©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Amyloid Peptide Produced in Vitro Is Degraded by Proteinases Released by Cultured Cells (*)

(Received for publication, June 30, 1994; and in revised form, November 10, 1994)

Asha Naidu Diana Quon Barbara Cordell (§)

From the From Scios Nova Inc., Mountain View, California 94043

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The primary histopathological feature of Alzheimer's disease is the accumulation of beta-amyloid in the brains of afflicted individuals. This peptide has been shown to be produced and liberated both in vitro and in vivo by normal physiological processes. The mechanism by which beta-amyloid is formed, as well as that by which it may be cleared, are events likely to impact on the development and progression of this disease. Thus, the fate of beta-amyloid peptides secreted by cultured mammalian cells was investigated. It was found that levels of the soluble peptide are reduced over time due to the activity of multiple types of proteinases including those from the metallo, aspartyl, and thiol classes. Inhibitors to each class of proteinase can only partially block beta-amyloid degradation, but, if used in combination, they can fully prevent its catabolism. The Kunitz serine proteinase inhibitor domain, present on two beta-amyloid precursor protein isoforms, was found to be an effective inhibitor of beta-amyloid peptide degradation. These data indicate that modulations in expression of secreted proteinases and/or beta-amyloid precursor isoforms may influence levels of beta-amyloid.


INTRODUCTION

Deposits of beta-amyloid peptide are characteristic of aging and Alzheimer's disease. The beta-amyloid peptide, at sufficiently high concentrations, has a propensity to aggregate into insoluble precipitates. This 39-43 amino acid peptide is derived from a larger precursor, the beta-amyloid precursor protein (beta-APP) (^1)which can be expressed in multiple isoforms. The two major isoforms are distinguished by the presence (751 amino acids) or absence (695 amino acids) of a serine proteinase inhibitor domain of the Kunitz family (KPI)(1, 2, 3, 4) . Alzheimer's disease-specific increases in the expression of KPI domain-bearing beta-APP isoforms have been observed in hippocampal and cortical neurons(5) , as well as in whole brain(6, 7, 8) . These data suggest that an imbalance in beta-APP isoform expression and/or an excess of KPI may be pathogenic.

Recently, it has been shown that beta-amyloid peptide is a naturally generated soluble protein. The peptide has been detected in the medium of a variety of cultured mammalian cells(9, 10, 11) , as well as in serum and cerebrospinal fluid(12) . The beta-amyloid peptide seems to be a minor degradation product of beta-APP catabolism, the production of which appears directly correlated with the expression of excess or aberrant beta-APPs(13) .

While the biology of beta-amyloid formation in vitro has been a focus of study, the fate of this peptide after it is secreted from the cell has not been examined. Both the synthesis and clearance of this protein may potentially contribute to increased levels of beta-amyloid and to the development of insoluble deposits. Therefore, to begin to understand the catabolic aspects of beta-amyloid biology we characterized the turnover of this protein produced in vitro.


EXPERIMENTAL PROCEDURES

Cell Culture

The CP-6 cell line was established by transfecting the human cDNA encoding the 695-amino acid isoform of beta-APP, driven by the beta-actin promoter, into Chinese hamster ovary fibroblasts using calcium phosphate according to standard procedures. Individual transfectants were isolated, subcloned, and characterized for beta-APP expression. CP-6 cells were propagated in a 1:1 mixture of Dulbecco's minimum essential medium and Coon's F12 medium with 10% fetal bovine serum in 10-cm dishes (1 times 10^6 cells/dish). The SK-N-MC cell line was obtained from the American Type Culture Collection (Rockville, MD) and was cultured on 10-cm dishes in Eagle's minimal essential medium with 10% fetal bovine serum. To prepare conditioned medium, cell monolayers were washed three times with saline and then placed in 2 ml of serum-free medium and incubated at 37 °C for either 4 or 24 h. After the conditioned medium was harvested, it was clarified by centrifugation at 1500 rpm for 5 min. To metabolically radiolabel protein with [S]methionine/cysteine (TranS-label, ICN), CP-6 cell monolayers were washed three times with saline, placed in serum-, methionine-, and cysteine-free medium for 30 min after which time 167 µCi/ml [S]methionine/cysteine was added. Culture dishes were radiolabeled for various times depending on the experimental design. For pulse-chase experiments, 10-cm dishes of CP-6 cell cultures (1 times 10^6 cells) were labeled for 60 min with 167 µCi/ml [S]methionine/cysteine in 2 ml of methionine/cysteine-free medium after which time the cell monolayers were washed, placed in complete serum-free medium lacking isotope, and incubated for varying lengths of time prior to harvesting. Clarified media were kept at 4 °C prior to analysis of the samples. Equivalent amounts of total radioactivity (2 ml of medium) were used for immunoprecipitation of beta-amyloid peptides.

