©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Degradation of Amyloid -Protein by a Serine Protease--Macroglobulin Complex (*)

(Received for publication, September 13, 1995; and in revised form, December 11, 1995)

Wei Qiao Qiu (1) (2) Wolfgang Borth (4) Zhen Ye (1) (2) Christian Haass (1) (2) David B. Teplow (1) (3) (2) Dennis J. Selkoe (1) (2)(§)

From the  (1)Department of Neurology and Program in Neuroscience, Harvard Medical School, (2)Center for Neurologic Diseases, and the (3)Biopolymer Laboratory, Brigham and Women's Hospital, Boston, Massachusetts 02115 and the (4)Department of Hematology, Mount Sinai Medical Center, New York, New York 10029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Progressive cerebral deposition of the amyloid beta-peptide (Abeta) is an early and constant feature of Alzheimer's disease. Abeta is derived by proteolysis from the beta-amyloid precursor protein. beta-Amyloid precursor protein processing and the generation of Abeta 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 Abeta 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, alpha(2)-macroglobulin (alpha(2)M), that retains activity against small substrates such as Abeta. Its NH(2)-terminal sequence suggests that the protease is previously unidentified. Our results indicate that the Abeta-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 Abeta-bearing plaques in Alzheimer's disease brain contain both alpha(2)M and receptors of alpha(2)M-protease complexes, the same or a similar alpha(2)M-protease complex could arise in vivo and play a role in Abeta clearance.


INTRODUCTION

All patients with Alzheimer's disease (AD) (^1)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 beta-protein (Abeta)(2) , a proteolytic fragment of the beta-amyloid precursor protein (betaAPP)(3) . betaAPP 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 Abeta and a large, soluble ectodomain fragment designated APP(s)(1) .

Several lines of evidence support the hypothesis that Abeta deposition is an early and critical event in AD pathogenesis. First, missense mutations in and around the Abeta region of betaAPP 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 Abeta peptides(5, 6, 7, 8) . Second, patients with trisomy 21 (Down's syndrome) have enhanced expression of betaAPP due to increased gene dosage and gradually develop the typical histopathological lesions of AD(9) . Third, trisomy 21 patients develop immature, non-neuritic Abeta deposits (diffuse plaques) prior to any other histopathological feature of AD(9, 10) . Fourth, transgenic mice expressing a mutant betaAPP protein develop Abeta deposits, surrounding neuritic and glial dystrophy and synaptic loss resembling those of AD(11) .

Based on these and other observations, it is likely that Abeta 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 Abeta production, retard its aggregation into potentially cytotoxic amyloid fibrils, protect cells from Abeta-mediated neurotoxicity, or enhance Abeta clearance from the brain. Although recent research has focused almost entirely on the biosynthetic processing of betaAPP and the production and fibrillogenesis of Abeta, the degradation of the peptide is of potentially equal importance in the process leading to progressive amyloid deposition. Because Abeta is a normal product of betaAPP metabolism that is present in cerebrospinal fluid and plasma at 10 to 10M(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 Abeta-secreting transfected cells, we observed a time-dependent decrease in the amount of Abeta in the conditioned medium. We have determined that the factor principally responsible for the decrease in Abeta in our cultures is a serine protease that degrades Abeta efficiently and is proteolytically active in a stable 700-kDa complex with the protease inhibitor, alpha(2)-macroglobulin (alpha(2)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-alpha(2)M complexes can efficiently degrade secreted Abeta peptides under physiological conditions, raising the possibility that the regulation of Abeta levels in brain could involve a similar mechanism.


EXPERIMENTAL PROCEDURES

Cell Culture

Chinese hamster ovary (CHO) cells stably transfected with betaAPP cDNA were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM glutamine, and antibiotics. Monkey kidney COS cells cultured in 10 cm dishes were routinely passaged by adding 3 ml of trypsin (0.25%), and 1 mM EDTA (Life Technologies, Inc., catalog 25200-023), immediately aspirating it off, and then incubating the cells at 37 °C for 2-3 min. One fifth of the cells were passaged and cultured in DMEM, 10% FBS medium. Alternatively, sequence-grade trypsin (Boehringer Mannheim) or 50 mM EDTA were used instead of trypsin from Life Technologies, Inc. during the passage of the COS cells.

