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Cell-free assays for -secretase activity¹

Mayo Clinic Jacksonville, Department of Pharmacology, Jacksonville, Florida 32224, USA; and
^* The Rockefeller University, Laboratory for Mass Spectrometry, New York, NY 10021, USA

²Correspondence: Mayo Clinic Jacksonville, Department of Pharmacology, 4500 San Pablo Rd., Jacksonville, FL 32224, USA. E-mail: golde.todd{at}mayo.edu

SPECIFIC AIMS

In this study we have explored the development of in vitro assays for the -secretase activity that cleaves the amyloid ß protein (Aß) from the amyloid protein precursor (APP).

PRINCIPAL FINDINGS

1. Aß and -CTF can be produced from membrane preparations containing APP CTF
Initial in vitro experiments focused on detecting generation of Aß by ELISA from membrane preparations containing the APP CTF (C99) that is produced after cleavage of the APP by ß-secretase. For these experiments, we isolated total membranes from a Chinese hamster ovary (CHO) line stably overexpressing APP695NL,I-his, an amino-terminal polyhistidine-tagged 695 amino acid isoform of APP containing both the familial Alzheimer’s disease (AD) -linked ‘Swedish’ mutation and the ‘London’ mutation. Whereas Aß40 production could be detected by ELISA in both the membrane and supernatant fractions from membrane preparations resuspended in 150 mM Na citrate (pH 6.4) and incubated at 37°C for 2–4 h, the Aß40 levels were low (<50 fmol Aß40/mg protein) and the results inconsistent, with many experiments failing to reveal detectable Aß. We suspected that several factors were contributing both to the lack of reproducibility of the assay and the low levels of Aß produced, including limiting amount of substrate and possible degradation of the Aß produced or instability of -secretase activity. Therefore, we analyzed the effects of 1) including a protease inhibitor cocktail (PI, 1X Complete, Roche, Nutley, N.J.) in the resuspension buffer and 2) increasing the substrate APP CTF by pretreating the cells prior to nitrogen cavitation for 16 h with 50 µM cbz-I-L-CHO (z-IL-cho), a moderately potent reversible -secretase inhibitor. Pretreatment of cells with z-IL-cho markedly increased the amount of Aß that could be detected, increasing the amount of Aß after a 2 h incubation by greater than threefold. Inclusion of the PI cocktail in the incubation buffer was also important. Omitting PI resulted in a threefold decrease in Aß in z-IL-cho pretreated samples. In samples not pretreated with z-IL-cho, Aß could not be detected, indicating that either the proteins responsible for -secretase activity are susceptible to proteolysis, the Aß produced was susceptible to degradation by proteases, or both. No detectable Aß was released from untransfected CHO cells. When the resuspended membranes from these experiments were examined by Western blotting, it was clear that pretreatment with z-IL-cho greatly increases the amount of -substrate present (both C99 and the smaller APPCTF, C83, that is generated after -secretase cleavage of APP) and that inclusion of PI in the incubation buffer did not appear to alter CTF stability. Furthermore, upon longer exposure, a single, 6 kDa CT20 and Pf998 immunoreactive band is detected in those samples pretreated and incubated at 37°C. This band is the predicted size of the -CTF produced by cleavage of either C99 or C83 by -secretase and comigrates with a synthetic -CTF. These data demonstrate that both Aß 40 and Aß42 can be generated de novo and released from membranes containing APP CTF in vitro, and that during Aß production a cognate CTF likely to be the -CTF is also produced.