Antibodies

Preparation of the BA1 and BA2 antisera used in the radioimmunoassay and for immunoprecipitation, respectively, was identical and has been previously described for BA2(13) . The binding epitopes for both antisera were characterized by radioimmunoassay. Epitopes recognized by each antiserum map to the carboxyl terminus of beta-amyloid 1-40 but to different sites.

Radioimmunoassay

Preparation and purification of I-beta-amyloid 1-40 tracer was as follows. Ten micrograms of synthetic beta-amyloid peptide 1-40 (Bachem, Torrance, CA) was radiolabeled with 1 mCi of I by the chloramine-T method. At the completion of the 1-min reaction, the reaction was diluted with an equal volume of H(2)O and passed through a Sepak C18 cartridge to separate the labeled peptide from unincorporated isotope. The I-labeled beta-amyloid peptide was found to elute from Sepak C18 in 50% CH(3)CN. The specific activity of the I-beta-amyloid peptide was calculated to be 5 times 10^7 cpm/µg. The peptide was further purified by reverse-phase high pressure liquid chromatography (HPLC) using a C8 cartridge (4.6 mm times 3 cm, Brownlee). The HPLC column was run in a linear gradient from 5-45% CH(3)CN in 0.1% trifluoroacetic acid in 30 min at a flow rate of 0.5 ml/min. Gradient fractions containing the labeled peptide were pooled and used within 3 days. The radioimmunoassay was carried out by the following procedure. The BA1 antiserum was made to a final dilution of 1/900, a dilution previously determined to bind 30% of I-beta-amyloid peptide in the absence of competing ligand. The dilution was made in 0.1 M sodium phosphate, pH 7.4, containing 0.1% bovine serum albumin and 0.1% Triton X-100. Labeled tracer (7000-9000 cpm) was used for each assay. Unknown sample or standard (beta-amyloid 1-40 synthetic peptide) was incubated with tracer and antibody overnight at 4 °C. The assay was terminated by the addition of 50 µl of normal rabbit serum followed by the addition of 800 µl of polyethylene glycol (M(r) = 6000-8000, 15.8% in the antibody dilution buffer). The samples were incubated for 10 min at 4 °C, and bound from free radiolabeled material was separated by low speed centrifugation. Pellets containing bound material were counted in a counter. Quantification of beta-amyloid protein in an unknown sample was made by comparison of the amount of bound material to a standard curve generated with dilutions of beta-amyloid 1-40 synthetic peptide. All determinations, unknowns and standards, were made in duplicate or triplicate. An IC of 1 nM for BA1 binding was obtained from the standard curve. Samples of conditioned medium (2-3 ml) required concentration and purification prior to assaying. To do so, the medium was bound and eluted from a Sepak C18 cartridge. Previous analyses determined that beta-amyloid protein and peptides elute from the Sepak C18 column in 50% CH(3)CN in 0.1% trifluoroacetic acid after washing with 5% CH(3)CN 0.1% trifluoroacetic acid and with 25% CH(3)CN, 0.1% trifluoroacetic acid. The 50% CH(3)CN eluate (2 ml) was dried and resuspended in 10% isopropyl alcohol in H(2)O to 250 µl. Synthetic beta-amyloid peptide standard was resuspended in 10% isopropyl alcohol and H(2)O. Aliquots of 50 µl were assayed in the radioimmunoassay.

