- Email: [email protected]
α2-Macroglobulin-Mediated Degradation of Amyloid β1–42: A Mechanism to Enhance Amyloid β Catabolism
Experimental Neurology 167, 385–392 (2001) doi:10.1006/exnr.2000.7569, available online at http://www.idealibrary.com on
␣2-Macroglobulin-Mediated Degradation of Amyloid 1– 42: A Mechanism to Enhance Amyloid  Catabolism D. Lauer, A. Reichenbach,* and G. Birkenmeier Institute of Biochemistry, University of Leipzig, Liebigstrasse 16, 04103 Leipzig, Germany; and *Paul-Flechsig-Institute for Brain Research, Department of Neurophysiology, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany Received May 25, 2000; accepted September 26, 2000; published online January 2, 2001 DEDICATED
TO
PROF. G KOPPERSCHLA¨ GER
Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid  (A), are considered to be central to the pathology of Alzheimer’s disease (AD). Soluble A is present in measurable concentrations in cerebrospinal fluid and blood. There are indications that soluble A present in circulation can cross the blood– brain barrier via transcytosis mediated by brain capillary endothelial cells. It implies that A originating from circulation may contribute to vascular and parenchymal A deposition in AD. Enhancing of A catabolism mediated by proteolytic degradation or receptor-mediated endocytosis could be a key mechanism to maintain low concentrations of soluble A. To launch A clearance we have exploited the A-degrading activity of diverse ␣2-macroglobulin (␣2-M)–proteinase complexes. Complexes with trypsin, ␣-chymotrypsin, and bromelain strongly degrade 125I-A1– 42 whereas complexes with endogenous proteinases, e.g., plasmin and prostate-specific antigen, were not effective. A degradation by the complexes was not inhibited by ␣1-antichymotrypsin and soybean trypsin inhibitor which normally would inactivate the free serine proteinases. A prerequisite for A degradation is its binding to specific binding sites in ␣2-M that may direct A to the active site of the caged proteinase. Ex vivo, enhanced degradation of 125 I-A1– 42 in blood could be achieved upon oral administration of high doses of proteinases to volunteers. These results suggest that up-regulation of A catabolism could probably reduce the risk of developing AD by preventing A accumulation in brain and vasculature. © 2001 Academic Press Key Words: Alzheimer’s disease; amyloid ; ␣2-macroglobulin; proteinase; proteinase inhibitor; oral enzyme therapy.
INTRODUCTION
Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid  (A)1– 40
ON THE OCCASION OF HIS
65TH
BIRTHDAY.
and A1– 42, are found in the amyloid of cerebral vasculature and senile plaques (30) and are considered to be central to the pathology of Alzheimer’s disease (AD) (9). Soluble A can be detected in brain parenchyma, cerebrospinal fluid, and blood plasma (31). The origin of A in cerebral vasculature and brain is still controversial. A may be produced locally or may originate from blood. Recently, it was shown that A1– 42 is bound to human brain capillary endothelial cells and transported from blood into brain by transcytosis (18). These findings support the hypothesis that A originating from circulation could contribute to neurotoxicity (36). The precise mechanism leading to accumulation of A in brain is yet unknown. However, a disturbed balance between amyloid production and its degradation is now considered to be important in AD pathology (15). There are lines of evidence that the human proteinase inhibitor ␣2-macroglobulin (␣2-M) is involved in AD pathology. The inhibitor accumulates in senile plaques and its expression seems to be regulated in AD by cytokines (12, 34). ␣2-M associates with A (11) and prevents fibril formation (10). Stable ␣2-M–A complexes are recognized and cleared via the ␣2-M receptor/low density lipoprotein-related receptor (␣2-M-R/ LRP) abundantly expressed in many different cells and tissues including brain (22, 28). Furthermore, recent results indicate a link of mutations in the ␣2-M gene and ␣2-M-R/LRP gene to late onset of AD in human (2, 16). It is well established that ␣2-M is capable of binding different proteinases. Bound proteinases are caged inside the inhibitor but are still active toward low-molecular-mass substrates and small peptides (32). Upon reaction with proteinases or reactive primary amines, e.g., methylamine (MA), the inhibitor undergoes a conformation change (transformation) conferring high affinity to its cellular receptor. Previous studies have shown that diverse cytokines, growth factors, and polypeptides are bound to and transported by ␣2-M
385
0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
386
LAUER, REICHENBACH, AND BIRKENMEIER
and can be to some extent degraded by caged proteinases (4, 25). Here we examined the capability of diverse ␣2-M–proteinase complexes to proteolytically degrade soluble A1– 42. We found that ␣2-M–proteinase complexes mediate catabolism of A in complex biological fluids. We could also show by ex vivo experiments that A-degrading activity in blood increases upon oral administration of a cocktail of proteinases. MATERIAL AND METHODS
Reagents Native ␣2-M, methylamine-treated ␣2-M (␣2-MMA), prostate-specific antigen, ␣1-antichymotrypsin, HRP-labeled anti-␣2-M-Ig from rabbit, and ELISA test kits MacroTrans and MacroNat for determination of the concentrations of total and transformed ␣2-M were obtained from BioMac GmbH (Leipzig, Germany). Trypsin, bromelain, ␣-chymotrypsin, and Phlogenzyme were obtained from Mucos Pharma GmbH (Geretsried, Germany). A1-42 was purchased from Bachem (Heidelberg, Germany). Radiolabeling A1-42 was labeled with I-Na (37 MBq) (Amersham-Pharmacia Biotech, Freiburg, Germany) using Iodo beads (Pierce Chemicals, Rockford, IL) according to the manufacturer’s instructions. Separation of the labeled peptide from free iodine was accomplished by NAP 05 columns (Amersham-Pharmacia Biotech) preabsorbed with bovine serum albumin. The labeled peptide was ⬍95% trichloroacetic acid precipitable. Removal of labeled aggregates from monomeric A was achieved by centrifugal filtration using a 10,000-kDa cutoff filter (Greiner, Frickenhausen, Germany). 125
Gel Electrophoresis SDS–polyacrylamide gel electrophoresis (SDS– PAGE) was performed in 4 –20 or 4 –30% gradient gels. Native (nondenaturing) gel electrophoresis was performed in the absence of SDS in 4 –20% gradient gels. Analysis of A1– 42 Degradation I-A1– 42 was incubated with diverse ␣2-M–proteinase complexes and free proteinases, respectively, in the presence or absence of proteinase inhibitors in phosphate-buffered saline (PBS) at 37°C for different times specified in the legends to the figures. Aliquots from each sample were taken and mixed with nonreducing sample buffer (SDS–PAGE) or glycerol (native PAGE) prior to electrophoresis. Staining of gels was accomplished by Coomassie brilliant blue R-250. After gel drying autoradiographic development was achieved using Hyperfilm MP (Amersham-Pharmacia Biotech). For quantitative analysis respective zones correspond125
ing to exposed areas on the X-ray films were cut off the gel and ␥-counted. Western Blotting Protein samples were subjected to nonreducing SDS–PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After blocking with defatted milk (5%) the membranes were incubated with rabbit HRP-labeled anti-␣2-M-Ig (10 g/ml) in PBS containing 0.1% Tween 20 (PBS-T) and 3% bovine serum albumin for 2 h at 25°C and than developed with diaminobenzidine and H 2O 2 as substrates. Methylamine Treatment of Plasma To generate transformed ␣2-M, 1 ml fresh human plasma was treated with 200 mM MA for 2 h at 25°C. Excess of methylamine was removed by intensive dialysis against PBS to obtain methylamine-treated plasma (MA plasma). Depletion of Plasma from ␣2-M Depletion of plasma from ␣2-M was achieved by extraction with antibody-coated magnetic beads. Activated magnetic beads (25 mg/ml) (Chemagen, Aachen, Germany) were reacted with rabbit anti-␣2-M-Ig following the procedure described by the manufacturer. The labeled beads (10 mg) were added to 1 ml MA plasma and incubated for 4 h at 25°C. After magnetic separation the incubation was repeated until total removal of ␣2-M as checked by ELISA. Preparation of ␣2-Macroglobulin–Proteinase Complexes Native ␣2-M was reacted with a two molar excess of different proteinases including prostate-specific antigen (PSA), trypsin, ␣-chymotrypsin, bromelain, and plasmin at 25°C for 2 min (trypsin, ␣-chymotrypsin, bromelain) or 15 min (PSA, plasmin), respectively. After reaction the samples were immediately loaded onto a BioSil SEC 125 HPLC column equilibrated with 50 mM sodium phosphate, 1 M NaCl, pH 7.4, for separation of the complex and free proteinases. The purity of the complexes was analyzed by electrophoresis and Western blotting. The success of transformation of ␣2-M by proteinases was assessed by ELISA. Enzyme Treatment Healthy volunteers were given daily 3 ⫻ 3 tablets of Phlogenzym (48 mg trypsin, 90 mg bromelain, 100 mg rutoside each) for 7 days followed by a bolus ingestion of 20 tablets. Blood was withdrawn from the patients before and at certain time intervals after treatment and plasma was subjected to further analysis.
