A Novel gamma -Secretase Assay Based on Detection of the Putative C-terminal Fragment-gamma of Amyloid beta  Protein Precursor*

Inga Pinnix, Usha Musunuru, Han Tun, Arati SridharanDagger, Todd Golde, Christopher Eckman, Chewki Ziani-Cherif, Luisa Onstead, and Kumar Sambamurti§

From the Mayo Clinic, Jacksonville, Florida 32224

Received for publication, July 7, 2000, and in revised form, October 13, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta  peptide (Abeta ). The Abeta protein precursor (APP) is cleaved by beta -secretase to generate a C-terminal fragment, CTFbeta , which in turn is cleaved by gamma -secretase to generate Abeta . Alternative cleavage of the APP by alpha -secretase at Abeta 16/17 generates the C-terminal fragment, CTFalpha . In addition to Abeta , endoproteolytic cleavage of CTFalpha and CTFbeta by gamma -secretase should yield a C-terminal fragment of 57-59 residues (CTFgamma ). However, CTFgamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of Abeta as well as an ~6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTFgamma , and we describe an in vitro assay for gamma -secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTFgamma fragment. Three compounds previously identified as gamma -secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2,2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTFgamma , providing additional evidence that the fragment arises from gamma -secretase cleavage. Consistent with reports that presenilins are the elusive gamma -secretases, subcellular fractionation studies showed that presenilin-1, CTFalpha , and CTFbeta are enriched in the CTFgamma -generating fractions. The in vitro gamma -secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma -secretase inhibitors.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The Abeta 1 that is invariably deposited in Alzheimer's disease (AD) is a 38-43-residue peptide derived from a larger precursor, APP, as summarized below (Fig. 1) (1-6). Normal neuronal cells constitutively secrete Abeta , which is detected in cerebrospinal fluid and blood (7, 8). The length of most of the secreted Abeta molecules is 40 residues (Abeta 40) (9-11), but a small fraction (~10%) is 42 residues long (Abeta 42) (12). Mutations in APP (12), presenilin-1 (PS1), and presenilin-2 (PS2) (13-15) linked to familial AD (FAD) invariably increase the levels of either Abeta 42 alone or both Abeta 40 and Abeta 42, providing evidence that it plays an important causative role in the pathogenesis of AD (16, 17). Furthermore, there is evidence that in typical late onset AD, as in FAD, there are genetic determinants that increase levels of Abeta 42 (18). To generate Abeta , APP is sequentially cleaved by proteases referred to as beta - and gamma -secretase as described below. beta -Secretase cleaves at the N-terminal end of the Abeta sequence, producing a secreted derivative, sAPPbeta (19), and the Abeta -bearing C-terminal fragment of 99 residues, CTFbeta , which is subsequently cleaved by gamma -secretase to release Abeta (2). An alternative activity, alpha -secretase, cleaves APP within Abeta (between residues 16 and 17) to the larger secreted derivative, sAPPalpha (20, 21), and membrane-associated 83-residue fragment, CTFalpha (22). Cleavage of CTFbeta and CTFalpha by gamma -secretase generates Abeta and a smaller fragment of 24-26 residues called P3, respectively (4). In addition, gamma -secretase cleavage should theoretically yield an ~6-kDa fragment of 57-59 residues, CTFgamma (Fig. 1). Although the focus of the field has been on Abeta , it is important to recognize that Abeta 42 is naturally linked to CTFgamma -57. Expression of CTFbeta was shown to be toxic to neurons (23, 24), and more recently, it was suggested that the 31 C-terminal residues of APP are responsible for at least part of the toxicity (25). Since CTFgamma can also serve as a precursor for this toxic 31-residue peptide, its production and turnover are important parameters that can influence the course of AD.