Pulse-Chase Experiments and Immunoprecipitations

To assess Abeta levels in the cultures, confluent monolayers of betaAPP-transfected CHO cells in 10 cm dishes were preincubated for 30 min in methionine-free medium and labeled for 2 h with 300 µCi of [S]methionine. After washing, cold medium containing FBS was added and the cells chased for various times. The conditioned media were then precipitated with antibody R1280 (to synthetic Abeta-(1-40) peptide) as described previously(13) . Protease inhibition assays were carried out by adding different protease inhibitors at the indicated concentrations to the chase media. All inhibitors shown in Table 1were purchased from Sigma, except that 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc) was obtained from Boehringer Mannheim, Inc.



Peptides

Human Abeta-(1-40) peptide was synthesized on an Applied Biosystems model 430A automated peptide synthesizer. Abeta-(1-42) peptide, amylin, epidermal growth factor (EGF), and neuropeptide Y were purchased from Bachem (Torrance, CA). Peptides were aliquoted, lyophilized, and then resuspended in reaction buffer before experiments. I-Labeled Abeta-(1-40) (specific activity = 7.6 times 10 cpm/µg) was the kind gift of Dr. John Maggio.

Assays of Abeta-degrading Activity

Abeta degradation in cultures was quantified by incubating 30 µl of conditioned medium with I-labeled Abeta-(1-40) (3.3 times 10^4 cpm) at 37 °C for 4 h. A 20-µl aliquot was electrophoresed on Tris-Tricine SDS-PAGE gels followed by autoradiography. Alternatively, 5 ml of conditioned medium was incubated with I-labeled Abeta-(1-40) (6.6 times 10^6 cpm), followed by precipitation with R1280 and autoradiography.

During purification of the protease (below), Abeta-degrading activity was monitored by incubating 0.5-7 µl of the fractions obtained at each purification step with 7 µg of cold synthetic Abeta-(1-40) in 14 µl of reaction buffer (187 mM NaCl, 0.02 M NaHPO(4), 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 Abeta-(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 Abeta-(1-40) at 37 °C for 2 h. After a 10,000 times g spin for 3 min, the supernatants were collected, quenched, and analyzed by acid-urea-PAGE.

Acid-Urea-Polyacrylamide Gel Electrophoresis

Acid-urea-PAGE (pH 4.0) was carried out by combining two previous methods(15, 16) . Briefly, the running gel solution consisted of a final concentration of 15% (v/v) acrylamide, 6 M urea, 88 mM KOH, 3% (v/v) glacial acetic acid, 5.4% (v/v) TEMED, and 0.375% (v/v) ammonium persulfate. A minigel apparatus was used, and the gels were prerun at 100 V for 1 h and run at 150 V in a buffer containing 0.035 M beta-alanine and 52 ml/liter glacial acetic acid (pH 4.0). Gels were stained in 0.1% (w/v) Coomassie Brilliant Blue R-250 in 50% methanol and destained in deionized H(2)O.

Purification of an Abeta-degrading Protease

COS cells were cultured in DMEM, 2.5% FBS in 15-cm dishes for 4-5 days. Pooled CM (1 liter) was filtered through 0.22-µm cellulose acetate and loaded on an Affi-Gel Blue column (Bio-Rad), which had been equilibrated in PBS, 10 mM Tris-HCl, pH 7.5. The flow-through medium was assayed for protease activity (above) and then applied to a Q-Sepharose anion exchange column (Pharmacia) equilibrated in PBS, 10 mM Tris-HCl, pH 7.5. After extensive washing with this buffer, bound proteins were eluted with 0.64 M NaCl, 10 mM Tris-HCl, pH 7.5. After protease assay, the eluant was precipitated (4 h) with 50% NH(4) SO(4). The pellet was resuspended in 10 ml of 1.2 M KI, 10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 0.5 mM dithiothreitol, centrifuged at 10,000 times g for 10 min and loaded onto a Bio-Gel A15 200-400-mesh agarose column (2.5 times 100 cm), which had been equilibrated in 0.6 M KCl, 10 mM Tris-HCl, pH 7.5, 10 mM EGTA, 1 mM dithiothreitol. The sample was eluted in the latter buffer at a flow rate of 0.5 ml/min. Fractions (1.5 ml) were collected and monitored by UV absorbance at 280 nm. After assaying protease activity and assessing protein composition by native PAGE, a subset of active fractions was pooled and concentrated on a Centricon 30 (Amicon). The concentrated sample (250 µl) was loaded on a Mono Q column (1 times 5 cm) (Pharmacia) pre-equilibrated in PBS, 10 mM Tris-HCl, pH 7.5, and the column washed with 10 column volumes of the latter buffer. The column was eluted with a 20-ml linear gradient from 10 mM to 1 M NaCl run at a flow rate of 0.5 ml/min. Aliquots of fractions (0.5 ml) were assayed for protease activity and for protein composition by PAGE and stored at -70 °C. All purification steps were carried out at 4 °C, except that the Mono Q-FPLC run was at room temperature. Protein concentrations were determined by the Bio-Rad assay.