␣2-MACROGLOBULIN-MEDIATED CATABOLISM OF AMYLOID 
FIG. 1. Degradation of radiolabeled A1– 42 by free proteinases. I-A1– 42 (120,000 cpm) was incubated with increasing concentrations of trypsin (Trp), bromelain (Br), and ␣-chymotrypsin (CT) in a final volume of 50 l PBS-T for 3 h at 37°C and separated by nonreducing SDS–PAGE (4 –30%) followed by autoradiography. The first lane reflects the radioactivity of 125I-A in the absence of proteinases. The 4- to 8-kDa zone comprises the position of A in the gel. 125
RESULTS
A contains a number of putative amino acid sequences providing vulnerability to proteolysis. We examined the degradation of A by the proteinases trypsin, ␣-chymotrypsin, bromelain, plasmin, and prostatespecific antigen and by their complexes with the ␣2-M. To test the capability of free proteinases to degrade A, radiolabeled A was incubated with increasing concentrations of different proteinases (Fig. 1). Different patterns of degradation were obtained demonstrating increasing vulnerability of A-degradation toward enzymes in the order of bromelain ⬍ trypsin ⬍ ␣-chymotrypsin. In contrast to these proteinases, plasmin and PSA which were also tested showed very little or no A-degrading activity, respectively, under the conditions used (results not shown). To characterize A-degrading activity of these proteinases when caged by ␣2-M, free proteinases were reacted with native ␣2-M to form stable ␣2-M–proteinase complexes. 125 I-A was incubated with equivalent concentrations of the different complexes (800 nM) and the binding of 125I-A to ␣2-M–proteinase complexes was examined by nondenaturing PAGE (Fig. 2). The results clearly demonstrate the association of 125I-A to all ␣2-M–proteinase complexes as well as to methylamine-treated ␣2-M (720-kDa protein band). No radioactivity was found in association with native ␣2-M. There are clear indications from additional experiments that native ␣2-M does not bind 125I-A. Obviously, 125I-A seems to bind less to ␣2-M-␣-chymotrypsin (CT) and ␣2-M-trypsin (Trp) under the conditions applied. However, shortening the incubation time resulted in an increased association of 125I-A to ␣2-MTrp and ␣2-M-CT which is equivalent to other ␣2-M– proteinase complexes. Therefore, we assumed that the bound 125I-A might be degraded during the time of incubation by the caged trypsin and ␣-chymotrypsin.
387
FIG. 2. Binding of radiolabeled A1– 42 to ␣2-M–proteinase complexes. 125I-A1– 42 (230,000 cpm) was incubated with different ␣2M–proteinase complexes (800 nM) in a final volume of 50 l PBS-T for 5 h at 37°C and separated by nondenaturing PAGE in gradient gels (4 –20%) followed by autoradiography. The arrow indicates the position of ␣2-M (720 kDa) in the gel.
To address this question, we incubated 125I-A with increasing concentrations of ␣2-M–proteinase complexes and analyzed the degradation of 125I-A (4 – 8 kDa) by nonreducing SDS–PAGE. The control comprises 125I-A without added complexes. It was assured by preliminary experiments that the ␣2-M–proteinase complexes used were essentially free of uncomplexed proteinases. The results in Fig. 3 clearly show that caged proteinases preserved their enzymatic activity to degrade A. The A-degrading activity of the complexes followed the order of ␣2-M-CT ⬎ ␣2-M-Trp ⬎ ␣2-M-bromelain (Br). Again, ␣2-M-plasmin (Pl) and ␣2-M-PSA were unable to break down A (not shown). This is similar to the results with free proteinases shown in Fig. 1. Further experiments were conducted to elaborate the effect of serine proteinase inhibitors on A-degradation by free proteinases and ␣2-M–proteinase complexes. Thus, 125I-A was incubated with trypsin, ␣-chymotrypsin, ␣2-M-Trp, and ␣2-M-CT, respectively, in the absence and presence of soybean trypsin inhibitor (STI) and ␣1-antichymotrypsin (ACT) for 2 h at
FIG. 3. Degradation of radiolabeled A1-42 by ␣2-M–proteinase complexes. 125I-A1– 42 (120,000 cpm) was incubated with increasing concentrations of ␣2-M-trypsin, ␣2-M-bromelain, and ␣2-M-chymotrypsin, respectively, in a final volume of 50 l PBS-T for 3 h at 37°C and separated by nonreducing SDS–PAGE (4 –30%) followed by autoradiography. The 4- to 8-kDa zone comprises the position of A1– 42 in the gel.
388
LAUER, REICHENBACH, AND BIRKENMEIER
TABLE 1 Influence of Proteinase Inhibitors on Degradation of A by Free Proteinases and ␣2-M–Proteinase Complexes Proteins
Relative radioactivity (%)
125 I-A1–42 Additives Trypsin Trypsin ⫹ STI Chymotrypsin Chymotrypsin ⫹ ACT Bromelain Bromelain ⫹ STI ␣2-M-trypsin ␣2-M-trypsin ⫹ STI ␣2-M-chymotrypsin ␣2-M-chymotrypsin ⫹ ACT ␣2-M-bromelain ␣2-M-bromelain ⫹ STI
100 50 ⫾ 4.2 98 ⫾ 15 55 ⫾ 8 110 ⫾ 4 61 ⫾ 7 76 ⫾ 4 65 ⫾ 12 58 ⫾ 2.6 60 ⫾ 12 52 ⫾ 5.9 53 ⫾ 6.9 56 ⫾ 8.6
Note. I-A1– 42 (190,000 cpm) was incubated with different ␣2-M– proteinase complexes (450 nM) or free proteinases (500 nM) in the absence or presence of a 6 molar excess (3 M) of STI and ␣1antichymotrypsin (ACT) for 2 h at 37°C and separated by SDS– PAGE (4 –30%) followed by autoradiography. The areas corresponding to the respective spots of radiolabeled A1– 42 on the X-ray films were cut out of the gels and subjected to ␥ counting. The analyses were performed in triplicate. The radioactivity of 125I-A1– 42 alone was set 100%.
37°C followed by nonreducing SDS–PAGE and autoradiography. Quantitative analysis was achieved by cutting respective segments out of the gels which were subjected to ␥ counting (Table 1). Degradation of 125 I-A by free proteinases was almost completely blocked by STI and ACT. As expected, no effect of STI was found in samples with the cysteine proteinase bromelain. In contrast, STI as well as ACT failed to inhibit the degradation by ␣2-M-Trp and ␣2-M-CT of 125 I-A (Table 1 and Fig. 4). It indicates that (i) degradation by ␣2-M–proteinase complexes is not attributed to a contamination with free proteinases and (ii) the catalytic site of the caged enzyme is not accessible to exogenous proteinase inhibitors. We hypothesized that degradation by ␣2-M–proteinase complexes of biologically active peptides requires binding to specific binding sites in ␣2-M. Furthermore, peptides specifically bound to ␣2-M may be shielded to prevent degradation by proteinases in the surrounding. To demonstrate this, we incubated 125I-A with ␣2-M-MA and ␣2-M-PSA to allow complex formation. These different forms of transformed ␣2-M were selected as it is known from previous experiments that both species bind A but neither ␣2-M-MA nor ␣2-MPSA possess A-degrading activity. Free trypsin was added and degradation of ␣2-M-associated A was monitored by nondenaturing PAGE and autoradiography (Fig. 5). Even at prolonged period of incubation no degradation of ␣2-M-associated A was observed. In contrast, radiolabeled insulin which does not bind to
FIG. 4. Effect of serine proteinase inhibitors on the degradation of A1– 42 by ␣2-M–proteinase complexes. 125I-A1– 42 (120,000 cpm) was incubated with different ␣2-M–proteinase complexes (500 nM) in the absence or presence of a 6 molar excess (3 M) of STI or ACT in a final volume of 50 l PBS-T for 3 h at 37°C and separated by nonreducing SDS–PAGE in gradient gels (4 –30%) followed by autoradiography. The 4- to 8-kDa zone comprises the position of A1– 42 in the gel. Lane 1, 125I-A1– 42; lane 2, 125I-A1– 42 ⫹ ␣2-MTrp; lane 3, 125I-A1– 42 ⫹ ␣2-M-Trp ⫹ STI; lane 4, 125I-A1– 42 ⫹ ␣2-M-Trp ⫹ ACT; lane 5, 125I-A1– 42 ⫹ ␣2-M-CT; lane 6, ( 125I-A1– 42 ⫹ ␣2-M-CT ⫹ STI; lane 7, 125I-A1– 42 ⫹ ␣2-M-CT ⫹ ACT.
transformed ␣2-M is completely degraded by added trypsin (not shown). The results let us assume the existence of ␣2-M species which obviously can bind A but do not degrade the peptide and thus provide full protection to proteolytic degradation by enzymes present in the surrounding. To simulate in vivo conditions degradation by ␣2-MTrp of 125I-A was assessed in whole human plasma (Fig. 6). Prior to the incubation experiments human plasma was treated with methylamine (MA plasma) to generate an A-binding form of ␣2-M being proteolyti-
FIG. 5. Protection of A1– 42 bound to ␣2-M against proteolytic degradation. ␣2-M-MA (800 nM) and ␣2-M-PSA (800 nM) dissolved in PBS-T were incubated with 125I-A1– 42 (185,000 cpm) in a final volume of 50 l PBS-T for 16 h at 37°C. Then, trypsin (200 nM) was added and the incubation was continued for an additional 2 h (lanes 3 and 6) or 4 h (lanes 4 and 7), respectively. The samples were separated by nondenaturing PAGE in gradient gels (4 –20%), followed by autoradiography. The arrow indicates the position of ␣2-M (720 kDa) in the gel.