The secretases that ultimately cleave APP to Abeta and CTFgamma have been the subject of intense research as potential targets for the treatment of AD. A novel pepstatin A-insensitive aspartyl protease, BACE/Asp2, was recently identified as the beta -secretase that cleaves at positions 1 and 11 of the Abeta sequence (26-29). In addition to BACE, thimet oligopeptidase, a metalloprotease, was shown to be involved in beta -secretase activity in COS7 cells (30). We recently showed that GPI-anchored proteins play an important role in beta -secretase activity (31). Recently, a family of disintegrin metalloproteases, the adamalysins such as tumor necrosis factor alpha -converting enzyme and ADAM 10, has been implicated in alpha -secretase cleavage of APP (32-34). However, tumor necrosis factor alpha -converting enzyme was shown to be specific for the phorbol ester-induced alpha -secretase activity (33). Although ADAM 10 appeared to be involved in both constitutive and inducible pathways, inhibition of this activity by a dominant-negative mutation indicated that it also plays a more important role in the inducible alpha -secretase pathway (34).

The final step in Abeta biogenesis is gamma -secretase cleavage and is the activity responsible for generating CTFgamma in vivo. This cleavage is particularly interesting as the scissile bond lies within the transmembrane domain (35, 36). Since the discovery that a PS1 knockout mutant mouse was deficient in gamma -secretase activity, the possibility was raised that PS1 and PS2 are the elusive gamma -secretases (37). The finding that PS1 and PS2 show conserved and essential aspartate residues within their respective transmembrane domains led to the suggestion that they are unusual aspartyl proteases with active sites within or close to the membrane (38). Recently, an in vitro assay for gamma -secretase was described, and PS1 was found to be a part of the active protease (39). By using this assay, a biotinylated inhibitor was cross-linked to PS1 and PS2, showing that the presenilins are indeed the active subunits of gamma -secretase (40, 41). However, the active enzyme was shown to be a large complex, presumably with many unidentified subunits, of which some may be also essential for gamma -secretase activity. To understand this interesting cleavage event, it is important to tease out each component of the gamma -secretase and examine its individual role in detail by a combination of biochemical and genetic methods. To address these issues, it is necessary to have a robust in vitro gamma -secretase assay in both the membrane-associated and soluble states. To develop such an assay, we incubated membranes from guinea pig and cow brains as well as from cultured cells to look for production of Abeta and CTFgamma . The CTFgamma fragment was detected in all systems tested, but guinea pig brain was used because the entire APP sequence is known, and the sequence of Abeta is identical to human allowing the use of human-specific reagents for its analysis. After appropriate fractionation, we observed a consistent time-dependent generation of a putative CTFgamma fragment from these membranes. In addition to describing a useful robust cell-free assay for gamma -secretase, this study presents the initial characterization of CTFgamma from brain membranes.


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Materials-- All buffers and reagents were obtained from VWR Scientific, Sigma, Life Technologies, Inc., or Fisher Scientific unless otherwise stated. Guinea pig brain and cow brain were obtained from Cocalico Biologicals Inc. Fetal calf serum and newborn calf serum were obtained from HyClone. Bis-Tris precast gels, MES buffer concentrate, and molecular weight markers were from Invitrogen Corp. Maleimide-activated keyhole limpet hemocyanin (KLH), Supersignal West Pico substrate, the BCA protein assay kit, and Brij 35 were from Pierce. The previously reported substrate-based difluoroketone inhibitor (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2,2-difluoropentanoyl-Val-Ile-OMe) of gamma -secretase that we are calling Wolfe-1 (42) was synthesized by Dr. Ziani-Cherif at the Mayo Clinic Chemical Core Facility. Additional reagents such as pepstatin A (Peptides International), 1,10-phenanthroline (PNT; J. T. Baker Co.), thiorphan (ICN), phosphatidylinositol-specific phospholipase C (PI-PLC; Molecular Probes), human recombinant caspase-3, Z-Val-Ala-Asp(OMe)-CH2F (Z-VAD-FMK), MG132, phenylmethylsulfonyl fluoride, E64, leupeptin, N-acetyl-Leu-Leu-Nle-CHO (ALLN), CHAPSO, and Big CHAP (Calbiochem) were purchased as indicated. The Chinese hamster ovary (CHO) cell line, G9PLAP, expressing the human placental alkaline phosphatase (PLAP) cDNA, a kind gift from Dr. Victoria Stevens (Emory University, Atlanta, GA), was maintained in F12HAMS medium supplemented with 10% fetal calf serum and 200 µg/ml geneticin (31).

