-degrading
activity we describe.
Figure 3:
The
A-degrading activity requires the presence of a pancreatic trypsin
preparation with fetal bovine serum during the passage of COS cells. A,
APP
-transfected CHO cells were
pulse-labeled with [
S]methionine for 2 h and
chased in DMEM, 10% FBS (DMEM/S, lane 1), CM of COS
detached for passaging by the pancreatic trypsin preparation from Life
Technologies, Inc. (COS/T, lane 2), CM of COS cells
detached for passaging by sequence-grade trypsin from Boehringer
Mannheim (COS/T*, lane 3), CM of COS cells detached
by EDTA (COS/EDTA, lane 4) or the DMEM, 10% FBS
incubated with the Life Technologies trypsin preparation without cells (DMEM/S+T, lane 5). Labeled A
and p3
present in the chase media were analyzed by precipitation with R1280,
Tris-Tricine SDS-PAGE, and autoradiography. B, A
secreted
by
APP
-transfected CHO cells was analyzed in
pulse-chase experiments as described in A, using either DMEM,
10% FBS (lane 1) or COS CM (lanes 2-4) for the
chase. The trypsin protease inhibitor, TLCK (lane 3), and the
chymotrypsin protease inhibitor, TPCK (lane 4), were added to
the chase CM. C, proteolytic products of A
-(1-40)
peptide after incubation (37 °C, 4 h) in the CM of COS cells
passaged with the Life Technologies trypsin preparation (lane
1), in plain DMEM preincubated with Life Technologies trypsin (DMEM+T, lane 2) or in DMEM, 10% FBS
preincubated with Life Technologies trypsin (DMEM/S+T, lane 3). Proteins were resolved by 15% acid-urea-PAGE and
stained with Coomassie. * indicates the A
cleavage
products.
To investigate further the origin of the
A-degrading activity we detected in CM, we preincubated DMEM with
the same small amount of Life Technologies trypsin preparation as used
for cell passage at 37 °C for 3 days either in the presence or
absence of fetal bovine serum and then incubated synthetic
A
-(1-40) in this solution at 37 °C for 16 h and detected
products by acid-urea-PAGE. Only the DMEM, 10% FBS incubated with the
Life Technologies trypsin gave rise to the pattern of A
fragments
that we originally detected in COS CM (Fig. 3C),
whereas Life Technologies trypsin without FBS hydrolyzed A
differently and FBS alone produced no degradation. These results
clearly indicated that the A
degradating activity detected in COS
CM (Fig. 1) required the presence of the Life Technologies
pancreatic trypsin preparation and FBS during cell passage.
Figure 4:
Purification of an active A-degrading
protease from conditioned medium of COS cells. A, the
A
-degrading protease was purified as summarized in Table 2and ``Results'' and detailed under
``Experimental Procedures.'' Fractions at various
purification steps were assessed for enzyme enrichment by native
(non-denaturing) polyacrylamide gel electrophoresis and Coomassie Blue
staining. Aliquots from starting CM of COS cells (48 µg, 10.8
units) (lane 1) and a proteolytically active fraction from the
final Mono Q column (3.6 µg, 13 units of A
-degrading activity) (lane 2) are shown. The position of the presumptive
A
-degrading protease is indicated. B, aliquots of
fractions at various purification steps were assayed for the presence
of [
H]DFP-labeled serine protease. After native
gel electrophoresis and autoradiography, the proteins present in DMEM,
10% FBS (lane 1), COS CM (COSM, lane 2), an
active fraction after Affi-Gel Blue chromatography (AB, lane 3), an active fraction eluted from the Q-Sepharose column (QS, lane 4) and then precipitated with ammonium
sulfate (AS, lane 5), pooled active fractions from
the Bio-Gel A15 column (BA15, lane 6), and an active
fraction from the final Mono Q chromatograph (lane 7) are
illustrated. The position of the [
H]DFP-bound
A
-degrading protease is indicated. The minor
[
H]DFP-bound bands shown above and below the
substantially enriched A
-degrading protease (lanes 6 and 7) are forms apparently derived by aggregation or partial
dissociation (see ``Results''). C, A
-degrading
activity obtained during the purification was monitored by incubating
A
-(1-40) peptide with an aliquot from each fraction at 37
°C for 1 h, followed by acid-urea-PAGE and Coomassie staining. Lane 1, A
-(1-40) peptide alone; lane 2,
A
-(1-40) peptide plus an active aliquot from Mono Q
chromatography without DFP; lane 3, as in lane 2 with
DFP. Arrow, intact A
-(1-40) peptide. *, cleavage
products of A
-(1-40) peptide. D, confirmation that
the protease shown in A and B mediates the
proteolytic degradation of A
. An active Mono Q fraction was
incubated with (lanes 2, 4, and 6) or
without (lanes 1, 3, and 5)
[
H]DFP at 37 °C for 30 min. The proteins were
analyzed by native gel electrophoresis and Coomassie staining (lanes 1 and 2), followed by autoradiography (lanes 3 and 4). Small gel pieces containing the
single protein band representing the protease were excised and added to
a buffer (50 µl) containing 25 µg of synthetic
A
-(1-40). After incubation at 37 °C for 2 h, 0.2 volume
of the supernatant was loaded on an acid-urea-PAGE gel and stained with
Coomassie (lanes 5 and 6). Arrow, intact
A
-(1-40); *, A
-(1-40) degradation
products.