␣2-MACROGLOBULIN-MEDIATED CATABOLISM OF AMYLOID 
389
ases appear in blood and form complexes with ␣2-M (7). Volunteers were given high doses of trypsin and bromelain over a time period of 7 days followed by a bolus ingestion of the same proteinases. Plasma was withdrawn after certain time intervals and its capability to degrade A was investigated (Fig. 7). Proteinase-conditioned plasma was found to metabolize A with a maximum between 1 and 2 h after bolus ingestion. Between 40 and 50% of A became degraded by proteinase-conditioned plasma when compared to plasma obtained prior to proteinase treatment. DISCUSSION
FIG. 6. Degradation of 125I-A1– 42 in human plasma. 125IA1– 42 (185,000 cpm) was added to PBS-T (lane 1), to ␣2-M-MAplasma (2 l) (lane 2), and to plasma depleted of ␣2-M-MA (2 l) (lanes 3 and 4) in a total volume of 20 l PBS-T. In addition, purified ␣2-M-trypsin (400 nM) was added to depleted plasma (lane 4). All samples were incubated for 4 h at 37°C. Afterward, the samples were analyzed by nondenaturing PAGE in gradient gels (4 –20%) followed by autoradiography (A), nonreducing SDS–PAGE in gradient gels (4 –30%) in combination with Western blot (B), and by nonreducing SDS–PAGE in gradient gels (4 –30%) followed by autoradiography (C). After autoradiography of the gel in C, the corresponding segments were cut out of the gel and ␥-counted. The measured counts (D) related to the blank (100%) (lane 1) represent the residual radioactivity of nondegraded A1– 42.
cally inactive. Plasma depleted of endogenous ␣2-M by means of antibody-coated magnetic beads was used as control. 125I-A was added to the plasma samples and binding/degradation was checked by PAGE and autoradiography. As expected, a fraction of 125I-A was bound to transformed ␣2-M (Fig. 6A, lane 2). Compared to the blank only a small decrease of radioactivity in the 4- to 8-kDa zone comprising free 125I-A in MA plasma was found (Figs. 6C and 6D, lane 2). This may be either due to the presence of low concentrations of endogenous ␣2-M-proteinase complexes (normally 0.5 to 2% of total ␣2-M) in fresh plasma or due to the binding of A to ␣2-M-MA. No radioactivity is seen at the ␣2-M position in depleted plasma due to the absence of ␣2-M as verified by Western blot (Fig. 6B, lane 3). Noteworthy was that when depleted plasma was reconstituted with purified ␣2-M-Trp an increased degradation of A was observed (Fig. 6C, lane 4). The degree of degradation was comparable to a sample containing the same amount of purified ␣2-M-Trp in the absence of plasma components. The data indicate that degradation of A present in plasma is mediated by proteinases caged in ␣2-M which seems to be an important route of proteolytic processing of A in biological fluids and tissue. In further studies we designed experiments to corroborate these results by ex vivo investigations. There are lines of evidences that orally administered protein-
␣2-M is a proteinase inhibitor with broad-range specificity. Binding of proteinases involves the caging mechanism after specific cleavage of peptide bonds in the bait region. Concomitant hydrolysis of an internal thioester induces a conformation change leading to formation of new binding sites for small biologically active peptides and triggers the clearance of transformed ␣2-M by the ␣2-M-R/LRP. The proteinases caged in the inhibitor have preserved their proteolytic activity toward accessible peptides or small proteins (32). There are strong indications that ␣2-M and its receptor are involved in pathophysiology of AD. At first
FIG. 7. Degradation of 125I-A1– 42 by proteinase-conditioned human plasma. A volunteer was given daily 3 ⫻ 3 tablets of Phlogenzyme (48 mg trypsin, 90 mg bromelain, 100 mg rutoside each) for 7 days followed by a bolus ingestion of 20 tablets. Plasma (10 l) withdrawn from the patients before and after treatment was mixed with 125I-A1– 42 (200,000 cpm) and incubated at 37°C for 22 h. The degrading activity was analyzed by nonreducing SDS–PAGE in gradient gels (4 –30%). Gel segments corresponding to the spots at the X-ray films were cut out and quantified by ␥-counting. Each sample was analyzed in triplicate. Lane 1, 125I-A1– 42 used as blank; lane 2, 125 I-A1– 42 ⫹ plasma before treatment; lane 3, 125I-A1– 42 ⫹ plasma after 7 days treatment; lane 4, 125I-A1-42 ⫹ plasma 1 h after bolus ingestion; lane 5, 125I-A1-42 ⫹ plasma 2 h after bolus ingestion; lane 6, 125I-A1-42 ⫹ plasma 5 h after bolus ingestion. SEM is given by the error bars. Data were treated by t test yielding sample 2 vs 3 (P ⬍ 0.01), sample 2 vs 4 (P ⬍ 0.01), sample 2 vs 5 (P ⬍ 0.022), and sample 2 vs 6 (P ⬍ 0.014).
390
LAUER, REICHENBACH, AND BIRKENMEIER
Qiu et al. (25) reported the mediation of A degradation by a serine proteinase–␣2-M complex with possible impact on amyloidosis in brain. This prompted us to study the A-degrading activity of diverse proteinases and ␣2-M–proteinase complexes. The complexes investigated either could be found normally in blood and tissue (␣2-M-PSA, ␣2-M-Pl) or can be induced by orally administered proteinases (␣2-M-Trp, ␣2-M-Br, ␣2-MCT). An important observation of this study is that all proteinases tested formed complexes with ␣2-M which mediated binding of A. However, significant differences with respect to the A-degrading activity of these complexes was found. In contrast to naturally occurring complexes such as ␣2-M-Pl and ␣2-M-PSA (24) which obviously do not degrade A, the complexes formed with trypsin, ␣-chymotrypsin, and bromelain were found to be proteolytically active. Inhibition studies revealed that the A-degrading activity of caged proteinases in contrast to noncomplexed proteinases cannot be abolished by inhibitors such as STI or ACT. That means that even in presence of high concentrations of specific inhibitors such as ␣1-proteinase inhibitor or ACT the caged proteinases are shielded from inactivation and can continue to degrade bound A. Furthermore, we supplied evidence that bound A due to the high-affinity constant between A and transformed ␣2-M of 10 ⫺10 M (11) is strongly protected from proteolysis by exogenous proteinases. This might be important as ␣2-M may aid the presentation of the amyloid peptide only to that enzymes being caged inside the inhibitor for further processing. It implies that the A-binding site on the surface of ␣2-M must be adjacent to the active site of the caged proteinase. Because most proteinases react with ␣2-M and only a few seem to be capable of proteolytically modifying A the specificity of this process might be of biological relevance. There are several possibilities how ␣2-M may influence the catabolism of A. At first it may promote A clearance. This can be achieved by nonproteolytically transformed ␣2-M created by activation through primary amines present in the brain parenchyma or by reaction with enzymes possessing no A-degrading activity. A cleared by this way may accumulate in lysosomes as it follows the endocytosis pathway of ␣2-MR/LRP. Thus, the activity of ␣2-M-R/LRP and the regulation of its expression in brain tissue seem to be important for that pathway (6, 26). In this sense, mutations in ␣2-M-R/LRP and ␣2-M which may impair the A clearance in brain might be predisposing factors of amyloidogenesis (2, 16). Another mechanism involves binding and site-directed proteolysis of A by ␣2-M–proteinase complexes. This may be an effective way to directly attenuate the A-induced toxicity in neuronal cells by lowering the concentration of soluble A in the extracellular space (25).
In early-onset AD cases A accumulation is caused by an increase in A anabolism due to presence of predisposing factors, e.g., mutations in presenilin 1 and 2 genes and amyloid precursor protein gene. However, in sporadic AD cases which lack significant overproduction of A an age-related reduction of A catabolism could be responsible for A accumulation in senile brain. Several recent studies stress that improper catabolism plays as large a role as A synthesis in amyloidogenesis. Degradation by the endopeptidase neprilysin of extracellular A, for instance, increased the catabolism of A in brain (15). On the other hand, inhibition of neprilysin activity by infused proteinase inhibitors led to pathological deposition of endogenous A in rat brain. Similarly, a membrane-bound insulindegrading enzyme was found to mediate degradation and clearance of A by neurons and microglia (27). These data suggest that defective degradation of A either by down-regulation of responsible proteinases or by up-regulation of proteinase inhibitors could be a risk factor for the development of AD disease in some subjects. Recently, plasmin was shown to degrade A1– 40 and, interestingly, the proteinase was capable of resolving even amyloid- aggregates (33). Contrarily, we found only small degradation of A by plasmin when compared to trypsin or ␣-chymotrypsin. This may be due to different experimental conditions such as enzyme/substrate ratios or level of activity of plasmin preparation used. Furthermore, plasmin may be less active toward A1– 42 as used in our study than toward A1– 40 used by others (33). In light of theses findings we have studied the mechanism of ␣2-M-mediated degradation of A. For the first time, we could show that ␣2-M-associated Adegrading complexes could be induced in a complex biological fluid such as blood by oral administration of proteinases. Trypsin and bromelain, the constituents of the proteinase cocktail, applied rapidly react with ␣2-M when appearing in blood despite the presence of other proteinase inhibitors (7). A similar mechanism is discussed for the proteinase PSA when liberated form the prostate into blood (23). High does of proteinases must be delivered to the intestine as enteric-coated formulation to allow crossing the mucosal barrier (17). The absorption of proteolytic enzymes from the gastrointestinal tract has been well documented in the past (1). It concerns endogenous proteolytic enzymes, e.g., pancreatic enzymes (13) and exogenous proteinases as well (35). Furthermore, proteinases have been successfully administered for treatment of diverse diseases (14). Although the precise mechanism of this unusual intestinal absorption phenomenon remains unclear, recent data favor absorption of proteolytic enzymes by a self-enhanced paracellular transport (3). However, transcytosis of proteinases as complex with intestinal ␣2-M should also be considered as the ␣2-M-R/LRP is
␣2-MACROGLOBULIN-MEDIATED CATABOLISM OF AMYLOID 
expressed in the intestinal epithelium (21) and ␣2-M is present in the intestinal lumen (5). We could clearly show that proteinase-conditioned plasma accelerated the degradation of A in blood. Would this be of potential interest with respect to amyloidogenesis in AD? Soluble A is produced by different cell types (20) and is present in measurable concentrations in cerebrospinal fluid and blood at 10 ⫺8–10 ⫺10 M (31). Although A concentration in circulation does not correlate with AD stages, a significant increase in A concentration was found in plasma of mutation carriers (8). The synthesis of APP outside the central nervous system and the deposition of A proteins in nonneuronal tissues suggest that AD might be a systemic disorder (20). Recent studies strongly indicate that soluble A1– 40 present in circulation can cross the blood– brain barrier via transcytosis mediated by brain capillary endothelial cells (19, 18). Similarly, A1– 42 is suggested to cross stereoisomer specifically the blood– brain barrier by receptor-mediated transport (24). The presence of cerebrovascular endothelial receptors for A is likely to influence the development of cerebrovascular amyloidosis and vascular injury. The latter may sustain additional macromolecules to pass the blood– brain barrier which normally could not, e.g., antibodies (29). Although it remains unproven, it is not unreasonable to expect that administration of proteinases may enhance A catabolism in blood as well as in nonneuronal and neuronal tissues which could be of clinical benefit. ACKNOWLEDGMENTS This work was supported by the Bundesministerium fu¨r Bildung, Forschung und Technologie (BMB⫹F) and the Interdisciplinary Center for Clinical Research at the University of Leipzig (01KS9504, Project C5).