[^3H]DFP Binding

[^3H]DFP was purchased from DuPont NEN at a specific activity of 6.0 Ci/mmol. 0.25 mCi/ml [^3H]DFP (final concentration) were incubated with protein samples at 37 °C for 30 min. [^3H]DFP-bound proteins were visualized by either native PAGE or Tris-Tricine SDS-PAGE, followed by autoradiography.

Sucrose Density Gradient Centrifugation

The partially purified material (200 µl) obtained after the Q-Sepharose chromatography (above) was overlaid onto a 13-ml 10%-40% (w/w) continuous sucrose gradient in PBS, 10 mM Tris-HCl, pH 7.5, and spun for 15 h at 40,000 rpm and 1 times 10 ^2t rad^2 s in a Beckman SW40 Ti rotor at 4 °C. Fractions (0.6 ml) were collected from the top of the gradient and stored at -70 °C until analyzed.

Immunoblotting

Following separation on either 10-20% or 16% Tris-Tricine SDS-PAGE gels, proteins were transferred to PVDF filters in 25 mM Tris, 192 mM glycine, 20% methanol at 0.4 mA for 4 h at 4 °C. PVDF sheets were soaked in methanol and rinsed in H(2)O, after which they were blocked in 5% dry milk resuspended in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20 (TBST) for 1 h at room temperature or overnight at 4 °C. Blots were incubated with primary antibody (anti-alpha(2) macroglobulin at 1:100 or R1280 at 1:500) in 1% bovine serum albumin, PBS, 0.02% NaN(3), 0.1% Tween 20 for 1.5 h at room temperature. Thereafter, blots were washed in TBST (3 times 10 min) and incubated for 30 min with alkaline phosphatase-conjugated goat anti-rabbit IgG at 1:7500 in 25 mM Tris HCl, pH 9.5, 25 mM NaCl, 5 mM MgCl(2). Blots were washed again and developed with 5-bromo-4-chloro-3indolyl phosphate/nitro blue tetrazolium phosphatase substrate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). For analyses of substrate specificity, 10 µg of Abeta-(1-42) or Abeta-(1-40) were incubated with 15 units of the purified Abeta-degrading protease in 150 µl of DMEM, 10% FBS at 37 °C for 12 h. Aliquots (20 µl) were run on 10-20% Tris-Tricine SDS-PAGE gels and immunoblotted with R1280.

Protein Sequencing

Samples were spotted onto Polybrene-coated glass fiber discs and sequenced on an Applied Biosystems 475/900/120 system. Data analysis was done by visual inspection of the resulting amino acid-phenylthiohydantoin HPLC chromatograms.

HPLC and Mass Spectrometry of Abeta Degradative Products

Protease reaction products were acidified with neat trifluoroacetic acid, filtered through 0.2-µm polysulfone filters (Alltech, Deerfield, IL), and analyzed by reverse phase HPLC on a Rainin Instruments (Woburn, MA) system consisting of two UD-XL pumps, dynamic and static mixers, a Rheodyne (Cotati, CA) 7161 injector, a UV-1 variable wavelength detector, an FC-1 fraction collector, and an Apple Macintosh Plus controller running Dynamax software. A trifluoroacetic acid/acetonitrile gradient was run at 1 ml/min using a 4.6 times 650-mm Rainin Dynamax C18 column, a 4.6 times 15-mm guard column of identical chemistry, and detection at 214 nm. The gradient was 0-70% B for 50 min, then 70-100% B for 10 min. Solvent ``A'' was 0.1% trifluoroacetic acid (v/v) in HPLC-grade water (Fisher, Fair Lawn, NJ). Solvent ``B'' was 0.09% trifluoroacetic acid (v/v) in 70% (v/v) acetonitrile (Fisher) in HPLC-grade water. One-ml fractions were collected for protein sequencing or for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on a VG TofSpec instrument, as described by the manufacturer. Data represent the masses of the predominant singly protonated molecular ions found in each sample. In cases where both the singly and doubly protonated molecular ions were observed, the final masses are the arithmetic average masses. The mass accuracy of the instrument is typically 0.2%. The isotopically averaged calculated masses were obtained using MacProMass 1.0, kindly provided by Dr. Lee (Beckman Institute, Duarte, CA).