Immunoprecipitation

Sample preparation, immunoprecipitation with BA2 antiserum, and analysis of the recovered products by 16.5% Tris/Tricine/polyacrylamide gel electrophoresis was performed as described by Zhong et al.(13) . Quantification of immunoprecipitated beta-amyloid peptides was made by densitometry of films using a Lynx 4000 (Applied Imaging Corp.) and by a PhosphorImager (Molecular Dynamics).

Proteinase Protection

Equal volumes (1.5 ml) of 4-h and 24-h conditioned medium were mixed and incubated for 25 h at 37 °C. As an experimental control, the media were mixed but not incubated. For another control, 4-h medium was mixed with serum-free medium never exposed to cells and incubated for 25 h at 37 °C. Some incubations were carried out in the presence or absence of proteinase inhibitors. Proteinase inhibitors were obtained from the following sources: both p-aminobenzamidine and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) from Sigma, phenylmethylsulfonyl fluoride from Dade (Cambridge, MA), and aprotinin, leupeptin, antipain, pepstatin, E64, and phosphoramidon from Boehringer Mannheim. The 57-amino acid KPI domain, generated by recombinant methods, was purified as described previously(14) . For boiling experiments, the 24-h medium was incubated at 100 °C for 40 min, cooled, mixed with 4-h conditioned medium, and incubated as described above. Potential effects of buffers and organic solutions used to solubilize several inhibitors were examined in the protection assay and were found to be negligible at the concentrations employed.


RESULTS

A clonal line of cultured Chinese hamster ovary fibroblasts which was stably transfected and expresses the human 695-amino acid isoform of beta-APP (termed CP-6) was used as a source of beta-amyloid protein. Based on recent reports, cultured mammalian cells have been demonstrated to produce and secrete beta-amyloid peptides which include an intact 4-kDa beta-amyloid peptide and an amino-terminally truncated 3-kDa peptide derived from the secretory cleavage of beta-APP(5, 6, 7, 15) . The single methionine residue present in the carboxyl-terminal domain of the beta-amyloid peptide permits metabolic labeling with [S]methionine of both the 3- and 4-kDa beta-amyloid peptides.

To examine the temporal profile of beta-amyloid expression, we employed a pulse-chase experimental format. Multiple cultures of CP-6 cells were metabolically radiolabeled for 1 h by addition of [S]methionine/cysteine to the medium. Following the pulse period, the isotope-containing medium was removed, and the cell cultures were replenished with fresh medium lacking isotope and incubated. At 2, 4, 6, 8, 10, and 12 h post-pulse, the conditioned medium was harvested. The fate of [S]methionine-labeled beta-amyloid peptides secreted into the culture medium over the 12-h incubation period was followed by immunoprecipitation with a beta-amyloid-specific antiserum and electrophoresis on a 16.5% Tris/Tricine/polyacrylamide gel. Fig. 1displays the results from the pulse-chase experiment. Maximum accumulation of both the 4- and 3-kDa beta-amyloid peptides was observed 6 h into the chase which remained high for 2 h then declined. By 12 h after pulse-labeling, modest amounts of the beta-amyloid peptides remained. When the chase period was extended to include a 24-h time point, little beta-amyloid was detected (data not shown). It should be noted that CP-6 cells were viable and metabolically active during the 24-h serum-free treatment, and, therefore, this was not a factor in the observed reduction of beta-amyloid peptides. In fact, levels of several labeled cell-associated proteins remained unchanged over the 24-h chase period indicating general cellular proteolysis mediated by serum deprivation was not responsible for the observed decrease in beta-amyloid peptides. The time to eliminate the bulk of [S]methionine-labeled beta-amyloid peptides from the serum-free medium after maximum levels are reached was determined using densitometry and found to be >12 h. Similar results were obtained for identical experiments performed with CP-6 cells of different passage.


Figure 1: beta-Amyloid peptide turnover in cell-conditioned medium. Multiple dishes of CP-6 cells were pulse-labeled with [S]methionine/cysteine for 1 h after which label was removed and isotope-free medium was added. At 2, 4, 6, 8, 10, and 12 h into the chase period, medium was harvested and analyzed for [S]methionine-labeled beta-amyloid using immunoprecipitation, polyacrylamide gel electrophoresis (PAGE), and autoradiography.