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
REFERENCES 1.
2.
3.
4. 5.
6.
Ambrus, J. L., H. B. Lassman, and J. J. DeMarchi. 1967. Absorption of exogenous and endogenous proteolytic enzymes. Clin. Pharmacol. Ther. 8: 362–368. Blacker, D., M. A. Wilcox, N. M. Laird, L. Rodes, S. M. Horvath, R. C. Go, R. Perry, B. Watson, Jr., S. S. Bassett, M. G. McInnis, M. S. Albert, B. T. Hyman, and R. E. Tanzi. 1998. Alpha-2 macroglobulin is genetically associated with Alzheimer disease. Nature Genet. 19: 357–360. Bock, U., C. Kolac, G. Borchard, K. Koch, R. Fuchs, P. Streichhan, and C. M. Lehr. 1998. Transport of proteolytic enzymes across Caco-2 cell monolayers. Pharm. Res. 15: 1393–1400. Borth, W. 1992. ␣2-Macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J. 6: 3345–3352. Burgess, J. W., and K. K. Stanley. 1997. Estrogen-stimulated transcytosis of desialylated ligands and alpha2 macroglobulin in rat liver. Biochim. Biophys. Acta 1359: 48 –58. Businaro, R., C. Fabrizi, T. Persichini, G. Storace, M. G. Ennas, L. Fumagalli, and G. M. Lauro. 1997. Modulation of the alpha 2 macroglobulin receptor/low density lipoprotein receptor related
17.
18.
19.
20.
21.
391
protein by interferon-gamma in human astroglial cells. J. Neuroimmunol. 72: 75– 81. Castell, J. V., G. Friedrich, C. S. Kuhn, and G. E. Poppe. 1997. Intestinal absorption of undegraded proteins in men: Presence of bromelain in plasma after oral intake. Am. J. Physiol. 273: 139 –146. De Jonghe, C., P. Cras, H. Vanderstichele, M. Cruts, I. Vanderhoeven, I. Smouts, E. Vanmechelen, J. J. Martin, L. Hendriks, and C. Van Broeckhoven. 1999. Evidence that Abeta42 plasma levels in presenilin-1 mutation carriers do not allow for prediction of their clinical phenotype. Neurobiol. Dis. 6: 280 –287. Dickson, D. W. 1997. The pathogenesis of senile plaques. J. Neuropathol. Exp. Neurol. 56: 321–339. Du, Y., K. R. Bales, R. C. Dodel, X. Liu, M. A. Glinn, J. W. Horn, S. P. Little, and S. M. Paul. 1998. Alpha2-macroglobulin attenuates beta-amyloid peptide 1– 40 fibril formation and associated neurotoxicity of cultured fetal rat cortical neurons. J. Neurochem. 70: 1182–1188. Du, Y., B. Ni, M. Glinn, R. C. Dodel, K. R. Bales, Z. Zhang, P. A. Hyslop, and S. M. Paul. 1997. Alpha2-macroglobulin as a betaamyloid peptide-binding plasma protein. J. Neurochem. 69: 299 –305. Ganter, U., S. Strauss, U. Jonas, A. Weidemann, K. Beyreuther, B. Volk, M. Berger, and J. Bauer. Alpha 2-macroglobulin synthesis in interleukin-6-stimulated human neuronal (SH-SY5Y neuroblastoma) cells: Potential significance for the processing of Alzheimer beta-amyloid precursor protein. FEBS Lett. 282: 127–131. Go¨tze, H., and S. S. Rothman. 1975. Enterohepatic circulation of digestive enzymes as a conservation mechanism. Nature 257: 607– 609. Heidland, A., K. Sebekova, L. Paczek, M. Teschner, J. Dammrich, and Z. Gaciong. 1997. Renal fibrosis: Role of impaired proteolysis and potential therapeutic strategies. Kidney Int. Suppl. 62: 32–35. Iwata, N., S. Tsubuki, Y. Takaki, K. Watanabe, M. Sekiguchi, E. Hosoki, M. Kawashima-Morishima, H. J. Lee, E. Hama, Y. Sekine-Aizawa, and T. C. Saido. 2000. Identification of the major Abeta1– 42-degrading catabolic pathway in brain parenchyma: Suppression leads to biochemical and pathological deposition. Nature Med. 6: 143–150. Kang, D. E., T. Saitoh, X. Chen, Y. Xia, E. Masliah, L. A. Hansen, R. G. Thomas, L. J. Thal, and R. Katzman. 1997. Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), an apolipoprotein E receptor, with late-onset Alzheimer’s disease. Neurology 49: 56 – 61. Kolac, C., P. Streichhan, and C.-M. Lehr. 1996. Oral biovailability of proteolytic enzymes. Eur. J. Biopharmacol. 42: 222– 232. Mackic, J. B., M. Stins, J. G. McComb, M. Calero, J. Ghiso, K. S. Kim, S. D. Yan, D. Stern, A. M. Schmidt, B. Frangione, and B. V. Zlokovic. 1998. Human blood– brain barrier receptors for Alzheimer’s amyloid-beta 1– 40: Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer. J. Clin. Invest. 102: 734 –743. Martel, C. L, J. B. Mackic, J. G. McComb, J. Ghiso, and B. V. Zlokovic. 1996. Blood– brain barrier uptake of the 40 and 42 amino acid sequences of circulating Alzheimer’s amyloid beta in guinea pigs. Neurosci. Lett. 206: 157–160. Miklossy, J., K. Taddei, R. Martins, G. Escher, R. Kraftsik, O. Pillevuit, D. Lepori, and M. Campiche. 1999. Alzheimer disease: Curly fibers and tangles in organs other than brain. J. Neuropathol. Exp. Neurol. 58: 803– 814. Moestrup, S. K., J. Gliemann, and G. Pallesen. 1992. Distribution of the alpha 2-macroglobulin receptor/low density lipopro-
392
22.
23.
24.
25.
26.
27.
28.
LAUER, REICHENBACH, AND BIRKENMEIER tein receptor-related protein in human tissues. Cell. Tissue Res. 269: 375–382. Narita, M., D. M. Holtzman, A. L. Schwartz, and G. Bu. 1997. Alpha2-macroglobulin complexes with and mediates the endocytosis of beta-amyloid peptide via cell surface low-density lipoprotein receptor-related protein. Neurochemistry 69: 1904 – 1911. Otto, A., J. Ba¨r, and G. Birkenmeier. 1998. Prostate-specific antigen forms complexes with human alpha 2-macroglobulin and binds to the alpha 2-macroglobulin receptor/LDL receptorrelated protein. J. Urol. 159: 297–303. Poduslo, J. F., G. L. Curran, B. Sanyal, and D. J. Selkoe. 1999. Receptor-mediated transport of human amyloid beta-protein 1– 40 and 1– 42 at the blood– brain barrier. Neurobiol. Dis. 6: 190 –199. Qiu, W. Q., W. Borth, Z. Ye, C. Haass, D. B. Teplow, and D. J. Selkoe. 1996. Degradation of amyloid beta-protein by a serine protease-alpha2-macroglobulin complex. J. Biol. Chem. 271: 8443– 8451. Qiu, Z., D. K. Strickland, B. T. Hyman, and G. W. Rebeck. 1999. Alpha2-macroglobulin enhances the clearance of endogenous soluble beta-amyloid peptide via low-density lipoprotein receptor-related protein in cortical neurons. Neurochemistry 73: 1393–1398. Qiu, W. Q., D. M. Walsh, Z. Ye, K. Vekrellis, J. Zhang, M. B. Podlisny, M. R. Rosner, A. Safavi, L. B. Hersh, and D. J. Selkoe. 1998. Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J. Biol. Chem. 273: 32730 –32738. Rebeck, G. W., S. D. Harr, D. K. Strickland, and B. T. Hyman 1995. Multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein E receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein. Ann. Neurol. 37: 211–217.
29.
30. 31.
32.
33.
34.
35.
36.
Schenk, D., R. Barbour, W. Dunn, G. Gordon, H. Grajeda, T. Guido, K. Hu, J. Huang, K. Johnson-Wood, K. Khan, D. Kholodenko, M. Lee, Z. Liao, I. Lieberburg, R. Motter, L. Mutter, F. Soriano, G. Shopp, N. Vasquez, C. Vandevert, S. Walker, M. Wogulis, T. Yednock, D. Games, and P. Seubert. 1999. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400: 173–177. Selkoe, D. J. 1994. Alzheimer’s disease: A central role for amyloid. J. Neuropathol. Exp. Neurol. 53: 438 – 447. Seubert, P., C. Vigo-Pelfrey, F. Esch, M. Lee, H. Dovey, D. Davis, S. Sinha, M. Schlossmacher, J. Whaley, C. Swindlehurst, R. McCormack, R. Wolfert, D. Selkoe, I. Lieberburg, and D. Schenk. 1992. Isolation and quantification of soluble Alzheimer’s beta-peptide from biological fluids. Nature 359: 325–327. Sottrup-Jensen, L. 1989. Alpha2-macroglobulins: Structure, shape and mechanism of proteinase complex formation. J. Biol. Chem. 264: 11539 –11542. Tucker, H. M., M. Kihiko, J. N. Caldwell, S. Wright, T. Kawarabayashi, D. Price, D. Walker, S. Scheff, J. P. McGillis, R. E. Rydel, and S. Estus. 2000. The plasmin system is induced by and degrades amyloid-beta aggregates. J. Neurosci. 20: 3937–3946. Thal, D. R., R. Schober, and G. Birkenmeier. 1997. The subunits of alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein, native and transformed alpha2-macroglobulin and interleukin 6 in Alzheimer’s disease. Brain Res. 777: 223–227. Wrba, H., K. Kleine, K. Miehlke, F.-W. Dittmar, and R. E. Weissenbacher. 1996. Systemische Enzymtherapie-aktueller Stand und Fortschritte. MMV Medizin Verlag, Mu¨nchen. Zlokovic, B. V. 1997. Can blood– brain barrier play a role in the development of cerebral amyloidosis and Alzheimer’s disease pathology? Neurobiol. Dis. 4: 23–26.