The protease obtained from the final Mono Q step was
incubated with synthetic A-(1-40) (Fig. 4C).
The characteristic degradation of A
and appearance of its
proteolytic fragments were confirmed, and this was inhibited by DFP. To
show directly that the single, DFP-labeled band in the native
polyacrylamide gel of the final fraction contained the A
-degrading
activity, this band was excised and incubated with 25 µg of
synthetic A
1-40. The characteristic A
degradation
pattern on acid-urea gels was obtained. Preincubation with DFP blocked
the degradation (Fig. 4D, lanes 5 and 6). We conclude that the single protein band on the native
polyacrylamide gel corresponds to the A
-degrading serine protease.
Figure 5:
Analysis of the A-degrading protease
by SDS-PAGE electrophoresis. An active fraction of the A
-degrading
protease from the final Mono Q chromatography (Table 2), which
was shown to be a single band on a native polyacrylamide gel (lane
1), was separated into multiple components by Tris-Tricine
SDS-PAGE electrophoresis (Coomassie-stained) (lane 2). The
same fraction was incubated with [
H]DFP at 37
°C for 30 min, and the labeled proteins (185 and 28 kDa) identified
by Tris-Tricine SDS-PAGE and autoradiography (lane 3). Marker
proteins are indicated.
Figure 6:
-Macroglobulin is present
in the A
-degrading protease complex. A, the purified
protease obtained after Mono Q chromatography was characterized by
immunoblotting with an anti-
M antiserum (lane
1). Purified
M served as a control (lane
2); charateristic heat-labile fragments of
M are
also seen. B, [
S]Met-labeled A
secreted by
APP
-transfected CHO cells was analyzed
in a pulse-chase experiment as in Fig. 1. During the chase
period, plain DMEM, 10% FBS was used as a control (lane 1).
COS CM was preincubated with either normal rabbit serum (lane
2) or anti-
M antibody (lane 3), followed
by precipitation with protein A-Sepharose, after which the supernatants
were harvested and added to the chase medium. After the chase, the
media were immunoprecipitated with R1280. C, COS CM was
precipitated with either anti-
M antibody (lane
1) or normal rabbit serum (lane 2). The precipitates were
washed and incubated in a buffer containing synthetic
A
-(1-40) (25 µg) at 37 °C for 2 h. Supernatants were
collected after spinning for 5 min, and proteolytic products of
A
-(1-40)(*) detected by acid-urea-PAGE and Coomassie
staining.
Figure 7:
The purified A-degrading protease is
a multi-component complex. To confirm that the A
-degrading
protease is a stable, high molecular weight complex, the partially
purified protein after ammonium sulfate (Table 2) was overlaid
onto a 10-40% continuous sucrose gradient and centrifuged for 15
h in a swinging bucket rotor. Fractions (0.6 ml) were collected from
the top of the gradient. A, the amount of protein per fraction
was determined by UV spectrophotometry at 280 nm. The approximate
positions of the marker proteins albumin (67 kDa), catalase (232 kDa),
and thyroglobulin (669 kDa) were estimated in a parallel gradient. B, A
-degrading activity was monitored by incubating with
the synthetic A
-(1-40) at 37 °C for 1 h followed by
acid-urea-PAGE and Coomassie staining. *, characteristic A
degradation products seen in lanes 12-18. C,
aliquots of fractions were assayed for DFP bound-proteins by incubating
with [
H]DFP at 37 °C for 30 min and analyzing
by Tris-Tricine SDS-PAGE and autoradiography. D, total
proteins were resolved by Tris-Tricine SDS-PAGE and silver staining.
Because large amounts of albumin occur in fractions 4-7, the
[
H]DFP binding in these fractions is nonspecific
and shows no A
-degrading activity (compared to B). E, the proteins in fractions 7-22 were examined by
immunoblotting with anti-
M antibody.
indicates
the fragments of
M in lanes with A
-degrading
activity (compare to B and C).