RESULTS

A Serine Protease Degrades Amyloid beta-Peptide

During experiments in which Chinese hamster ovary (CHO) cells stably transfected with betaAPP695 cDNA (CHO695) were co-cultured with monkey kidney COS cells, we noticed a marked decrease in the amount of Abeta present in the conditioned medium (CM) compared to that found in CM of the CHO transfectants alone. We therefore cultured CHO in the COS CM alone, using a pulse-chase paradigm. Two hr labeling of CHO with [S]Met followed by a 4-h chase in COS CM (versus in just DMEM, 10% FBS) revealed a marked and consistent decrease in the amounts of the CHO-derived Abeta and p3 peptides, as demonstrated by precipitation of the medium with an Abeta antibody (R1280) followed by Tris-Tricine SDS-PAGE and autoradiography (Fig. 1A). In contrast, the amount of secreted APP(s) (precipitated by antibodies specific for this betaAPP derivative) changed very little or not at all, and the profile of total labeled proteins in the CM did not change (data not shown). To determine whether the COS CM contained an activity that degraded Abeta or a factor that decreased the expression or amyloidogenic processing of betaAPP in the CHO cells, we examined the stability of radiolabeled synthetic Abeta (I-Abeta-(1-40)) in COS CM versus DMEM, 10% FBS by gel autoradiography. The amount of synthetic Abeta declined substantially in the COS CM (Fig. 1B). To detect the products of this apparent Abeta degradation, we used acid-urea-PAGE to analyze unlabeled synthetic Abeta after incubation in COS medium. We observed a doublet of protein products that was not present after incubation in DMEM, 10% FBS alone (Fig. 1C). These results indicated that Abeta underwent proteolytic degradation in COS CM, generating specific peptide products.


Figure 1: Degradation of Abeta by COS conditioned medium. A, CHO cells transfected with betaAPP695 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 Abeta antibody, R1280, and electrophoresed on Tris-Tricine SDS-PAGE gels, followed by autoradiography. Arrows indicate APP(s), Abeta, and p3. Protein size markers are shown. B, I-labeled synthetic Abeta-(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. Abeta remaining after this in vitro reaction was detected by Tris-Tricine SDS-PAGE and autoradiography. C, proteolytic products of the Abeta-(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 Abeta-(1-40) is shown. * indicates the Abeta 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 Abetadegrading serine protease was present in the medium (Table 1). Both DFP and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc) virtually abolished the degradation of secreted Abeta and partially inhibited that of secreted p3 (Fig. 2). Because Pefabloc actually decreased the levels of secreted Abeta 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 Abeta from betaAPP, in addition to its inhibition of the Abeta-degrading protease. Two other serine protease inhibitors, elastatin and aprotinin, produced only 10-15% inhibition of Abeta degradation by the COS medium.


Figure 2: Degradation of Abeta mediated by COS conditioned medium is blocked by certain serine protease inhibitors. Abeta secreted by betaAPP-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 Abeta in DMEM, 10% FBS, it may have additional effects on betaAPP processing (see ``Results''). Abeta and p3 (arrows) were precipitated by R1280.



In further experiments, we observed that the Abeta-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 Abeta 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''). Abeta-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 Abeta-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 Abeta-degrading activity in this paradigm, while DMEM, 10% FBS incubated with the trypsin from Life Technologies without the COS cells degraded Abeta (Fig. 3A), indicating that the major Abeta-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 Abeta degradation, TLCK, an inhibitor of trypsin, was included during the chase period. Abeta 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 Abeta degradation, it was not trypsin per se that degraded Abeta 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 Abeta degradation was not inhibited (Fig. 3B). In addition, purified chymotrypsin was incubated with synthetic Abeta-(1-40) in DMEM, 10% FBS, but a different pattern of Abeta fragmentation was observed on acid-PAGE, and this was inhibitable by TPCK (data not shown). Thus, chymotrypsin is not responsible for the Abeta-degrading activity we describe.