The secretion and elimination of the beta-amyloid peptides by CP-6 cells was analyzed using a different assay, a radioimmunoassay. This radioimmunoassay employs a second beta-amyloid-specific antiserum from that used for immunoprecipitation. Each serum recognizes a different epitope within the carboxyl-terminal region of the beta-amyloid sequence. Consequently, both the 4- and 3-kDa peptides are scored in both assays. The secretion of soluble beta-amyloid peptides into serum-free culture medium was followed utilizing the radioimmunoassay. Identical with the results obtained by immunoprecipitation, the radioimmunoassay revealed peak levels of peptide 6 h into the time course followed by a substantial decrease in levels by 24 h (Fig. 2). Some variation from experiment to experiment was noticed, however, in the beta-amyloid peptide level at 24 h. Generally, 10-30% of maximum levels were present at 24 h; however, in one experiment, as much as 50% remained. Approximately 2.5 ng/ml beta-amyloid protein was present in the conditioned medium at peak times (6 h) which is roughly equivalent to the level described that was released from a human brain primary cell culture(12) . Similar to a previous report(7) , the absolute level of beta-amyloid protein, however, fluctuated with cell type. Levels also varied slightly with cell passage.


Figure 2: Quantitation of beta-amyloid peptide turnover in cell-conditioned medium. beta-Amyloid in CP-6 cultured cell media which were prepared in Fig. 1was quantitated by film densitometry and phosphorimaging (open circles). An identical time course experiment was performed as in Fig. 1except that radioisotope was omitted and media samples were scored for beta-amyloid using a radioimmunoassay (filled diamonds).



These experiments, assayed by either immunoprecipitation or radioimmunoassay, indicated that the secreted beta-amyloid peptides were depleted from the culture medium over time. To determine whether the peptides were removed due to cellular uptake or to proteolytic degradation, we investigated the potential catabolic effects of conditioned medium on beta-amyloid peptides in the absence of cells by performing mixing experiments. Medium which had been conditioned by CP-6 cells for 24 h was mixed with either unlabeled or [S]methionine/cysteine-labeled serum-free medium harvested 4 h after addition to the cell monolayer. 4-h conditioned medium represents a time when levels of soluble beta-amyloid peptide were high. The combined 24- and 4-h media (1:1 volume) were incubated at 37 °C, in the presence or absence of proteinase inhibitors, after which the mixed media were assayed for beta-amyloid protein. As a control treatment, 4-h conditioned medium was mixed (1:1 volume) with medium never exposed to cells. By performing a time course for the 37 °C treatment, it was determined that 25 h was a desirable incubation period (data not shown) and was, consequently, used for all experiments.

The data presented in Fig. 3are a compilation of multiple mixing experiments using unlabeled media followed by radioimmunoassay scoring of beta-amyloid protein. When 4-h CP-6 cell-conditioned medium was combined with control medium and incubated at 37 °C for 0 or for 25 h, the level of beta-amyloid protein was unchanged and remained at 3 ng/ml. In contrast, when the 4-h medium was incubated with 24-h conditioned medium, the amount of beta-amyloid protein was reduced by greater than 50% after the 25-h incubation. No depletion was observed with 4- and 24-h media which were mixed but not incubated. An increase of beta-amyloid protein (1 ng/ml) was frequently measured for the unincubated 4- plus 24-h sample over the level present in only 4-h medium. It is probable that this increase was derived from residual beta-amyloid peptides present in the 24-h medium which, when mixed with the 4-h medium, resulted in slightly higher initial amounts. Significantly, a reduction in beta-amyloid protein after incubation with 24-h conditioned medium was prevented by the addition of a collection of proteinase inhibitors. The mixture of inhibitors (mix-C) used and the concentration of each is summarized in Table 1. Since protection of beta-amyloid peptides was seen in the presence of proteinase inhibitors, these data suggest that proteolytic enzymes were involved in the observed reduction of this protein. It should be noted that on occasion some samples of 4-h CP-6 conditioned media showed slight beta-amyloid peptide catabolic activity; however, most preparations lacked this effect. This suggests that the degrading proteolytic enzymes were not released or sufficiently accumulated in the medium until after 4 h.