␣2-Macroglobulin-Mediated Degradation of Amyloid 1– 42: A Mechanism to Enhance Amyloid  Catabolism D. Lauer, A. Reichenbach,* and G. Birkenmeier Institute of Biochemistry, University of Leipzig, Liebigstrasse 16, 04103 Leipzig, Germany; and *Paul-Flechsig-Institute for Brain Research, Department of Neurophysiology, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany Received May 25, 2000; accepted September 26, 2000; published online January 2, 2001 DEDICATED
TO
PROF. G KOPPERSCHLA¨ GER
Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid  (A), are considered to be central to the pathology of Alzheimer’s disease (AD). Soluble A is present in measurable concentrations in cerebrospinal fluid and blood. There are indications that soluble A present in circulation can cross the blood– brain barrier via transcytosis mediated by brain capillary endothelial cells. It implies that A originating from circulation may contribute to vascular and parenchymal A deposition in AD. Enhancing of A catabolism mediated by proteolytic degradation or receptor-mediated endocytosis could be a key mechanism to maintain low concentrations of soluble A. To launch A clearance we have exploited the A-degrading activity of diverse ␣2-macroglobulin (␣2-M)–proteinase complexes. Complexes with trypsin, ␣-chymotrypsin, and bromelain strongly degrade 125I-A1– 42 whereas complexes with endogenous proteinases, e.g., plasmin and prostate-specific antigen, were not effective. A degradation by the complexes was not inhibited by ␣1-antichymotrypsin and soybean trypsin inhibitor which normally would inactivate the free serine proteinases. A prerequisite for A degradation is its binding to specific binding sites in ␣2-M that may direct A to the active site of the caged proteinase. Ex vivo, enhanced degradation of 125 I-A1– 42 in blood could be achieved upon oral administration of high doses of proteinases to volunteers. These results suggest that up-regulation of A catabolism could probably reduce the risk of developing AD by preventing A accumulation in brain and vasculature. © 2001 Academic Press Key Words: Alzheimer’s disease; amyloid ; ␣2-macroglobulin; proteinase; proteinase inhibitor; oral enzyme therapy.
INTRODUCTION
Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid  (A)1– 40
ON THE OCCASION OF HIS
65TH
BIRTHDAY.
and A1– 42, are found in the amyloid of cerebral vasculature and senile plaques (30) and are considered to be central to the pathology of Alzheimer’s disease (AD) (9). Soluble A can be detected in brain parenchyma, cerebrospinal fluid, and blood plasma (31). The origin of A in cerebral vasculature and brain is still controversial. A may be produced locally or may originate from blood. Recently, it was shown that A1– 42 is bound to human brain capillary endothelial cells and transported from blood into brain by transcytosis (18). These findings support the hypothesis that A originating from circulation could contribute to neurotoxicity (36). The precise mechanism leading to accumulation of A in brain is yet unknown. However, a disturbed balance between amyloid production and its degradation is now considered to be important in AD pathology (15). There are lines of evidence that the human proteinase inhibitor ␣2-macroglobulin (␣2-M) is involved in AD pathology. The inhibitor accumulates in senile plaques and its expression seems to be regulated in AD by cytokines (12, 34). ␣2-M associates with A (11) and prevents fibril formation (10). Stable ␣2-M–A complexes are recognized and cleared via the ␣2-M receptor/low density lipoprotein-related receptor (␣2-M-R/ LRP) abundantly expressed in many different cells and tissues including brain (22, 28). Furthermore, recent results indicate a link of mutations in the ␣2-M gene and ␣2-M-R/LRP gene to late onset of AD in human (2, 16). It is well established that ␣2-M is capable of binding different proteinases. Bound proteinases are caged inside the inhibitor but are still active toward low-molecular-mass substrates and small peptides (32). Upon reaction with proteinases or reactive primary amines, e.g., methylamine (MA), the inhibitor undergoes a conformation change (transformation) conferring high affinity to its cellular receptor. Previous studies have shown that diverse cytokines, growth factors, and polypeptides are bound to and transported by ␣2-M
385
0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
386
LAUER, REICHENBACH, AND BIRKENMEIER
and can be to some extent degraded by caged proteinases (4, 25). Here we examined the capability of diverse ␣2-M–proteinase complexes to proteolytically degrade soluble A1– 42. We found that ␣2-M–proteinase complexes mediate catabolism of A in complex biological fluids. We could also show by ex vivo experiments that A-degrading activity in blood increases upon oral administration of a cocktail of proteinases. MATERIAL AND METHODS
Reagents Native ␣2-M, methylamine-treated ␣2-M (␣2-MMA), prostate-specific antigen, ␣1-antichymotrypsin, HRP-labeled anti-␣2-M-Ig from rabbit, and ELISA test kits MacroTrans and MacroNat for determination of the concentrations of total and transformed ␣2-M were obtained from BioMac GmbH (Leipzig, Germany). Trypsin, bromelain, ␣-chymotrypsin, and Phlogenzyme were obtained from Mucos Pharma GmbH (Geretsried, Germany). A1-42 was purchased from Bachem (Heidelberg, Germany). Radiolabeling A1-42 was labeled with I-Na (37 MBq) (Amersham-Pharmacia Biotech, Freiburg, Germany) using Iodo beads (Pierce Chemicals, Rockford, IL) according to the manufacturer’s instructions. Separation of the labeled peptide from free iodine was accomplished by NAP 05 columns (Amersham-Pharmacia Biotech) preabsorbed with bovine serum albumin. The labeled peptide was ⬍95% trichloroacetic acid precipitable. Removal of labeled aggregates from monomeric A was achieved by centrifugal filtration using a 10,000-kDa cutoff filter (Greiner, Frickenhausen, Germany). 125
Gel Electrophoresis SDS–polyacrylamide gel electrophoresis (SDS– PAGE) was performed in 4 –20 or 4 –30% gradient gels. Native (nondenaturing) gel electrophoresis was performed in the absence of SDS in 4 –20% gradient gels. Analysis of A1– 42 Degradation I-A1– 42 was incubated with diverse ␣2-M–proteinase complexes and free proteinases, respectively, in the presence or absence of proteinase inhibitors in phosphate-buffered saline (PBS) at 37°C for different times specified in the legends to the figures. Aliquots from each sample were taken and mixed with nonreducing sample buffer (SDS–PAGE) or glycerol (native PAGE) prior to electrophoresis. Staining of gels was accomplished by Coomassie brilliant blue R-250. After gel drying autoradiographic development was achieved using Hyperfilm MP (Amersham-Pharmacia Biotech). For quantitative analysis respective zones correspond125
ing to exposed areas on the X-ray films were cut off the gel and ␥-counted. Western Blotting Protein samples were subjected to nonreducing SDS–PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After blocking with defatted milk (5%) the membranes were incubated with rabbit HRP-labeled anti-␣2-M-Ig (10 g/ml) in PBS containing 0.1% Tween 20 (PBS-T) and 3% bovine serum albumin for 2 h at 25°C and than developed with diaminobenzidine and H 2O 2 as substrates. Methylamine Treatment of Plasma To generate transformed ␣2-M, 1 ml fresh human plasma was treated with 200 mM MA for 2 h at 25°C. Excess of methylamine was removed by intensive dialysis against PBS to obtain methylamine-treated plasma (MA plasma). Depletion of Plasma from ␣2-M Depletion of plasma from ␣2-M was achieved by extraction with antibody-coated magnetic beads. Activated magnetic beads (25 mg/ml) (Chemagen, Aachen, Germany) were reacted with rabbit anti-␣2-M-Ig following the procedure described by the manufacturer. The labeled beads (10 mg) were added to 1 ml MA plasma and incubated for 4 h at 25°C. After magnetic separation the incubation was repeated until total removal of ␣2-M as checked by ELISA. Preparation of ␣2-Macroglobulin–Proteinase Complexes Native ␣2-M was reacted with a two molar excess of different proteinases including prostate-specific antigen (PSA), trypsin, ␣-chymotrypsin, bromelain, and plasmin at 25°C for 2 min (trypsin, ␣-chymotrypsin, bromelain) or 15 min (PSA, plasmin), respectively. After reaction the samples were immediately loaded onto a BioSil SEC 125 HPLC column equilibrated with 50 mM sodium phosphate, 1 M NaCl, pH 7.4, for separation of the complex and free proteinases. The purity of the complexes was analyzed by electrophoresis and Western blotting. The success of transformation of ␣2-M by proteinases was assessed by ELISA. Enzyme Treatment Healthy volunteers were given daily 3 ⫻ 3 tablets of Phlogenzym (48 mg trypsin, 90 mg bromelain, 100 mg rutoside each) for 7 days followed by a bolus ingestion of 20 tablets. Blood was withdrawn from the patients before and at certain time intervals after treatment and plasma was subjected to further analysis.