2. Solubilization of the -secretase activity
Using membranes isolated from CHO APP695NL,I-his cells pretreated with 50 µM cbz-IL-CHO prior to disruption, we evaluated the effects of a number of detergents on Aß production by adding these detergents at concentrations above their CMC to the resuspended membrane preparations. Two detergents, Tween 80 and BRIJ 35, enhanced Aß production four- to fivefold. Subsequent experiments focused on optimization of activity in the presence BRIJ 35, and after a number of experiments it was found that incubation of the sample with 2% BRIJ 35 in 150 mM NaCl on ice for 30 minutes reliably solubilized the activity as assessed by the amount of total Aß per mg of protein generated and the lack of -secretase activity in the BRIJ 35 insoluble pellet. Once we found that we could solubilize activity in BRIJ 35, we explored whether -secretase assays could be performed simply in 2% BRIJ 35 cell extracts. For these experiments, cells pretreated with z-IL-cho were washed 3x in ice-cold phosphate-buffered saline, then lysed directly in 150 mM Na citrate (pH 6.4), 2% BRIJ 35 with PI. Aß and the putative -CTF were produced in total cell extracts incubated for 1–4 h at 37°C (Fig. 1A , B ). As in the experiments on resuspended membranes, sufficient Aß was produced to detect Aß42, and the relative amount of Aß42 produced (10–20%) was similar to the ratio of Aß secreted by cells transfected with this construct. Significantly, both the production of Aß and the production of the -CTF could be blocked by inclusion of the -secretase inhibitor 50 µM z-IL-cho in the incubation buffer (Fig. 1A , B ).

View larger version (32K): [in this window] [in a new window]   Figure 1. Solubilization of -secretase activity. A) 2% BRIJ 35 lysates of whole cells were prepared as described from CHOAPP695NL,I-his cells pretreated with z-IL-cho. Aß40 and Aß42 produced during incubation at 37°C were assayed by ELISAs. Addition of the -secretase inhibitor z-IL-cho decreases production markedly (+z-IL-cho). No Aß42 could be detected in samples treated with the inhibitor. B) Western blot analysis. 10 µg of protein from the BRIJ 35 lysates was separated on a 16% Tris-Tricine gel. The top panel shows a 1 s exposure, the lower panel a 20 s exposure. Detection of the APP CTF was with CT20, which recognizes an epitope within the last 20 amino acids of APP. 50 µM z-IL-cho markedly inhibits the time-dependent production of the -CTF seen upon longer exposure. Similar results are seen with Pf998, an antibody raised against the juxtamembranous region of the cytoplasmic tail of APP. C) Mass spectral analysis of Aß produced from BRIJ 35 solubilized membrane preparations. Aß1–34, 1–38, 1–40, and 1–42 can be detected in the sample incubated at 37°C. The peak at 5733.6 m/z is from insulin. D) -Secretase activity is present in CEMs. -Secretase activity was assessed in various fractions of CHO-APP695NL,I-his cells pretreated with z-IL-cho. A minus indicates that the fractions were resuspended in 150 mM Na citrate pH 6.4 +PI; a plus sign indicates the fractions were solublized in 2% BRIJ. S1 is the postnuclear supernatant. TM indicates total membranes and CEM are carbonate-extracted membranes. Aß40 and Aß42 were analyzed using 3160/BA27 and 3160/BC05 ELISA, respectively. E) Effect of pH on activity. The effect of pH was examined on Aß40 production from BRIJ 35 solubilized CHO-APP695NL,I-his whole cell lysates. CHO-APP695NL,I-his pretreated with z-IL-cho were resuspended in 2% BRIJ 35, 150 mM Na citrate buffers at the pHs indicated + PI. Samples were divided in half: one half incubated on ice and the other incubated at 37°C for 2 h. Prior to Aß detection, the samples were neutralized to pH 7.0. Aß40 and Aß42 were detected using 3160/BA27 and 3160/BC05 ELISAs, respectively.

3. Aß and P3 are produced in vitro
Because our ELISA systems detect full-length or nearly full-length Aß peptides and not the smaller P3 fragments generated by -secretase cleavage of the C83, we examined whether P3 was generated. We metabolically labeled CHO APP695NL+I-his cells overnight in the presence of the IL inhibitor and then immunoprecipitated Aß from BRIJ 35 lysates with 4G8, an antibody that recognizes an epitope from Aß17–24 and thus is capable of detecting both Aß and P3. Both a 4 kDa Aß and a 3 kDa band were detected in the immunoprecipitates from samples incubated at 37°C, but not from samples incubated at 4°C. These bands comigrated with the 4 kDa Aß and 3 kDa P3 immunoprecipitated from the conditioned media of metabolically labeled cells. Attempts to detect the P3 produced in vitro by immunoprecipitation mass spectrometry (IP/MS) analysis using 4G8 as the immunoprecipitating antibody were unsuccessful; however, IP/MS analysis did confirm production of authentic Aß1–40, Aß1–42 and two other Aß peptides, Aß1–34 and Aß1–38 (Fig. 1C ). No Aß peptides were detected by IP/MS in the sample incubated at 4°C.