Figure 3: Degradation of beta-amyloid peptides by cell-conditioned medium and protection by proteinase inhibitors. Medium conditioned by CP-6 cells for 4 h was incubated at 37 °C with control medium (EMEM) or medium conditioned by CP-6 cells for 24 h, either in the presence or absence of proteinase inhibitor mix-C (PI mix) (refer to Table 1). Incubations were for 0 h (thin-lined columns) or for 25 h (large line cross-hatched columns) after which beta-amyloid was measured by radioimmunoassay.





The above result was confirmed and extended in experiments using 4-h conditioned media containing [S]methionine-labeled beta-amyloid peptides and an immunoprecipitation assay (Fig. 4). Again, the beta-amyloid peptides were depleted after incubation at 37 °C for 25 h with 24-h CP-6 cell-conditioned medium but, if incubated in the presence of the proteinase inhibitor mixture, protection against degradation was observed. Both the 4- and 3-kDa peptides were degraded in the absence of proteinase inhibitors. No significant reduction in the [S]methionine-labeled peptides was seen when the sample was identically incubated with control medium.


Figure 4: Degradation of [S]methionine-labeled beta-amyloid peptides by cell-conditioned medium and protection by proteinase inhibitors. [S]Methionine-labeled beta-amyloid peptides present in 4-h CP-6 cell-conditioned medium (CM) was incubated at 37 °C for 25 h with control medium (control CM) or with 24-h unlabeled CP-6 cell-conditioned medium (24hr CM), either in the presence or absence of proteinase inhibitor mix-C (+PI mix-C) (refer to Table 1). After incubation, beta-amyloid protein remaining in media samples was analyzed by immunoprecipitation, PAGE, and autoradiography.



The proteolytic activity released by CP-6 fibroblasts which degrades the beta-amyloid peptides was found to be present in media conditioned by other cell types. Fig. 5illustrates this point in which 24-h conditioned medium was prepared from a human neuroblastoma cell line, SK-N-MC, and tested. The SK-N-MC conditioned medium was mixed with 4-h CP-6 medium, incubated at 37 °C for 25 h in either the presence or absence of proteinase inhibitors described in Table 1(mix-C). The SK-N-MC medium promoted a 50% reduction in beta-amyloid peptides which was prevented by the addition of the collection of proteinase inhibitors.


Figure 5: Degradation of beta-amyloid peptides by SK-N-MC cell-conditioned medium. Medium conditioned for 4 h by CP-6 cells was incubated at 37 °C for 0 h (darker columns) or for 25 h (light cross-hatched columns) with medium conditioned for 24 h by SN-N-MC neuroblastoma cells either in the presence (PI Mix) or absence of proteinase inhibitors described in Table 1under mix-C. The beta-amyloid present after incubation was measured by radioimmunoassay.



To identify the type of proteinase(s) responsible for the clearance of the beta-amyloid peptides, we examined the inhibitory effects of individual proteinase inhibitors present in the mixture. This inhibitor mixture consisted of a number of commonly used proteinase inhibitors covering the four major classes of proteinases; serine, metallo, aspartyl, and thiol. A standard concentration of each inhibitor was used for the mixture. When each inhibitor was individually evaluated for its ability to block beta-amyloid peptide degradation using analysis by radioimmunoassay, none was effective (data not shown). This result suggested that multiple proteinases may be involved in the degradation of these proteins. Therefore, we studied the effects of inhibitor combinations. These combinations were based on class or classes of enzyme target. The inhibitor combinations tested are outlined in Table 1. We repeatedly found that a mixture of leupeptin and pepstatin partially protected the breakdown of beta-amyloid and that the addition of EDTA and phosphoramidon to this mixture fully protected the beta-amyloid peptides ( Fig. 6and Table 2). Other proteinase inhibitor combinations proved ineffective ( Fig. 6and data not shown). To provide further evidence that enzymes are responsible for the protein depletion, we characterized the effect of boiled 24 h conditioned CP-6 medium on the catabolism of the beta-amyloid peptides. No degradation was seen when 4-h medium was incubated at 37 °C for 25 h with boiled 24-h medium (Table 2).