Antibodies, Peptides, and Standards-- Antibodies against marker proteins syntaxin-6 (43) (Transduction Laboratories) and synaptophysin (44) (Roche Molecular Biochemicals) were kind gifts from Drs. Sevlever (Mayo Clinic, Jacksonville, FL) and Lahiri (Indiana University, IN). The anti-cross-reacting determinant (anti-CRD) antibody (Oxford Glycosciences) reacts with a neoepitope generated after the cleavage of GPI-anchored proteins by PI-PLC. Pf998, a rabbit polyclonal antibody against the juxtamembrane cytoplasmic domain of APP (726-744 of APP770) and the synthetic C-terminal 57 residues of APP (CTFgamma -57) were kind donations from Pfizer. CTFgamma -57 includes 10 residues from the transmembrane domain and the entire 47-residue cytoplasmic domain of APP and corresponds to one of the predicted CTFgamma fragments. The monoclonal antibodies BNT77, BA27, and BC05 utilized for the quantification of Abeta 40 and Abeta 42 were kind gifts of Dr. Nobu Suzuki and have been described previously (12).

The rabbit antibody O443 was raised against a maleimide-activated KLH-conjugated synthetic peptide (CKMQQNGYENPTYKFFEQMQN) prepared at the Mayo Clinic Protein Core Facility, which corresponds to the C-terminal 20 residues of APP. The animal care, injections, and bleeds were carried out by Cocalico Biologicals, Inc. (Animal approval number A3669-01), and the sera were characterized in our laboratory. The antibody detects less than 0.1 fmol (0.6 pg) of synthetic CTFgamma by Western blot analysis. The anti-PS1 antibody was a copy of that reported by Duff et al. (45) and was prepared against a KLH-conjugated synthetic peptide corresponding to residues 2-13 (CRKTELPAPLSYF).

The LC99 construct in the pCEP4 vector consisting of the signal sequence of APP fused to its C-terminal 99 residues, starting at the Abeta sequence, was transfected into CHO cells (CHO C99) for use as a standard for CTFbeta .

Preparation and Incubation of Active Membrane Fractions-- Brains were minced and homogenized for 1 min on ice in 10 volumes of buffer A (50 mM HEPES, 150 mM NaCl, and 5 mM EDTA, pH 7.4). All subsequent steps were carried out at 4 °C unless otherwise indicated. Homogenates were sequentially fractionated by centrifugation at 2500 × g for 15 min to collect unbroken cells and nuclei (F1P), and the post-nuclear supernatant was spun at 10,000 × g for 15 min (F2P) followed by 100,000 × g for 1 h (F3P). The supernatant (F3S) was discarded, and the three pellets were washed once in buffer A and resuspended in 200 µl/g initial brain weight of buffer B (50 mM HEPES buffer, 150 mM NaCl, pH 7.0). For Abeta measurements, the F2P fraction was incubated in buffer B supplemented with 5 mM EDTA, 5 mM PNT, and 1 mM thiorphan. The G9PLAP cell line was similarly prepared, except that the initial homogenate was directly centrifuged at 10,000 × g for 15 min to obtain a F2P pellet that included membranes from F1P as well. For most assays, fraction F2P was rehomogenized for 10 s and incubated at 37 °C for the indicated time. The reactions were stopped by chilling on ice, and the membranes were removed by centrifugation at 10,000 × g for 15 min unless otherwise specified. Finally, the supernatants were analyzed for Abeta and the released C-terminal fragments of APP.