4. Peptide based -secretase inhibitors are effective in cell-free systems, further evidence for distinct -40 and -42 secretase activities
In previous studies on cultured cells, our group and others have shown that pepstatin and both peptide aldehyde based and difluoroketone based -secretase inhibitors are often more potent inhibitors of Aß40 production than Aß42 production (i.e., selective -40 inhibitors). We therefore performed detailed dose response studies with several of our peptide aldehyde and epoxide based -secretase inhibitors, as well as pepstatin, to determine if they showed selectivity in vitro. The in vitro studies were carried out in BRIJ 35 solubilized membrane preparations isolated from cells pretreated with 50 µM z-IL-cho and incubated at 37°C for 2 h. Corresponding doses of inhibitors were also used in cell culture studies on CHO 2b-7 cells that stably overexpress APP695. In these studies, in addition to the Complete PI cocktail, 25 µM phosphoramidon was included in the incubation buffer to further eliminate any ongoing proteolysis of newly generated Aß. IC₅₀s for the -40 and -42 activities are listed in Table 1 . The in vitro analysis demonstrated that pepstatin is actually a reasonably potent -secretase inhibitor and is marginally selective for -40. Our more selective -40 inhibitors in cultured cells, z-C(tBu)IL-cho and boc-K(Dnp)IL-epoxide, also show selectivity for -40 inhibition in vitro, while a less selective inhibitor in cultured cells, z-IL-cho, exhibited the least amount of selectivity in vitro. Significantly, at concentrations of inhibitor slightly above the IC₅₀ for -40, the selective effect of these inhibitors in vitro is most pronounced. For example, Aß40 production is inhibited 69% and Aß42 production only 25% in the presence of 0.5 µM z-C(tBu)IL-cho, and 1 µM boc-K(Dnp)IL-epoxide does not inhibit Aß42 production but inhibits Aß40 production by 32%. In cultured cell studies the selective effects of treatment with 6.25 µM boc-K(Dnp)IL-epoxide are extremely marked; Aß40 production is inhibited by 52% but Aß42 is increased by 80%. At this concentration, boc-K(Dnp)IL-epoxide causes a marked accumulation of APP CTF; thus, the increase in Aß42 is likely the result of combined effects of increasing substrate due to inhibition of Aß40 production and lack of inhibition of the activity that generates Aß42.

View this table: [in this window] [in a new window]   Table 1. IC50s of -secretase inhibitorsa

5. -Secretase is an integral membrane protease with maximal activity at pH 6.8
Sodium carbonate extraction of total membranes solubilizes both peripheral membrane proteins and membrane-enclosed contents; thus, membranes extracted with sodium carbonate contain only integral membrane proteins or proteins very tightly bound to an integral membrane protein. Therefore, to further characterize -secretase activity we examined the effect of sodium carbonate extraction (0.1M, pH 11.0) on -secretase activity. Significant -secretase activity as assessed by Aß production could be detected in the BRIJ 35 solubilized carbonate extracted membranes (Fig. 1D ). As expected, APP CTF and PS1 and PS2 were present in these CEMs. We also examined the activity of -secretase at pHs varying from 5.6 to 8.4. -Secretase activity could be detected from pH 6.0 to pH 8.4, with highest levels of both Aß40 and Aß42 being produced at pH 6.8 (Fig. 1E ). Aß40 and Aß42 production are proportional at pH 7.6, but above pH 8.0 Aß42 production increases slightly whereas Aß40 production decreases.

DISCUSSION

We have developed a solubilized in vitro assay for -secretase catalyzed cleavages of APP CTF. By showing de novo production of Aß1–40, Aß1–42, a putative P3 derivative, and -CTF from C99 and C83 as well as inhibition of production of these -secretase cleavage products by known -secretase inhibitors, we demonstrated that we are monitoring the same -secretase activity that generates Aß in living cells. Using this assay, we also showed that -secretase is present in CEMs and therefore is likely to be an integral membrane protease or proteases. Moreover, our finding that selective -secretase inhibitors that preferentially inhibit Aß40 production in cultured cells also preferentially inhibit Aß40 production in vitro essentially rules out the possibility that these inhibitors selectively inhibit one activity because of differential organelle penetrance and strongly supports the notion that there are indeed at least two pharmacologically distinct -secretase activities: a -40 activity that generates Aß40 and smaller Aß peptides (e.g., Aß 38), and a -42 activity that generates Aß42 (Fig. 2 ). Because some inhibitors show little or no preference for the -40 and -42 activities and because all -40 selective inhibitors do inhibit -42 activity at higher concentrations, it is likely that the differential activities can be attributed either to the action of 1) two closely related proteases or 2) a single protease with two distinct active conformations.