Figure 6: Protection of beta-amyloid peptide degradation by select proteinase inhibitors. CP-6 cell medium conditioned for 4 h (4hr) was incubated at 37 °C for 0 h or for 25 h with control medium (EMEM) or with CP-6 cell medium conditioned for 24 h (24hr) to which combinations of proteinase inhibitors (PI mix) were added. Refer to Table 2for composition of individual inhibitor mixes. beta-Amyloid protein remaining in each sample was measured by radioimmunoassay. Values are expressed as the percentage of beta-amyloid protein present in each sample after 25 h of incubation compared to no incubation or ``0 hr'' incubation.





The inhibitory effects seen with leupeptin, pepstatin, EDTA, and phosphoramidon indicated that thiol, aspartyl, and metalloproteinases together comprise the beta-amyloid peptide degrading activity. Since leupeptin can also inhibit select serine proteinases, in addition to thiol proteinases, we were interested in determining whether the KPI domain, a serine proteinase inhibitor, harbored within the 751- and 770-amino acid isoforms of beta-APP could block the breakdown of beta-amyloid peptides. We have previously described the recombinant production, purification, and activity of the 57-amino acid KPI domain derived from beta-APP(14, 16) . A mixing experiment was performed in which [S]methionine-labeled beta-amyloid peptides present in 4-h conditioned CP-6 medium were incubated with 24-h conditioned medium in the absence or presence of 50 µM KPI. After the standard incubation, the radiolabeled beta-amyloid peptides were immunoprecipitated and analyzed by polyacrylamide gel electrophoresis (Fig. 7). Partial protection (quantified at 50%) was found upon addition of the beta-APP Kunitz inhibitor domain to the mixed media. In the absence of KPI inhibitor, the 3- and 4-kDa beta-amyloid peptides were completely eliminated.


Figure 7: Partial protection of beta-amyloid peptide degradation by beta-APP KPI domain. [S]Methionine-labeled beta-amyloid peptides present in 4-h medium conditioned and labeled by CP-6 cells was mixed with control medium (control CM) or with unlabeled medium conditioned by CP-6 cells for 24 h (24hr CM). Replica samples of the 4-h plus 24-h mixture were incubated in the presence (24hr CM + KPI) or absence (24hr CM) of 50 µM KPI domain. Following incubation of all samples at 37 °C for 25 h, beta-amyloid protein was analyzed by immunoprecipitation, PAGE, and autoradiography.




DISCUSSION

Accumulation of beta-amyloid peptide appears to have a central role in the pathogenesis of Alzheimer's disease. Substantial evidence, both direct and indirect, implicate this protein as a major contributing factor in the initiation and progression of this disorder (reviewed in (17) and (18) ). Several properties of the beta-amyloid peptide are of particular relevance to Alzheimer's disease, notably the ability to self-polymerize (reviewed in (19) and (20) ) and to induce neurotoxicity (21; reviewed in (20) and (22) ). These two properties are also inter-related in that beta-amyloid neurotoxicity is directly correlated with the aggregation state of the peptide(23, 24) . It has been shown that beta-amyloid peptide is generated as part of normal cellular physiology (9, 10, 11, 12) and presumably as a result of beta-APP catabolism(13) . As part of this cellular process, the beta-amyloid peptide is secreted from the cell as a soluble protein. In addition to the extracellular soluble nature of beta-amyloid peptide, the local concentration of this protein in the microenvironment is likely to be an important factor in eliciting neuronal degeneration and plaque formation.

Because increases in soluble beta-amyloid peptide are likely to enhance its propensity for self-polymerization, it is important to understand the fate of beta-amyloid peptide after it is released from the cell. Heretofore, studies have primarily focused on the processes of beta-amyloid peptide formation and not on its clearance. Therefore, to begin to understand the overall biology of beta-amyloid peptide, we investigated the turnover profile of this protein produced in vitro.