To determine the pH optimum of fragment generation, aliquots of F2P were resuspended and incubated either in 50 mM sodium citrate, 150 mM NaCl, pH 5.0-6.5, buffer B, pH 7.0-7.5, or 50 mM Tris, 150 mM NaCl, pH 8.0-9.0. To make sure that the alternative buffers were not interfering with the assay, we also carried out the pH optimum analysis in buffer B adjusted to the various pH levels and essentially obtained the same results (data not shown).

Sucrose Density Gradient Fractionation of F2P-- Step gradients were set up using 25, 27.5, 30, 32.5, 35, 37.5, and 40% sucrose in buffer A containing 5 mM PNT in 7 layers of 0.6 ml each. The F2P obtained from 0.75 g of guinea pig brain was resuspended in buffer A containing 20% sucrose, overlaid onto the gradient, and centrifuged for 20 h at 48,000 rpm in a Beckman SW 50.1 rotor. Eight fractions (0.6 ml) were collected from the top of the gradient and diluted 5-fold in buffer A. Membrane pellets were collected by centrifugation at 100,000 × g for 1 h and then resuspended in equal volumes of buffer B. The protein concentration peaked in fractions 5 and 8. Fractions 1 and 2 containing low levels of protein were pooled for further analysis. The fractions were incubated, and the supernatants were analyzed for the release of CTFgamma as described earlier.

The membrane pellets from the fractions were analyzed for the presence of marker proteins. Markers of the Golgi, syntaxin-6 (31 kDa), synaptic vesicles and synaptophysin (38 kDa), were readily identified by their size. The anti-CRD antibody detects multiple GPI-anchored proteins as broad patches of ~38 and 62 kDa. The bands were considered as specific as they were only detected after PI-PLC treatment (data not shown).

Immunoassays-- Western blotting and immunoprecipitation were carried out as described previously (46), except that Hammerstein grade casein (1%) in 25 mM Na2HPO4, 0.2 M NaCl, pH 7.0 was used as a blocking agent for some of the antibodies, and Bis-Tris gels with the MES running buffer system was used for protein separation. Where relevant, scanned images were quantified using the ImageQuant software (Molecular Dynamics). For comparison of treatments between experiments, treated samples were compared with controls adjusted to 100%. Abeta was measured as reported earlier by a specific and sensitive sandwich ELISA using a monoclonal antibody BNT77 against Abeta 11-28 for capture and horseradish peroxidase-labeled end-specific antibodies BA27 (Abeta 40) or BC05 (Abeta 42) for detection (12, 31).


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Generation of a Novel C-terminal APP Fragment from Guinea Pig Brain Membranes-- To assay for gamma -secretase activity, guinea pig brains were homogenized and fractionated as described under "Experimental Procedures" into F1P, F2P, and F3P and resuspended in equal volumes of buffer B. Since the yield of membranes from CHO cells was low, these membranes were only fractionated into 10,000 and 100,000 × g pellets (F2P and F3P). Equal volumes of each fraction were incubated for 0 and 2 h at 37 °C, chilled on ice, and centrifuged at 100,000 × g to remove the membranes. Supernatants were examined by Western blotting with the O443 antibody raised against the C-terminal 20 residues of APP (Fig. 1). Western blots detected a soluble C-terminal fragment of ~6 kDa from the F1P and F2P of guinea pig brain and the F2P fraction of CHO cells, which increased considerably after 2 h at 37 °C (Fig. 2A). The fragment was not detected upon incubation of F3P (Fig. 2A). For further characterization, we immunoprecipitated the supernatant obtained from the F2P fraction of guinea pig brain with Pf988, an antibody against the juxtamembrane domain of APP (residues 726-744; Fig. 1), and we analyzed the recovered APP C-terminal fragments by Western blotting with O443 as above. The capacity to immunoprecipitate the putative CTFgamma with Pf998 shows that it includes epitopes from the juxtamembrane region on the cytoplasmic domain of APP (Fig. 2B).