Figure 3: The Abeta-degrading activity requires the presence of a pancreatic trypsin preparation with fetal bovine serum during the passage of COS cells. A, betaAPP-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 Abeta and p3 present in the chase media were analyzed by precipitation with R1280, Tris-Tricine SDS-PAGE, and autoradiography. B, Abeta secreted by betaAPP-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 Abeta-(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 Abeta cleavage products.



To investigate further the origin of the Abeta-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 Abeta-(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 Abeta fragments that we originally detected in COS CM (Fig. 3C), whereas Life Technologies trypsin without FBS hydrolyzed Abeta differently and FBS alone produced no degradation. These results clearly indicated that the Abeta degradating activity detected in COS CM (Fig. 1) required the presence of the Life Technologies pancreatic trypsin preparation and FBS during cell passage.

Purification of a Serine Protease That Degrades Abeta

Because these data suggested that an unknown serine protease could efficiently degrade secreted Abeta and p3 but not larger proteins under culture conditions, we purified and further characterized the putative serine protease from 1 liter of CM of COS cells grown in DMEM, 2.5% FBS and passaged routinely with the Life Technologies trypsin preparation (see ``Experimental Procedures''). The CM was subjected to sequential fractionation by Affi-Gel Blue chromatography, Q-Sepharose anion exchange chromatography, ammonium sulfate precipitation, size exclusion chromatography, and a final Mono Q anion exchange column (Table 2). Abeta-degrading activity was monitored at each step by either the pulse-chase method on CHO cells (above) or the degradation of cold synthetic Abeta-(1-40) peptide followed by acid-urea-PAGE. ^3H-Labeled DFP was incubated with an aliquot from each step to visualize the amount and migration of the serine protease by autoradiography of non-denaturing polyacrylamide gels. One unit of enzyme was defined as that which degrades 1 µg of synthetic Abeta-(1-40) in 14 µl of reaction buffer at 37 °C in 1 h. The complete protocol enriched the Abeta-degrading activity >40-fold and yielded a fraction containing 0.4% of the total protein in the starting medium. Gel filtration chromatography (step 4) showed that the protease-containing moiety had a molecular mass of >700 kDa. The purification produced a single Coomassie- and DFP-reactive high molecular weight band in native gels of the final fraction (Fig. 4, A and B). This band was not present in DMEM, 10% FBS (Fig. 4B). In the last two purification steps, minor radiolabeled bands were observed above and below the major DFP-positive band (Fig. 4B, lanes 6 and 7), suggesting that the protease undergoes both limited aggregation and some dissociation during purification (see below).




Figure 4: Purification of an active Abeta-degrading protease from conditioned medium of COS cells. A, the Abeta-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 Abeta-degrading activity) (lane 2) are shown. The position of the presumptive Abeta-degrading protease is indicated. B, aliquots of fractions at various purification steps were assayed for the presence of [^3H]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 [^3H]DFP-bound Abeta-degrading protease is indicated. The minor [^3H]DFP-bound bands shown above and below the substantially enriched Abeta-degrading protease (lanes 6 and 7) are forms apparently derived by aggregation or partial dissociation (see ``Results''). C, Abeta-degrading activity obtained during the purification was monitored by incubating Abeta-(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, Abeta-(1-40) peptide alone; lane 2, Abeta-(1-40) peptide plus an active aliquot from Mono Q chromatography without DFP; lane 3, as in lane 2 with DFP. Arrow, intact Abeta-(1-40) peptide. *, cleavage products of Abeta-(1-40) peptide. D, confirmation that the protease shown in A and B mediates the proteolytic degradation of Abeta. An active Mono Q fraction was incubated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) [^3H]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 Abeta-(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 Abeta-(1-40); *, Abeta-(1-40) degradation products.



The protease obtained from the final Mono Q step was incubated with synthetic Abeta-(1-40) (Fig. 4C). The characteristic degradation of Abeta 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 Abeta-degrading activity, this band was excised and incubated with 25 µg of synthetic Abeta1-40. The characteristic Abeta 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 Abeta-degrading serine protease.