Our studies, using cultured mammalian cells as a source of beta-amyloid peptide, indicate that this protein is degraded within 12 to 24 h of its release from the cell. Two different methods and immunoreagents were employed to measure beta-amyloid peptide, a radioimmunoassay and an immunoprecipitation assay. The data obtained from both assays were in complete agreement. Catabolism of both the 4-kDa beta-amyloid peptide and the 3-kDa truncated form of this protein was found to be due to proteolytic activity released by the cultured cells. Chinese hamster ovary fibroblastic and human SK-N-MC neuroblastoma cell lines were characterized as having proteolytic activities which degrade the beta-amyloid peptides. Serum-free medium which had been conditioned by the cultured mammalian cells was the source of the beta-amyloid degrading activity employed in these studies. Therefore, the elimination of beta-amyloid was not due to reinternalization by the cell. Cellular uptake of exogenously supplied beta-amyloid peptide has been described(25) .

The beta-amyloid catabolic activity was shown to be of enzymatic nature in that it could be fully inhibited by heat denaturation or by the addition of proteinase inhibitors. Several classes of proteinases were identified as being responsible for the breakdown of the beta-amyloid peptide based on the types of inhibitors found to be effective in preventing degradation. Of the active families of proteinases secreted by the cells studied, thiol, aspartyl, and metalloproteinase members appeared responsible for beta-amyloid catabolism. Select inhibitors of each class were only partially able to prevent beta-amyloid degradation. Only when these multiple types of proteinase inhibitors are used collectively could beta-amyloid catabolism be fully blocked within the selected incubation period. In addition to the above mentioned proteinases, it appeared that a serine proteinase(s) also contributed to beta-amyloid turnover since the KPI domain of beta-APP could partially block this process. However, this KPI-responsive activity appears unique in that other serine proteinase inhibitors such as aprotinin, TLCK, and benzamidine were incapable of blocking beta-amyloid peptide degradation. Likewise, one thiol proteinase inhibitor was ineffective in protecting beta-amyloid clearance, whereas another inhibitor of this class was effective.

As mentioned, the serine proteinase inhibitor domain, KPI, contained in some beta-APP isoforms was tested and was found to partially protect (50%) the degradation of beta-amyloid. That the KPI domain of beta-APP can partially inhibit the degradation of beta-amyloid peptide in vitro may hold relevance to the increase in KPI containing beta-APP isoforms specifically observed in Alzheimer's disease brain tissue(5, 6, 7, 8) . Furthermore, in one study, the increase in beta-APP isoforms containing the KPI domain was directly correlated with the density of plaques(5) . Since beta-APP has been shown to be secreted from the cell both in vitro(26) and in vivo(27) , such an increase in expression of KPI bearing beta-APP isoforms in the disease state might lead to increased levels of secreted KPI beta-APPs. Higher levels of soluble KPI-containing beta-APP might retard the clearance of beta-amyloid peptide and, in turn, contribute to increased local concentrations of beta-amyloid and to the development of insoluble aggregates. It may be relevant that secreted beta-APP has been reported to harbor a metalloproteinase inhibitor domain (28) which might also serve to retard beta-amyloid catabolism and promote deposit formation.

Many factors might influence the normal clearance rate of beta-amyloid protein in addition to the possible contribution of increased expression of beta-APP and its resident serine and metalloproteinase inhibitory activities. The binding of beta-amyloid to lipoproteins apoE (29) demonstrated with in vitro studies might sequester this protein, thereby arresting or retarding its degradation. In addition, it is possible that aberrant expression or bioactivity of any of the beta-amyloid degrading proteinases might have a pathological contribution by elevating levels of this protein. Hence, knowledge of the beta-amyloid degrading proteinases may be important to our complete understanding of beta-amyloidogenesis and Alzheimer's disease.


FOOTNOTES

*
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 and reprint requests should be addressed: Scios Nova, Inc., 2450 Bayshore Pkwy., Mountain View, CA 94043. Tel.: 415-940-6674; Fax: 415-968-2438.

(^1)
The abbreviations used are: beta-APP, beta-amyloid precursor protein; KPI, serine proteinase inhibitor domain of the Kunitz family; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We gratefully acknowledge Marion Merrell Dow, Inc. for sponsoring this research.


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