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Fig. 1.   Major APP processing pathways. The APP holoprotein is a type I integral membrane protein with a large N-terminal extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. Abeta depicted as a violet box is partly extracellular (28 residues) and partly embedded within the membrane (12-14 residues). Antibodies O443 and Pf998 are against the final 20 residues and 19 juxtamembrane residues, respectively. APP is cleaved to sAPPalpha and CTFalpha by alpha -secretase or sAPPbeta and CTFbeta by beta -secretase. The membrane-bound CTFalpha and CTFbeta are further cleaved within the transmembrane domain by gamma -secretase (green arrow) to generate P3 and Abeta , respectively. Most (90%) Abeta and P3 end at residue 40 and a small fraction (5-10%) after residue 42 of the Abeta sequence. In addition, gamma -secretase cleavage should yield a C-terminal fragment, CTFgamma , of 57-59 residues.



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Fig. 2.   A 6-kDa C-terminal fragment of APP is released on incubation of membrane fractions. Guinea pig brain was fractionated into 2,500 × g (F1P), 10,000 × g (F2P), and 100,000 × g (F3P) fractions, as described under "Experimental Procedures." CHO cells were processed into two fractions of 10,000 × g (F2P) and 100,000 × g (F3P). Fractions were incubated for 0 or 2 h in the presence of 5 mM PNT, and the membranes from the incubation mixture were removed by centrifugation. Supernatants were directly analyzed on Western blots using O443 antibody (A). B, the incubated guinea pig brain F2P fraction was immunoprecipitated using Pf998 and then analyzed by Western blotting with O443. Lysates from CHO cells expressing LC99 (C99) mark the positions of the CTFbeta and the 83-residue CTFalpha fragments.

We also measured the levels of Abeta in the supernatant using a well characterized sandwich ELISA (12). The data obtained from 10 independently processed brain samples in three experiments detected moderate levels of Abeta 40 (62.3 ± 3.3 pM; Fig. 3). Abeta 42 was also detected at low levels of 5.4 ± 1 pM consistent with the in vivo findings that Abeta 42 constitutes 5-10% of the secreted Abeta (12). The increase in Abeta 40 was highly significant (p = 4.7 × 10-12) but Abeta 42 was not (p = 0.18), presumably due to the large variance in background levels (two-tailed t test).



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Fig. 3.   Abeta 40 is generated during the incubation of the F2P membrane fraction. F2P membrane fractions were incubated for 0 h (squares) and 2 h (triangles) in the presence of 5 mM PNT, 5 mM EDTA, and 1 mM thiorphan. Membranes from the incubation mix were removed by centrifugation, and Abeta 40 in the supernatant was quantified by sandwich ELISA as described under "Experimental Procedures." The bar at each time point indicates the mean values of 24.8 ± 2.1 pM (0 h) and 87.2 ± 3.5 pM (2 h).

To determine the stability of CTFgamma , the supernatant obtained after removal of membranes from the incubation mix was incubated further for 2 and 4 h in the absence of membranes. The CTFgamma fragment was degraded by over 50% after 2 h and by over 90% after 4 h at 37 °C. Addition of PNT, a metalloprotease inhibitor, partially protected the fragment from degradation after 4 h (Fig. 4). In addition, PNT increased the yield of the C-terminal fragment by over 2-fold, suggesting that one or more metalloproteases in the preparation degrade the fragment and/or the enzyme responsible for its generation (Fig. 4). Although several protease inhibitors including phenylmethylsulfonyl fluoride, E64, leupeptin, and ALLN were tested, protection was only seen by EDTA and PNT, making metalloproteases the only identified class important for degrading the released CTFgamma in vitro (data not shown). The pH optimum of the observed gamma -secretase activity was between pH 7 and pH 7.5, but activity was detected in a broad range from pH 5 to pH 9 (Fig. 5). This pH optimum is similar to that recently reported for gamma -secretase activity using membranes purified from HeLa cells (39).