␣2-MACROGLOBULIN-MEDIATED CATABOLISM OF AMYLOID 
FIG. 1. Degradation of radiolabeled A1– 42 by free proteinases. I-A1– 42 (120,000 cpm) was incubated with increasing concentrations of trypsin (Trp), bromelain (Br), and ␣-chymotrypsin (CT) in a final volume of 50 l PBS-T for 3 h at 37°C and separated by nonreducing SDS–PAGE (4 –30%) followed by autoradiography. The first lane reflects the radioactivity of 125I-A in the absence of proteinases. The 4- to 8-kDa zone comprises the position of A in the gel. 125
RESULTS
A contains a number of putative amino acid sequences providing vulnerability to proteolysis. We examined the degradation of A by the proteinases trypsin, ␣-chymotrypsin, bromelain, plasmin, and prostatespecific antigen and by their complexes with the ␣2-M. To test the capability of free proteinases to degrade A, radiolabeled A was incubated with increasing concentrations of different proteinases (Fig. 1). Different patterns of degradation were obtained demonstrating increasing vulnerability of A-degradation toward enzymes in the order of bromelain ⬍ trypsin ⬍ ␣-chymotrypsin. In contrast to these proteinases, plasmin and PSA which were also tested showed very little or no A-degrading activity, respectively, under the conditions used (results not shown). To characterize A-degrading activity of these proteinases when caged by ␣2-M, free proteinases were reacted with native ␣2-M to form stable ␣2-M–proteinase complexes. 125 I-A was incubated with equivalent concentrations of the different complexes (800 nM) and the binding of 125I-A to ␣2-M–proteinase complexes was examined by nondenaturing PAGE (Fig. 2). The results clearly demonstrate the association of 125I-A to all ␣2-M–proteinase complexes as well as to methylamine-treated ␣2-M (720-kDa protein band). No radioactivity was found in association with native ␣2-M. There are clear indications from additional experiments that native ␣2-M does not bind 125I-A. Obviously, 125I-A seems to bind less to ␣2-M-␣-chymotrypsin (CT) and ␣2-M-trypsin (Trp) under the conditions applied. However, shortening the incubation time resulted in an increased association of 125I-A to ␣2-MTrp and ␣2-M-CT which is equivalent to other ␣2-M– proteinase complexes. Therefore, we assumed that the bound 125I-A might be degraded during the time of incubation by the caged trypsin and ␣-chymotrypsin.
387
FIG. 2. Binding of radiolabeled A1– 42 to ␣2-M–proteinase complexes. 125I-A1– 42 (230,000 cpm) was incubated with different ␣2M–proteinase complexes (800 nM) in a final volume of 50 l PBS-T for 5 h at 37°C and separated by nondenaturing PAGE in gradient gels (4 –20%) followed by autoradiography. The arrow indicates the position of ␣2-M (720 kDa) in the gel.
To address this question, we incubated 125I-A with increasing concentrations of ␣2-M–proteinase complexes and analyzed the degradation of 125I-A (4 – 8 kDa) by nonreducing SDS–PAGE. The control comprises 125I-A without added complexes. It was assured by preliminary experiments that the ␣2-M–proteinase complexes used were essentially free of uncomplexed proteinases. The results in Fig. 3 clearly show that caged proteinases preserved their enzymatic activity to degrade A. The A-degrading activity of the complexes followed the order of ␣2-M-CT ⬎ ␣2-M-Trp ⬎ ␣2-M-bromelain (Br). Again, ␣2-M-plasmin (Pl) and ␣2-M-PSA were unable to break down A (not shown). This is similar to the results with free proteinases shown in Fig. 1. Further experiments were conducted to elaborate the effect of serine proteinase inhibitors on A-degradation by free proteinases and ␣2-M–proteinase complexes. Thus, 125I-A was incubated with trypsin, ␣-chymotrypsin, ␣2-M-Trp, and ␣2-M-CT, respectively, in the absence and presence of soybean trypsin inhibitor (STI) and ␣1-antichymotrypsin (ACT) for 2 h at
FIG. 3. Degradation of radiolabeled A1-42 by ␣2-M–proteinase complexes. 125I-A1– 42 (120,000 cpm) was incubated with increasing concentrations of ␣2-M-trypsin, ␣2-M-bromelain, and ␣2-M-chymotrypsin, respectively, in a final volume of 50 l PBS-T for 3 h at 37°C and separated by nonreducing SDS–PAGE (4 –30%) followed by autoradiography. The 4- to 8-kDa zone comprises the position of A1– 42 in the gel.
388
LAUER, REICHENBACH, AND BIRKENMEIER
TABLE 1 Influence of Proteinase Inhibitors on Degradation of A by Free Proteinases and ␣2-M–Proteinase Complexes Proteins
Relative radioactivity (%)
125 I-A1–42 Additives Trypsin Trypsin ⫹ STI Chymotrypsin Chymotrypsin ⫹ ACT Bromelain Bromelain ⫹ STI ␣2-M-trypsin ␣2-M-trypsin ⫹ STI ␣2-M-chymotrypsin ␣2-M-chymotrypsin ⫹ ACT ␣2-M-bromelain ␣2-M-bromelain ⫹ STI
100 50 ⫾ 4.2 98 ⫾ 15 55 ⫾ 8 110 ⫾ 4 61 ⫾ 7 76 ⫾ 4 65 ⫾ 12 58 ⫾ 2.6 60 ⫾ 12 52 ⫾ 5.9 53 ⫾ 6.9 56 ⫾ 8.6
Note. I-A1– 42 (190,000 cpm) was incubated with different ␣2-M– proteinase complexes (450 nM) or free proteinases (500 nM) in the absence or presence of a 6 molar excess (3 M) of STI and ␣1antichymotrypsin (ACT) for 2 h at 37°C and separated by SDS– PAGE (4 –30%) followed by autoradiography. The areas corresponding to the respective spots of radiolabeled A1– 42 on the X-ray films were cut out of the gels and subjected to ␥ counting. The analyses were performed in triplicate. The radioactivity of 125I-A1– 42 alone was set 100%.
37°C followed by nonreducing SDS–PAGE and autoradiography. Quantitative analysis was achieved by cutting respective segments out of the gels which were subjected to ␥ counting (Table 1). Degradation of 125 I-A by free proteinases was almost completely blocked by STI and ACT. As expected, no effect of STI was found in samples with the cysteine proteinase bromelain. In contrast, STI as well as ACT failed to inhibit the degradation by ␣2-M-Trp and ␣2-M-CT of 125 I-A (Table 1 and Fig. 4). It indicates that (i) degradation by ␣2-M–proteinase complexes is not attributed to a contamination with free proteinases and (ii) the catalytic site of the caged enzyme is not accessible to exogenous proteinase inhibitors. We hypothesized that degradation by ␣2-M–proteinase complexes of biologically active peptides requires binding to specific binding sites in ␣2-M. Furthermore, peptides specifically bound to ␣2-M may be shielded to prevent degradation by proteinases in the surrounding. To demonstrate this, we incubated 125I-A with ␣2-M-MA and ␣2-M-PSA to allow complex formation. These different forms of transformed ␣2-M were selected as it is known from previous experiments that both species bind A but neither ␣2-M-MA nor ␣2-MPSA possess A-degrading activity. Free trypsin was added and degradation of ␣2-M-associated A was monitored by nondenaturing PAGE and autoradiography (Fig. 5). Even at prolonged period of incubation no degradation of ␣2-M-associated A was observed. In contrast, radiolabeled insulin which does not bind to
FIG. 4. Effect of serine proteinase inhibitors on the degradation of A1– 42 by ␣2-M–proteinase complexes. 125I-A1– 42 (120,000 cpm) was incubated with different ␣2-M–proteinase complexes (500 nM) in the absence or presence of a 6 molar excess (3 M) of STI or ACT in a final volume of 50 l PBS-T for 3 h at 37°C and separated by nonreducing SDS–PAGE in gradient gels (4 –30%) followed by autoradiography. The 4- to 8-kDa zone comprises the position of A1– 42 in the gel. Lane 1, 125I-A1– 42; lane 2, 125I-A1– 42 ⫹ ␣2-MTrp; lane 3, 125I-A1– 42 ⫹ ␣2-M-Trp ⫹ STI; lane 4, 125I-A1– 42 ⫹ ␣2-M-Trp ⫹ ACT; lane 5, 125I-A1– 42 ⫹ ␣2-M-CT; lane 6, ( 125I-A1– 42 ⫹ ␣2-M-CT ⫹ STI; lane 7, 125I-A1– 42 ⫹ ␣2-M-CT ⫹ ACT.
transformed ␣2-M is completely degraded by added trypsin (not shown). The results let us assume the existence of ␣2-M species which obviously can bind A but do not degrade the peptide and thus provide full protection to proteolytic degradation by enzymes present in the surrounding. To simulate in vivo conditions degradation by ␣2-MTrp of 125I-A was assessed in whole human plasma (Fig. 6). Prior to the incubation experiments human plasma was treated with methylamine (MA plasma) to generate an A-binding form of ␣2-M being proteolyti-
FIG. 5. Protection of A1– 42 bound to ␣2-M against proteolytic degradation. ␣2-M-MA (800 nM) and ␣2-M-PSA (800 nM) dissolved in PBS-T were incubated with 125I-A1– 42 (185,000 cpm) in a final volume of 50 l PBS-T for 16 h at 37°C. Then, trypsin (200 nM) was added and the incubation was continued for an additional 2 h (lanes 3 and 6) or 4 h (lanes 4 and 7), respectively. The samples were separated by nondenaturing PAGE in gradient gels (4 –20%), followed by autoradiography. The arrow indicates the position of ␣2-M (720 kDa) in the gel.
␣2-MACROGLOBULIN-MEDIATED CATABOLISM OF AMYLOID 
389
ases appear in blood and form complexes with ␣2-M (7). Volunteers were given high doses of trypsin and bromelain over a time period of 7 days followed by a bolus ingestion of the same proteinases. Plasma was withdrawn after certain time intervals and its capability to degrade A was investigated (Fig. 7). Proteinase-conditioned plasma was found to metabolize A with a maximum between 1 and 2 h after bolus ingestion. Between 40 and 50% of A became degraded by proteinase-conditioned plasma when compared to plasma obtained prior to proteinase treatment. DISCUSSION
FIG. 6. Degradation of 125I-A1– 42 in human plasma. 125IA1– 42 (185,000 cpm) was added to PBS-T (lane 1), to ␣2-M-MAplasma (2 l) (lane 2), and to plasma depleted of ␣2-M-MA (2 l) (lanes 3 and 4) in a total volume of 20 l PBS-T. In addition, purified ␣2-M-trypsin (400 nM) was added to depleted plasma (lane 4). All samples were incubated for 4 h at 37°C. Afterward, the samples were analyzed by nondenaturing PAGE in gradient gels (4 –20%) followed by autoradiography (A), nonreducing SDS–PAGE in gradient gels (4 –30%) in combination with Western blot (B), and by nonreducing SDS–PAGE in gradient gels (4 –30%) followed by autoradiography (C). After autoradiography of the gel in C, the corresponding segments were cut out of the gel and ␥-counted. The measured counts (D) related to the blank (100%) (lane 1) represent the residual radioactivity of nondegraded A1– 42.