The formation of complexes of
M with proteases is known to inhibit degradation of
large substrates but allow retention of activity against small
peptides(18, 19) . To show directly that
M forms a functional complex with the active
A
-degrading serine protease, we immunodepleted our COS CM with the
M antibody. The resultant supernatant no longer
contained A
-degrading activity (Fig. 6B). To
confirm this result, a partially purified protease fraction was
precipitated with anti-
M and incubated with synthetic
A
-(1-40). The
M immunoprecipitate induced
proteolysis of A
peptide, whereas an identical precipitation with
non-immune serum did not (Fig. 6C).
[
H]DFP labeling of the
M
immunoprecipitate confirmed that it contained the 185- and 28-kDa
proteins that are the DFP-labeled components of the purified protease
complex (data not shown). These results show unequivocally that the
purified protease is a complex that contains a serine protease and
M, and that the latter component does not abolish the
activity of the enzyme on A
.
On a continuous sucrose density
gradient, the A-degrading activity co-sedimented with a
700-kDa size standard (Fig. 7, A and B).
The active gradient fractions always contained the two DFP-labeled
proteins of 185 and 28 kDa, and variably contained weakly DFP-labeled
bands at 110 and 65 kDa (Fig. 7, C and D). The
185-, 110-, 80-, and 70- kDa proteins present in the proteolytically
active fractions were reactive with anti-
M antibody (Fig. 7E), but among these, only the 185- and 110-kDa
proteins were labeled by [
H]DFP. Full-length
M (185 kDa) and the characteristic heat-induced
fragments thereof (120 and 60 kDa) (18) that arise during
sample boiling were present in fractions of the sucrose gradient
lacking A
degradative activity and DFP binding (Fig. 7, D and E). These results suggested the possibility
that the DFP-positive 185-kDa moiety in the active fractions is an
SDS-,
-mercaptoethanol- and heat-resistant complex of a large
fragment of
M with a small serine protease, presumably
the 28-kDa component. To address this possibility, we attempted to
dissociate the 185-kDa moiety by acidic denaturation, a method used
previously to separate
M from tightly associated
proteins(20) . The purified complex was dialyzed against pH 3.0
acetate buffer and then against a neutral buffer. Using labeling with
[
H]DFP after the dialysis, this treatment
consistently produced a marked decrease in the 185-kDa band and an
increase in the 28-kDa band, as well as an increase of
M-immunoreactive fragments (Fig. 8). These
results strongly suggest a precursor-product relationship between the
185-kDa component on the one hand and the 28-kDa serine protease and
M fragments on the other. Pretreatment with DFP before
such acid treatment of the purified complex prevented these alterations
in the 185- and 28-kDa proteins (data not shown). The latter result
suggests that DFP-inhibitable autoproteolysis of the complex rather
than its dissociation occurs during the acid dialysis. We hypothesize
that the increased 28-kDa protein represents the serine protease that
is covalently bound to
M and is released from the
complex by proteolysis of the
M backbone (see
``Discussion'').
Figure 8:
The DFP-reactive A-degrading protease
is linked to an
M fragment in an acid-dissociable
complex. The purified A
-degrading protease was dialyzed (4 °C)
against a buffer of 0.1 M sodium acetate, pH 3.0, for 12 h,
followed by 0.1 M potassium phosphate, pH 7.5, for 12 h (lane
1) or was dialyzed solely against a buffer of 0.1 M potassium
phosphate, pH 7.5, for 24 h (lane 2). A, Tris-Tricine
SDS-PAGE and silver staining. B, labeling with
[
H]DFP at 37 °C for 30 min, followed by
Tris-Tricine SDS-PAGE and autoradiography.
Figure 9:
Specificity of the serine
protease-M complex toward low molecular peptides. A, synthetic A
(lanes
1-3) or A
(lanes
5-7) peptides in DMEM were incubated without (lanes 1 and 5) and with (lanes 2, 3, 6, and 7) purified A
-degrading protease at 37
°C for 16 h. DFP was added during incubation to the samples in lanes 3 and 7. In addition, A
-(1-40) (lane 4) and A
-(1-42) (lane 8) were
incubated in COS CM at 37 °C for 16 h. Samples were resolved on 16%
Tris-Tricine SDS-PAGE and immunoblotted with R1280. B,
proteolytic products of A
-(1-40) (lanes 1-3)
and A
-(1-42) (lanes 4-6) after incubation in
unconditioned medium (DMEM, lanes 1 and 4), in
unconditioned medium plus the purified enzyme (lanes 2 and 5) or in COS CM (lanes 3 and 6) at 37 °C
for 16 h were resolved by 15% acid-urea-PAGE and Coomassie staining. Arrow, intact A
. For unknown reasons, the intact
A
-(1-42) does not migrate well into acid-urea-PAGE gels. The
extent of proteolytic activity by purified protease and COS CM are not
directly comparable, because significantly different amounts were used.