The Abeta-degrading Protease Is a High Molecular Weight Complex That Contains alpha(2)-Macroglobulin

As noted above, the final purified fraction yielded a single band on a native polyacrylamide gel (Fig. 5, lane 1). However, when the fraction was analyzed by SDS-PAGE, five protein bands were resolved with approximate masses of 28, 70, 80, 110, and 185 kDa, respectively (Fig. 5, lane 2). A similar pattern was obtained when the single band from the native gel was excised, electroeluted, and electrophoresed on SDS-PAGE (data not shown). In a purified fraction that was preincubated with [^3H]DFP prior to SDS-PAGE, the 28- and 185-kDa proteins were found to bind DFP (Fig. 5, lane 3). Coupled with the fact that the single peak at 700-kDa position obtained in the gel filtration step was shown to degrade Abeta (Table 2), these results indicated that the purified protease was a multicomponent complex. Because the 70- and 80-kDa proteins were by far the major Coomassie-positive bands, they were resolved by SDS-PAGE, transferred to PVDF, excised, and sequenced. The first 20 amino acids of the 80-kDa band (SVSGKPQYMVLVPSLLHTET) were identical to the NH(2) terminus of human alpha(2)-macroglobulin (alpha(2)M). Sequencing the 70-kDa protein also revealed identity with the alpha(2)M NH(2) terminus. Immunoblotting with a polyclonal alpha(2)M antibody produced strong and specific labeling of the 70- and 80-kDa bands (Fig. 6A). Immunolabeling of the 105- and 185-kDa bands, the intensity of which varied considerably from preparation to preparation, was also observed (see below and Fig. 7E).


Figure 5: Analysis of the Abeta-degrading protease by SDS-PAGE electrophoresis. An active fraction of the Abeta-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 [^3H]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: alpha(2)-Macroglobulin is present in the Abeta-degrading protease complex. A, the purified protease obtained after Mono Q chromatography was characterized by immunoblotting with an anti-alpha(2)M antiserum (lane 1). Purified alpha(2)M served as a control (lane 2); charateristic heat-labile fragments of alpha(2)M are also seen. B, [S]Met-labeled Abeta secreted by betaAPP-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-alpha(2)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-alpha(2)M antibody (lane 1) or normal rabbit serum (lane 2). The precipitates were washed and incubated in a buffer containing synthetic Abeta-(1-40) (25 µg) at 37 °C for 2 h. Supernatants were collected after spinning for 5 min, and proteolytic products of Abeta-(1-40)(*) detected by acid-urea-PAGE and Coomassie staining.




Figure 7: The purified Abeta-degrading protease is a multi-component complex. To confirm that the Abeta-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, Abeta-degrading activity was monitored by incubating with the synthetic Abeta-(1-40) at 37 °C for 1 h followed by acid-urea-PAGE and Coomassie staining. *, characteristic Abeta degradation products seen in lanes 12-18. C, aliquots of fractions were assayed for DFP bound-proteins by incubating with [^3H]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 [^3H]DFP binding in these fractions is nonspecific and shows no Abeta-degrading activity (compared to B). E, the proteins in fractions 7-22 were examined by immunoblotting with anti-alpha(2)M antibody. indicates the fragments of alpha(2)M in lanes with Abeta-degrading activity (compare to B and C).



The formation of complexes of alpha(2)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 alpha(2)M forms a functional complex with the active Abeta-degrading serine protease, we immunodepleted our COS CM with the alpha(2)M antibody. The resultant supernatant no longer contained Abeta-degrading activity (Fig. 6B). To confirm this result, a partially purified protease fraction was precipitated with anti-alpha(2)M and incubated with synthetic Abeta-(1-40). The alpha(2)M immunoprecipitate induced proteolysis of Abeta peptide, whereas an identical precipitation with non-immune serum did not (Fig. 6C). [^3H]DFP labeling of the alpha(2)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 alpha(2)M, and that the latter component does not abolish the activity of the enzyme on Abeta.

On a continuous sucrose density gradient, the Abeta-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-alpha(2)M antibody (Fig. 7E), but among these, only the 185- and 110-kDa proteins were labeled by [^3H]DFP. Full-length alpha(2)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 Abeta 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-, beta-mercaptoethanol- and heat-resistant complex of a large fragment of alpha(2)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 alpha(2)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 [^3H]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 alpha(2)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 alpha(2)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 alpha(2)M and is released from the complex by proteolysis of the alpha(2)M backbone (see ``Discussion'').