cally inactive. Plasma depleted of endogenous ␣2-M by means of antibody-coated magnetic beads was used as control. 125I-A was added to the plasma samples and binding/degradation was checked by PAGE and autoradiography. As expected, a fraction of 125I-A was bound to transformed ␣2-M (Fig. 6A, lane 2). Compared to the blank only a small decrease of radioactivity in the 4- to 8-kDa zone comprising free 125I-A in MA plasma was found (Figs. 6C and 6D, lane 2). This may be either due to the presence of low concentrations of endogenous ␣2-M-proteinase complexes (normally 0.5 to 2% of total ␣2-M) in fresh plasma or due to the binding of A to ␣2-M-MA. No radioactivity is seen at the ␣2-M position in depleted plasma due to the absence of ␣2-M as verified by Western blot (Fig. 6B, lane 3). Noteworthy was that when depleted plasma was reconstituted with purified ␣2-M-Trp an increased degradation of A was observed (Fig. 6C, lane 4). The degree of degradation was comparable to a sample containing the same amount of purified ␣2-M-Trp in the absence of plasma components. The data indicate that degradation of A present in plasma is mediated by proteinases caged in ␣2-M which seems to be an important route of proteolytic processing of A in biological fluids and tissue. In further studies we designed experiments to corroborate these results by ex vivo investigations. There are lines of evidences that orally administered protein-
␣2-M is a proteinase inhibitor with broad-range specificity. Binding of proteinases involves the caging mechanism after specific cleavage of peptide bonds in the bait region. Concomitant hydrolysis of an internal thioester induces a conformation change leading to formation of new binding sites for small biologically active peptides and triggers the clearance of transformed ␣2-M by the ␣2-M-R/LRP. The proteinases caged in the inhibitor have preserved their proteolytic activity toward accessible peptides or small proteins (32). There are strong indications that ␣2-M and its receptor are involved in pathophysiology of AD. At first
FIG. 7. Degradation of 125I-A1– 42 by proteinase-conditioned human plasma. A volunteer was given daily 3 ⫻ 3 tablets of Phlogenzyme (48 mg trypsin, 90 mg bromelain, 100 mg rutoside each) for 7 days followed by a bolus ingestion of 20 tablets. Plasma (10 l) withdrawn from the patients before and after treatment was mixed with 125I-A1– 42 (200,000 cpm) and incubated at 37°C for 22 h. The degrading activity was analyzed by nonreducing SDS–PAGE in gradient gels (4 –30%). Gel segments corresponding to the spots at the X-ray films were cut out and quantified by ␥-counting. Each sample was analyzed in triplicate. Lane 1, 125I-A1– 42 used as blank; lane 2, 125 I-A1– 42 ⫹ plasma before treatment; lane 3, 125I-A1– 42 ⫹ plasma after 7 days treatment; lane 4, 125I-A1-42 ⫹ plasma 1 h after bolus ingestion; lane 5, 125I-A1-42 ⫹ plasma 2 h after bolus ingestion; lane 6, 125I-A1-42 ⫹ plasma 5 h after bolus ingestion. SEM is given by the error bars. Data were treated by t test yielding sample 2 vs 3 (P ⬍ 0.01), sample 2 vs 4 (P ⬍ 0.01), sample 2 vs 5 (P ⬍ 0.022), and sample 2 vs 6 (P ⬍ 0.014).
390
LAUER, REICHENBACH, AND BIRKENMEIER
Qiu et al. (25) reported the mediation of A degradation by a serine proteinase–␣2-M complex with possible impact on amyloidosis in brain. This prompted us to study the A-degrading activity of diverse proteinases and ␣2-M–proteinase complexes. The complexes investigated either could be found normally in blood and tissue (␣2-M-PSA, ␣2-M-Pl) or can be induced by orally administered proteinases (␣2-M-Trp, ␣2-M-Br, ␣2-MCT). An important observation of this study is that all proteinases tested formed complexes with ␣2-M which mediated binding of A. However, significant differences with respect to the A-degrading activity of these complexes was found. In contrast to naturally occurring complexes such as ␣2-M-Pl and ␣2-M-PSA (24) which obviously do not degrade A, the complexes formed with trypsin, ␣-chymotrypsin, and bromelain were found to be proteolytically active. Inhibition studies revealed that the A-degrading activity of caged proteinases in contrast to noncomplexed proteinases cannot be abolished by inhibitors such as STI or ACT. That means that even in presence of high concentrations of specific inhibitors such as ␣1-proteinase inhibitor or ACT the caged proteinases are shielded from inactivation and can continue to degrade bound A. Furthermore, we supplied evidence that bound A due to the high-affinity constant between A and transformed ␣2-M of 10 ⫺10 M (11) is strongly protected from proteolysis by exogenous proteinases. This might be important as ␣2-M may aid the presentation of the amyloid peptide only to that enzymes being caged inside the inhibitor for further processing. It implies that the A-binding site on the surface of ␣2-M must be adjacent to the active site of the caged proteinase. Because most proteinases react with ␣2-M and only a few seem to be capable of proteolytically modifying A the specificity of this process might be of biological relevance. There are several possibilities how ␣2-M may influence the catabolism of A. At first it may promote A clearance. This can be achieved by nonproteolytically transformed ␣2-M created by activation through primary amines present in the brain parenchyma or by reaction with enzymes possessing no A-degrading activity. A cleared by this way may accumulate in lysosomes as it follows the endocytosis pathway of ␣2-MR/LRP. Thus, the activity of ␣2-M-R/LRP and the regulation of its expression in brain tissue seem to be important for that pathway (6, 26). In this sense, mutations in ␣2-M-R/LRP and ␣2-M which may impair the A clearance in brain might be predisposing factors of amyloidogenesis (2, 16). Another mechanism involves binding and site-directed proteolysis of A by ␣2-M–proteinase complexes. This may be an effective way to directly attenuate the A-induced toxicity in neuronal cells by lowering the concentration of soluble A in the extracellular space (25).
In early-onset AD cases A accumulation is caused by an increase in A anabolism due to presence of predisposing factors, e.g., mutations in presenilin 1 and 2 genes and amyloid precursor protein gene. However, in sporadic AD cases which lack significant overproduction of A an age-related reduction of A catabolism could be responsible for A accumulation in senile brain. Several recent studies stress that improper catabolism plays as large a role as A synthesis in amyloidogenesis. Degradation by the endopeptidase neprilysin of extracellular A, for instance, increased the catabolism of A in brain (15). On the other hand, inhibition of neprilysin activity by infused proteinase inhibitors led to pathological deposition of endogenous A in rat brain. Similarly, a membrane-bound insulindegrading enzyme was found to mediate degradation and clearance of A by neurons and microglia (27). These data suggest that defective degradation of A either by down-regulation of responsible proteinases or by up-regulation of proteinase inhibitors could be a risk factor for the development of AD disease in some subjects. Recently, plasmin was shown to degrade A1– 40 and, interestingly, the proteinase was capable of resolving even amyloid- aggregates (33). Contrarily, we found only small degradation of A by plasmin when compared to trypsin or ␣-chymotrypsin. This may be due to different experimental conditions such as enzyme/substrate ratios or level of activity of plasmin preparation used. Furthermore, plasmin may be less active toward A1– 42 as used in our study than toward A1– 40 used by others (33). In light of theses findings we have studied the mechanism of ␣2-M-mediated degradation of A. For the first time, we could show that ␣2-M-associated Adegrading complexes could be induced in a complex biological fluid such as blood by oral administration of proteinases. Trypsin and bromelain, the constituents of the proteinase cocktail, applied rapidly react with ␣2-M when appearing in blood despite the presence of other proteinase inhibitors (7). A similar mechanism is discussed for the proteinase PSA when liberated form the prostate into blood (23). High does of proteinases must be delivered to the intestine as enteric-coated formulation to allow crossing the mucosal barrier (17). The absorption of proteolytic enzymes from the gastrointestinal tract has been well documented in the past (1). It concerns endogenous proteolytic enzymes, e.g., pancreatic enzymes (13) and exogenous proteinases as well (35). Furthermore, proteinases have been successfully administered for treatment of diverse diseases (14). Although the precise mechanism of this unusual intestinal absorption phenomenon remains unclear, recent data favor absorption of proteolytic enzymes by a self-enhanced paracellular transport (3). However, transcytosis of proteinases as complex with intestinal ␣2-M should also be considered as the ␣2-M-R/LRP is
␣2-MACROGLOBULIN-MEDIATED CATABOLISM OF AMYLOID 
expressed in the intestinal epithelium (21) and ␣2-M is present in the intestinal lumen (5). We could clearly show that proteinase-conditioned plasma accelerated the degradation of A in blood. Would this be of potential interest with respect to amyloidogenesis in AD? Soluble A is produced by different cell types (20) and is present in measurable concentrations in cerebrospinal fluid and blood at 10 ⫺8–10 ⫺10 M (31). Although A concentration in circulation does not correlate with AD stages, a significant increase in A concentration was found in plasma of mutation carriers (8). The synthesis of APP outside the central nervous system and the deposition of A proteins in nonneuronal tissues suggest that AD might be a systemic disorder (20). Recent studies strongly indicate that soluble A1– 40 present in circulation can cross the blood– brain barrier via transcytosis mediated by brain capillary endothelial cells (19, 18). Similarly, A1– 42 is suggested to cross stereoisomer specifically the blood– brain barrier by receptor-mediated transport (24). The presence of cerebrovascular endothelial receptors for A is likely to influence the development of cerebrovascular amyloidosis and vascular injury. The latter may sustain additional macromolecules to pass the blood– brain barrier which normally could not, e.g., antibodies (29). Although it remains unproven, it is not unreasonable to expect that administration of proteinases may enhance A catabolism in blood as well as in nonneuronal and neuronal tissues which could be of clinical benefit. ACKNOWLEDGMENTS This work was supported by the Bundesministerium fu¨r Bildung, Forschung und Technologie (BMB⫹F) and the Interdisciplinary Center for Clinical Research at the University of Leipzig (01KS9504, Project C5).