*, A
cleavage products. C, synthetic small peptides
A
1-40 (lanes 1 and 2), amylin (lanes 3 and 4), EGF (lanes 5 and 6), or
neuropeptide Y (lanes 7 and 8) were incubated in the
absence(-) (lanes 1, 3, 5, and 7) or the presence (+) (lanes 2, 4, 6, and 8) of the purified serine
protease-
M complex at 37 °C for 4 h. Products were
analyzed by acid-PAGE and Coomassie. Note that some fragments of a
peptide sometimes migrate slower than the intact peptide in this gel
system, depending on their charges.
The biosynthesis of APP and the posttranslational
processing required to generate its A
fragment have received
intense scrutiny. Because excessive A
deposition is a hallmark of
AD, it is also important to understand the mechanisms of A
clearance and search for methods to accelerate this process once A
is released from cells. Here, we describe a serine protease that binds
to
-macroglobulin to form a stable high molecular
weight complex capable of efficiently cleaving A
but not larger
proteins in tissue culture medium.
DFP and Pefabloc, both inhibitors
of serine proteases, almost completely blocked the loss of endogenous
A in COS CM, and the purified protease binds DFP and is
irreversibly inhibited by it. Selected inhibitors of the cysteine,
aspartyl, and metalloprotease classes produced little or no detectable
inhibition of A
degradation in COS CM under the conditions of our
experiments. The protease complex we describe appears to derive from
the incubation of a pancreatic trypsin preparation with fetal bovine
serum. However, four types of experiments indicate that the
A
-degrading serine protease we purified from CM of COS cells
passaged with this preparation is not trypsin or chymotrypsin. First,
fetal bovine serum, which contains trypsin inhibitors, did not prevent
the degradation of A
by the protease (Fig. 1). Second,
TLCK, a trypsin inhibitor, and TPCK, a chymotrypsin inhibitor, are
known to inhibit these proteases in protease-
M
complexes, but they produced no inhibition of the A
-degrading
protease (Fig. 3B). Third, the NH
-terminal
sequence of our purified 28-kDa protease is distinct from that of
either trypsin or chymotrypsin. Fourth, HPLC separation and structural
analysis of the proteolytic products of A
-(1-40) showed that
the principal cleavages made by the protease occur at hydrophobic
residues 17-19 and 32-34, whereas trypsin cleaves after
lysine or arginine. Taken together, these results strongly suggest that
the A
-degrading protease we have purified is a previously
unidentified enzyme. However, further experiments will be needed to
determine whether the protease is produced by the pancreas (and other
tissues) and is present as a minor contaminant in the trypsin
preparation or is produced by the activation of a zymogen in serum by a
protease in the latter preparation.
Several lines of evidence
support the conclusion that the A-degrading serine protease occurs
as a stable high molecular weight complex with
M.
First, the A
-degrading activity elutes from a gel filtration
column at an estimated size of >700 kDa (Table 2). Second, all
of the activity co-sediments with a
700-kDa size standard during
sucrose density gradient centrifugation (Fig. 7). Third,
immunodepletion of CM or a partially purified fraction thereof with
M antibodies removes the A
-degrading activity (Fig. 6B). Fourth, an
M
immunoprecipitate of the fraction actively degrades A
(Fig. 6C) and contains the 185- and 28-kDa proteins
(data not shown) that are the principal DFP-labeled components of the
purified protease complex. Fifth, purification of the activity results
in a single DFP-labeled band on native gels (Fig. 4) that, upon
SDS-PAGE, yields a 185-kDa DFP-positive protein that cross-reacts with
M and a 28-kDa DFP-positive protein that likely
represents the free protease ( Fig. 5and Fig. 6).
NH
-terminal
M fragments that are
DFP-negative and are produced by the cleavage of the
M
backbone were identified by protein sequencing.