View larger version (38K): [in this window] [in a new window]   Figure 2. Two pharmacologically distinct -secretase activities generate the Aß from APP CTF. Generation of Aß from APP requires two proteolytic events: a proteolytic cleavage at the amino terminus of the Aß sequence referred to as ß-secretase and a cleavage at the carboxyl terminus known as -secretase. Based on accumulating evidence that the -secretase activities that generate Aß40 and Aß42 are pharmacologically distinguishable, it is likely that -secretase activity is catalyzed by either 1) two closely related proteases or 2) a single protease with two distinct active conformations. Given the recent evidence indicating that PSs are the -secretases and that both PS1 and PS2 have -40 and -42 activities, it is likely that PSs assume distinct conformations within a larger high molecular weight -secretase complex and that this altered conformation accounts for this differential activity.

The studies described confirm the notion that -secretase has an atypical proteolytic activity, as its activity is not inhibited by classical serine, cysteine, and metalloprotease inhibitors (EDTA). With regard to the hypothesis that PSs are novel di-aspartyl -secretases, these data can be viewed as either neutral or supportive. The data demonstrating that pepstatin inhibits -secretase activity with an IC₅₀ of 1–2 µM clearly support the idea that -secretase is an aspartyl protease; however, it should not be viewed as definitive, as other proteolytic activities have been shown to be sensitive to pepstatin (albeit at fairly high concentrations). Furthermore, peptide aldehyde and epoxide protease inhibitors have been shown to inhibit serine, cysteine, and aspartyl proteases as well as atypical proteases such as the proteasome. As some aspartyl proteases are active even at neutral or slightly alkaline pH (e.g., renin, SIV), our data showing that -secretase is active over a broad pH range, with a peak activity at pH 6.8, are not inconsistent with the hypothesis that -secretase is an aspartyl protease. However, in contrast to the -secretase activity we have characterized, most aspartyl proteases are not active at alkaline pH and have optimal activity at fairly low pHs. Thus, if -secretase activity is catalyzed by an aspartyl protease or proteases, these studies suggest that they are atypical aspartyl proteases.

Given the unusual nature of the -secretase activity, including both its potential to cleave bonds that may lie within membranes and its determinant of cleavage of specificity (the position of the -cleavage site with respect to the membrane), the development of this in vitro assay should enable additional insights into the nature of the specificity underlying -secretase catalyzed cleavages. Furthermore, the ability to solubilize and readily monitor the activity should ultimately enable the definitive identification of the catalytic proteins as well as potential regulatory or accessory proteins.

Because inhibition of -secretase activity decreases Aß production, development of -secretase inhibitors is one of a number of rational therapeutic approaches for the treatment of Alzheimer’s disease. Based on a number of studies showing that Aß42 is the more pathogenic form of Aß, it appears that specific inhibition Aß42 production may be the most desirable Aß-lowering strategy. Indeed, our data indicate that it may be dangerous to use an Aß40 selective inhibitor, as such inhibitors may actually increase Aß42 production. Confirmation that the -40 and -42 activities are indeed pharmacologically distinct, both in cultured cells and in vitro, suggests that development of -42 selective inhibitors may be feasible. This in vitro assay should facilitate the development of both nonselective and selective -secretase inhibitors, as it could be readily adapted for high throughput screening of -secretase inhibitors.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0286fje To cite this article, use (October 6, 2000) FASEB J. 10.1096/fj.00-0286fje

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This Article Full Text (PDF) All Versions of this Article: 14/15/2383 00-0286fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MCLENDON, C. Articles by GOLDE, T. E. Search for Related Content PubMed PubMed Citation Articles by MCLENDON, C. Articles by GOLDE, T. E.