Figure 8: The DFP-reactive Abeta-degrading protease is linked to an alpha(2)M fragment in an acid-dissociable complex. The purified Abeta-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 [^3H]DFP at 37 °C for 30 min, followed by Tris-Tricine SDS-PAGE and autoradiography.



Substrate Specificity of the Serine Protease-alpha(2)M Complex

To examine the substrate specificity of the protease, the products of digestion of synthetic Abeta-(1-40) were characterized by a combination of HPLC, mass spectrometry, and NH(2)-terminal sequence analysis. The resultant data demonstrated that the protease is an endopeptidase that cleaves primarily after hydrophobic residues within contiguous stretches of hydrophobic amino acids (data not shown). Two such stretches within Abeta, residues 17-21 and 30-36, were the favored cleavage sites for the purified enzyme under the conditions we utilized. Next, we compared the activity of the purified complex on synthetic Abeta-(1-42) and Abeta-(1-40) peptides. Abeta-(1-40) was almost completely degraded in 16 h, but Abeta-(1-42), which comprises the major constituent of amyloid plaques in AD, underwent less degradation (Fig. 9A). Similar results were obtained when the two peptides were incubated in the starting COS CM that contains the protease complex. Because considerably less degradation of Abeta-(1-42) was seen by immunoblotting, we confirmed that proteolysis of this peptide had in fact occurred by both acid-urea-PAGE (Fig. 9B) and HPLC (data not shown). Several other low molecular weight peptides were also incubated with the purified enzyme at 37 °C for 4 h. A scrambled Abeta-(1-40) peptide, which contains only one segment of hydrophobic amino acids longer than three residues (AIGL), was cleaved by the enzyme (data not shown), consistent with the apparent peptide bond specificity shown on Abeta-(1-40). In contrast, amylin (3.9 kDa), which has a distinct sequence but similar secondary structure to that of Abeta, was resistant to hydrolysis (Fig. 9C). Epidermal growth factor (6 kDa) was also not degraded, whereas neuropeptide Y (4.1 kDa) was (Fig. 9C).


Figure 9: Specificity of the serine protease-alpha(2)M complex toward low molecular peptides. A, synthetic Abeta (lanes 1-3) or Abeta (lanes 5-7) peptides in DMEM were incubated without (lanes 1 and 5) and with (lanes 2, 3, 6, and 7) purified Abeta-degrading protease at 37 °C for 16 h. DFP was added during incubation to the samples in lanes 3 and 7. In addition, Abeta-(1-40) (lane 4) and Abeta-(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 Abeta-(1-40) (lanes 1-3) and Abeta-(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 Abeta. For unknown reasons, the intact Abeta-(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. *, Abeta cleavage products. C, synthetic small peptides Abeta1-40 (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-alpha(2)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.



NH(2)-terminal Sequence of the Purified Protease

Because the Abeta-degrading protease complex purified from CM appeared to derive from the incubation of the Life Technologies pancreatic trypsin preparation with fetal bovine serum, we investigated further the identity of the protease by performing NH(2)-terminal sequencing on the DFP-labeled 28-kDa protease in the complex purified from COS CM. The sequence obtained (NXDFVWFDDTSEPP) showed no significant homology to known proteins in GenBank. We next sequenced all four major protein bands migrating around 25-30 kDa in the Life Technologies trypsin preparation to determine if one of them had formed the complex with alpha(2)M. Their NH(2)-terminal sequences indicated that they are trypsin, chymotrypsin, and elastase. None of these showed homology to the NH(2)-terminal sequence of the 28-kDa protease, suggesting that the purified DFP-bound 28-kDa protease in the alpha(2)M complex is a previously unidentified serine protease present in trace quantities in the pancreatic extract.


DISCUSSION

The biosynthesis of betaAPP and the posttranslational processing required to generate its Abeta fragment have received intense scrutiny. Because excessive Abeta deposition is a hallmark of AD, it is also important to understand the mechanisms of Abeta clearance and search for methods to accelerate this process once Abeta is released from cells. Here, we describe a serine protease that binds to alpha(2)-macroglobulin to form a stable high molecular weight complex capable of efficiently cleaving Abeta but not larger proteins in tissue culture medium.