The ability of
M to form either covalent or non-covalent complexes
with a wide range of secreted endoproteases of all four classes has
been extensively documented. Human and bovine
M exists
as a
720-kDa tetramer composed of four identical
180-kDa
subunits(18, 19) . The mid-region of each
M subunit contains an amino acid stretch that is
readily cleavable by virtually any protease. Cleavage of this
``bait region'' results in an instantaneous conformational
change of
M and the trapping of the protease, thereby
excluding large but not small molecular weight substrates from
proteolysis. The residual activity against small substrates can still
be blocked by small inhibitors, including DFP. Thus, the trapping of a
protease by
M, although referred to as ``protease
inhibition,'' contrasts with the mechanisms of irreversible
protease-antiprotease reactions, which block the active site of a
protease(18) . The trapping can result in non-covalent or
covalent binding of the protease to the COOH-terminal region of
M, depending on the particular enzyme. Covalent
-lysyl-
-glutamyl cross-linking of the protease with the
COOH-terminal
M backbone occurs to a variable degree
between an
-amino lysyl group(s) of the protease and the glutamine
group of the cyclic internal thiolester in
M that
becomes activated following proteolysis of
M. In
addition, the cleavage of the ``bait region'' of
M produces NH
-terminal fragments of
variable molecular mass (
70-80 kDa). All of these moieties
can be identified by SDS-PAGE (19) . Our results suggest that
the active A
-degrading protease has formed a SDS-, reducing agent-
and heat-resistant complex of 185 kDa (and sometimes of 110 kDa) with a
fragment of
M (Fig. 5Fig. 6Fig. 7). Dialysis of the purified
protease complex at acidic pH markedly decreased the
[
H]DFP-labeled 185-kDa protein, removing its DFP
reactivity, and resulted in a marked increase in a 28-kDa DFP-positive
protein that is present at small and variable levels prior to acid
treatment (Fig. 8). However, if the complex was incubated with
DFP prior to acid dialysis, this change did not occur. These results
suggest that autoproteolysis of the 185-kDa complex is induced by the
acidic denaturation, thereby releasing the 28-kDa protease. Based on
all of these data, we propose that the protease-
M
complexes seen in SDS-PAGE (Fig. 5Fig. 6Fig. 7)
represent complexes of a
28-kDa protease covalently linked to
either one or two COOH-terminal
M fragments of
80
kDa, thus giving rise to DFP-labeled 110- and 185-kDa bands on
SDS-PAGE. This interpretation is consistent with analogous results
obtained by Van Leuven et al.(21) , who showed a
protease bound to the COOH-terminal region of
M using
a COOH-terminal specific antibody. As regards the actual size of the
free protease, we propose that the DFP-positive 28-kDa band present in
variable amounts represents protease molecules that are not covalently
bound to
M.
The A-degrading serine protease we
describe forms a tight complex with
M that allows
retention of significant degradative activity toward A
. The bound
A
-degrading protease also shows specificity, in that not all low
molecular peptides can serve as substrates. Whereas the scrambled
A
-(1-40) peptides and neuropeptide Y were degraded by this
protease complex, EGF, a small peptide growth factor, was resistant to
cleavage. Amylin, which shares similar secondary structure with A
and also induces neurotoxicity (22) was not cleaved by this
protease, suggesting that the first order sequence of small peptides
but not their secondary structure is crucial to the enzyme's
specificity. Our studies demonstrate that the serine
protease-
M complex is capable of proteolytically
clearing both A
-(1-40) and A
-(1-42). Importantly,
the cleavage results in disruption of the central region of the peptide
(residues 10-35), which is believed to mediate the conformational
change that underlies A
self-aggregation(23) .
A recent
report describes the inhibition of the loss of secreted A from the
conditioned medium of CHO cells by a mixture of all four major classes
of protease inhibitors(24) . Therefore, it is likely that
proteases of different classes and varying cellular origin will be
found to be capable of degrading secreted A
and A
-related
peptides in vivo. In view of our finding of an efficient
A
-degrading protease that forms tight complexes with
M, it is of particular interest that neuritic plaques
in AD brain show prominent
M
immunoreactivity(25, 26) . Moreover, the low density
lipoprotein receptor-related protein, which is the receptor of
M in brain tissue and can internalize both
M-protease complexes and apolipoprotein E, is also
up-regulated in plaques(27, 28) . In addition,
apolipoprotein E4 is a major genetic risk factor for the development of
AD(29) , and inheritance of the apolipoprotein E
4 allele
leads to a significant increase in the number of A
plaques in AD
brain(30, 31) . Furthermore, an
M-containing kallikrein-like protease (32, 33) and an
M/thrombin complex
have been isolated from cell culture media(34, 35) . A
serine protease complexed with
M has been isolated
from porcine gastric mucosa(20) . Therefore, the
A
-degrading protease-
M complex reported here,
which degrades A
-(1-40) more efficiently than
A
-(1-42), may exemplify other protease-
M
complexes capable of clearing A
under physiological conditions in
non-neural and neural tissues. It is important to determine whether
this or related
M-protease complexes degrade A
in vivo.