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
REFERENCES 1.
2.
3.
4. 5.
6.
Ambrus, J. L., H. B. Lassman, and J. J. DeMarchi. 1967. Absorption of exogenous and endogenous proteolytic enzymes. Clin. Pharmacol. Ther. 8: 362–368. Blacker, D., M. A. Wilcox, N. M. Laird, L. Rodes, S. M. Horvath, R. C. Go, R. Perry, B. Watson, Jr., S. S. Bassett, M. G. McInnis, M. S. Albert, B. T. Hyman, and R. E. Tanzi. 1998. Alpha-2 macroglobulin is genetically associated with Alzheimer disease. Nature Genet. 19: 357–360. Bock, U., C. Kolac, G. Borchard, K. Koch, R. Fuchs, P. Streichhan, and C. M. Lehr. 1998. Transport of proteolytic enzymes across Caco-2 cell monolayers. Pharm. Res. 15: 1393–1400. Borth, W. 1992. ␣2-Macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J. 6: 3345–3352. Burgess, J. W., and K. K. Stanley. 1997. Estrogen-stimulated transcytosis of desialylated ligands and alpha2 macroglobulin in rat liver. Biochim. Biophys. Acta 1359: 48 –58. Businaro, R., C. Fabrizi, T. Persichini, G. Storace, M. G. Ennas, L. Fumagalli, and G. M. Lauro. 1997. Modulation of the alpha 2 macroglobulin receptor/low density lipoprotein receptor related
17.
18.
19.
20.
21.
391
protein by interferon-gamma in human astroglial cells. J. Neuroimmunol. 72: 75– 81. Castell, J. V., G. Friedrich, C. S. Kuhn, and G. E. Poppe. 1997. Intestinal absorption of undegraded proteins in men: Presence of bromelain in plasma after oral intake. Am. J. Physiol. 273: 139 –146. De Jonghe, C., P. Cras, H. Vanderstichele, M. Cruts, I. Vanderhoeven, I. Smouts, E. Vanmechelen, J. J. Martin, L. Hendriks, and C. Van Broeckhoven. 1999. Evidence that Abeta42 plasma levels in presenilin-1 mutation carriers do not allow for prediction of their clinical phenotype. Neurobiol. Dis. 6: 280 –287. Dickson, D. W. 1997. The pathogenesis of senile plaques. J. Neuropathol. Exp. Neurol. 56: 321–339. Du, Y., K. R. Bales, R. C. Dodel, X. Liu, M. A. Glinn, J. W. Horn, S. P. Little, and S. M. Paul. 1998. Alpha2-macroglobulin attenuates beta-amyloid peptide 1– 40 fibril formation and associated neurotoxicity of cultured fetal rat cortical neurons. J. Neurochem. 70: 1182–1188. Du, Y., B. Ni, M. Glinn, R. C. Dodel, K. R. Bales, Z. Zhang, P. A. Hyslop, and S. M. Paul. 1997. Alpha2-macroglobulin as a betaamyloid peptide-binding plasma protein. J. Neurochem. 69: 299 –305. Ganter, U., S. Strauss, U. Jonas, A. Weidemann, K. Beyreuther, B. Volk, M. Berger, and J. Bauer. Alpha 2-macroglobulin synthesis in interleukin-6-stimulated human neuronal (SH-SY5Y neuroblastoma) cells: Potential significance for the processing of Alzheimer beta-amyloid precursor protein. FEBS Lett. 282: 127–131. Go¨tze, H., and S. S. Rothman. 1975. Enterohepatic circulation of digestive enzymes as a conservation mechanism. Nature 257: 607– 609. Heidland, A., K. Sebekova, L. Paczek, M. Teschner, J. Dammrich, and Z. Gaciong. 1997. Renal fibrosis: Role of impaired proteolysis and potential therapeutic strategies. Kidney Int. Suppl. 62: 32–35. Iwata, N., S. Tsubuki, Y. Takaki, K. Watanabe, M. Sekiguchi, E. Hosoki, M. Kawashima-Morishima, H. J. Lee, E. Hama, Y. Sekine-Aizawa, and T. C. Saido. 2000. Identification of the major Abeta1– 42-degrading catabolic pathway in brain parenchyma: Suppression leads to biochemical and pathological deposition. Nature Med. 6: 143–150. Kang, D. E., T. Saitoh, X. Chen, Y. Xia, E. Masliah, L. A. Hansen, R. G. Thomas, L. J. Thal, and R. Katzman. 1997. Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), an apolipoprotein E receptor, with late-onset Alzheimer’s disease. Neurology 49: 56 – 61. Kolac, C., P. Streichhan, and C.-M. Lehr. 1996. Oral biovailability of proteolytic enzymes. Eur. J. Biopharmacol. 42: 222– 232. Mackic, J. B., M. Stins, J. G. McComb, M. Calero, J. Ghiso, K. S. Kim, S. D. Yan, D. Stern, A. M. Schmidt, B. Frangione, and B. V. Zlokovic. 1998. Human blood– brain barrier receptors for Alzheimer’s amyloid-beta 1– 40: Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer. J. Clin. Invest. 102: 734 –743. Martel, C. L, J. B. Mackic, J. G. McComb, J. Ghiso, and B. V. Zlokovic. 1996. Blood– brain barrier uptake of the 40 and 42 amino acid sequences of circulating Alzheimer’s amyloid beta in guinea pigs. Neurosci. Lett. 206: 157–160. Miklossy, J., K. Taddei, R. Martins, G. Escher, R. Kraftsik, O. Pillevuit, D. Lepori, and M. Campiche. 1999. Alzheimer disease: Curly fibers and tangles in organs other than brain. J. Neuropathol. Exp. Neurol. 58: 803– 814. Moestrup, S. K., J. Gliemann, and G. Pallesen. 1992. Distribution of the alpha 2-macroglobulin receptor/low density lipopro-
392
22.
23.
24.
25.
26.
27.
28.
LAUER, REICHENBACH, AND BIRKENMEIER tein receptor-related protein in human tissues. Cell. Tissue Res. 269: 375–382. Narita, M., D. M. Holtzman, A. L. Schwartz, and G. Bu. 1997. Alpha2-macroglobulin complexes with and mediates the endocytosis of beta-amyloid peptide via cell surface low-density lipoprotein receptor-related protein. Neurochemistry 69: 1904 – 1911. Otto, A., J. Ba¨r, and G. Birkenmeier. 1998. Prostate-specific antigen forms complexes with human alpha 2-macroglobulin and binds to the alpha 2-macroglobulin receptor/LDL receptorrelated protein. J. Urol. 159: 297–303. Poduslo, J. F., G. L. Curran, B. Sanyal, and D. J. Selkoe. 1999. Receptor-mediated transport of human amyloid beta-protein 1– 40 and 1– 42 at the blood– brain barrier. Neurobiol. Dis. 6: 190 –199. Qiu, W. Q., W. Borth, Z. Ye, C. Haass, D. B. Teplow, and D. J. Selkoe. 1996. Degradation of amyloid beta-protein by a serine protease-alpha2-macroglobulin complex. J. Biol. Chem. 271: 8443– 8451. Qiu, Z., D. K. Strickland, B. T. Hyman, and G. W. Rebeck. 1999. Alpha2-macroglobulin enhances the clearance of endogenous soluble beta-amyloid peptide via low-density lipoprotein receptor-related protein in cortical neurons. Neurochemistry 73: 1393–1398. Qiu, W. Q., D. M. Walsh, Z. Ye, K. Vekrellis, J. Zhang, M. B. Podlisny, M. R. Rosner, A. Safavi, L. B. Hersh, and D. J. Selkoe. 1998. Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J. Biol. Chem. 273: 32730 –32738. Rebeck, G. W., S. D. Harr, D. K. Strickland, and B. T. Hyman 1995. Multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein E receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein. Ann. Neurol. 37: 211–217.
29.
30. 31.
32.
33.
34.
35.
36.
Schenk, D., R. Barbour, W. Dunn, G. Gordon, H. Grajeda, T. Guido, K. Hu, J. Huang, K. Johnson-Wood, K. Khan, D. Kholodenko, M. Lee, Z. Liao, I. Lieberburg, R. Motter, L. Mutter, F. Soriano, G. Shopp, N. Vasquez, C. Vandevert, S. Walker, M. Wogulis, T. Yednock, D. Games, and P. Seubert. 1999. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400: 173–177. Selkoe, D. J. 1994. Alzheimer’s disease: A central role for amyloid. J. Neuropathol. Exp. Neurol. 53: 438 – 447. Seubert, P., C. Vigo-Pelfrey, F. Esch, M. Lee, H. Dovey, D. Davis, S. Sinha, M. Schlossmacher, J. Whaley, C. Swindlehurst, R. McCormack, R. Wolfert, D. Selkoe, I. Lieberburg, and D. Schenk. 1992. Isolation and quantification of soluble Alzheimer’s beta-peptide from biological fluids. Nature 359: 325–327. Sottrup-Jensen, L. 1989. Alpha2-macroglobulins: Structure, shape and mechanism of proteinase complex formation. J. Biol. Chem. 264: 11539 –11542. Tucker, H. M., M. Kihiko, J. N. Caldwell, S. Wright, T. Kawarabayashi, D. Price, D. Walker, S. Scheff, J. P. McGillis, R. E. Rydel, and S. Estus. 2000. The plasmin system is induced by and degrades amyloid-beta aggregates. J. Neurosci. 20: 3937–3946. Thal, D. R., R. Schober, and G. Birkenmeier. 1997. The subunits of alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein, native and transformed alpha2-macroglobulin and interleukin 6 in Alzheimer’s disease. Brain Res. 777: 223–227. Wrba, H., K. Kleine, K. Miehlke, F.-W. Dittmar, and R. E. Weissenbacher. 1996. Systemische Enzymtherapie-aktueller Stand und Fortschritte. MMV Medizin Verlag, Mu¨nchen. Zlokovic, B. V. 1997. Can blood– brain barrier play a role in the development of cerebral amyloidosis and Alzheimer’s disease pathology? Neurobiol. Dis. 4: 23–26.