DFP and Pefabloc, both inhibitors of serine proteases, almost completely blocked the loss of endogenous Abeta 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 Abeta 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 Abeta-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 Abeta by the protease (Fig. 1). Second, TLCK, a trypsin inhibitor, and TPCK, a chymotrypsin inhibitor, are known to inhibit these proteases in protease-alpha(2)M complexes, but they produced no inhibition of the Abeta-degrading protease (Fig. 3B). Third, the NH(2)-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 Abeta-(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 Abeta-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 Abeta-degrading serine protease occurs as a stable high molecular weight complex with alpha(2)M. First, the Abeta-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 alpha(2)M antibodies removes the Abeta-degrading activity (Fig. 6B). Fourth, an alpha(2)M immunoprecipitate of the fraction actively degrades Abeta (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 alpha(2)M and a 28-kDa DFP-positive protein that likely represents the free protease ( Fig. 5and Fig. 6). NH(2)-terminal alpha(2)M fragments that are DFP-negative and are produced by the cleavage of the alpha(2)M backbone were identified by protein sequencing.

The ability of alpha(2)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 alpha(2)M exists as a 720-kDa tetramer composed of four identical 180-kDa subunits(18, 19) . The mid-region of each alpha(2)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 alpha(2)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 alpha(2)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 alpha(2)M, depending on the particular enzyme. Covalent -lysyl--glutamyl cross-linking of the protease with the COOH-terminal alpha(2)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 alpha(2)M that becomes activated following proteolysis of alpha(2)M. In addition, the cleavage of the ``bait region'' of alpha(2)M produces NH(2)-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 Abeta-degrading protease has formed a SDS-, reducing agent- and heat-resistant complex of 185 kDa (and sometimes of 110 kDa) with a fragment of alpha(2)M (Fig. 5Fig. 6Fig. 7). Dialysis of the purified protease complex at acidic pH markedly decreased the [^3H]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-alpha(2)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 alpha(2)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 alpha(2)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 alpha(2)M.

The Abeta-degrading serine protease we describe forms a tight complex with alpha(2)M that allows retention of significant degradative activity toward Abeta. The bound Abeta-degrading protease also shows specificity, in that not all low molecular peptides can serve as substrates. Whereas the scrambled Abeta-(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 Abeta 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-alpha(2)M complex is capable of proteolytically clearing both Abeta-(1-40) and Abeta-(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 Abeta self-aggregation(23) .

A recent report describes the inhibition of the loss of secreted Abeta 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 Abeta and Abeta-related peptides in vivo. In view of our finding of an efficient Abeta-degrading protease that forms tight complexes with alpha(2)M, it is of particular interest that neuritic plaques in AD brain show prominent alpha(2)M immunoreactivity(25, 26) . Moreover, the low density lipoprotein receptor-related protein, which is the receptor of alpha(2)M in brain tissue and can internalize both alpha(2)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 Abeta plaques in AD brain(30, 31) . Furthermore, an alpha(2)M-containing kallikrein-like protease (32, 33) and an alpha(2)M/thrombin complex have been isolated from cell culture media(34, 35) . A serine protease complexed with alpha(2)M has been isolated from porcine gastric mucosa(20) . Therefore, the Abeta-degrading protease-alpha(2)M complex reported here, which degrades Abeta-(1-40) more efficiently than Abeta-(1-42), may exemplify other protease-alpha(2)M complexes capable of clearing Abeta under physiological conditions in non-neural and neural tissues. It is important to determine whether this or related alpha(2)M-protease complexes degrade Abeta in vivo.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AG 06173 and AG 12749 and grants from the Richard Saltonstall Charitable Trust and the Adler Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Brigham and Women's Hospital, 221 Longwood Ave., LMRC 103, Boston, MA 02115. Tel.: 617-732-6454; Fax: 617-732-7787.

(^1)
The abbreviations used are: AD, Alzheimer's disease; Abeta, amyloid beta-peptide; betaAPP, beta-amyloid precursor protein; alpha(2)M, alpha(2)-macroglobulin; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; DFP, diisopropyl fluorophosphate; TEMED, N,N,N`,N`-tetramethylethylenediamine; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; CM, conditioned medium; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.


ACKNOWLEDGEMENTS

We thank Drs. Allen Taylor, Nicole Leclerc, Joachim Herz, Dale Schenk, and Sukanto Sinha for suggestions and helpful discussions.


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