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Fig. 4.   PNT increases the yield of the putative CTFgamma and partially protects it from degradation. Western blots probed with O443 (A) show F2P membranes incubated in the absence of PNT for 0 h (lane 1) or 2 h (lane 2) or in the presence of PNT for 0 h (lane 11) and 2 h (lane 12). Supernatants generated after a 2-h incubation without PNT were incubated further in the absence (lanes 3-6) or presence of PNT (lanes 7-10) for an additional 2 (lanes 3, 4, 7, and 8) and 4 h (lanes 5, 6, 9, and 10). Note the higher CTFgamma recovery after 4 h in the presence of PNT (lanes 9 and 10) than in its absence (lanes 5 and 6). Also note the 3-fold higher yield of CTFgamma with PNT (lane 12) than without PNT (lane 2). The graphical data shown in B represent the mean quantified by densitometry ± S.D. from three independent experiments.



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Fig. 5.   The yield of CTFgamma is optimal at close to neutral pH. The generation of CTFgamma at each pH was determined as an increase in the fragment yield as detected by Western blotting with O443 after a 2-h incubation compared with 0 h as blank.

We attempted to determine the cleavage site(s) that generate CTFgamma in collaboration with Dr. Rong Wang (Rockefeller University, New York). Presumably due to its low levels and unfavorable flying characteristics, the exact size of CTFgamma could not be detected by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy. However, the fragment comigrates with a 57-residue synthetic peptide corresponding to one of the predicted CTFgamma fragments on polyacrylamide gels (Fig. 6). Judging from the separation of CTFalpha from CTFbeta and the molecular weight markers, small changes of over five residues (0.5 kDa) should be readily detected in this size range. Thus, the cleavage size is likely to be close to the C-terminal end of the Abeta sequence and occur within the transmembrane domain of APP as predicted for gamma -secretase activity (Fig. 6).



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Fig. 6.   The 6-kDa fragment migrates with synthetic CTFgamma -57 and is degraded by caspase-3. Western blot probed with O443 shows F2P from guinea pig brain incubated at 0 (lane 1) and 2 h (lanes 2-5). Z-VAD-FMK was included in lanes 3 and 5, and caspase-3 was included in lanes 4 and 5. Note that caspase-3 reduces the yield of CTFgamma in lane 4, but the yield is recovered when Z-VAD-FMK is added in lane 5. The synthetic CTFgamma -57 (lane 6) migrates with the fragment generated from brain membranes (lanes 2-5). The synthetic CTFgamma is degraded by caspase-3 (lane 7), which is protected by Z-VAD-FMK (lane 8).

Caspase Inhibitor Does Not Reduce CTFgamma Production-- Previous studies have shown that caspase can cleave within the cytoplasmic domain of full-length APP in cells undergoing apoptosis (25, 47-49). This cleavage resulted in an ~3-kDa fragment and was inhibited by a broad spectrum caspase inhibitor, Z-VAD-FMK (49). It is unlikely that the observed 6-kDa fragments are generated by caspase, as the fragment is much larger than reported, and this cleavage occurs in the membrane pellet fraction after washing away the cytoplasm. However, we cannot rule out that some caspase is bound to the membrane pellet. To rule out the possibility that the CTFgamma -like fragment is a product of caspase cleavage, we included Z-VAD-FMK, a broad spectrum caspase inhibitor, in our incubations. Z-VAD-FMK did not inhibit the generation of the putative CTFgamma (Fig. 6). We also incubated the 57-residue synthetic CTFgamma as well as the brain membrane fraction with caspase ± Z-VAD-FMK. Caspase-3 cleaved the synthetic CTFgamma as shown by the reduction in band intensity, and Z-VAD-FMK blocked this reduction, indicating that the inhibitor was active under the conditions used (Fig. 6). Similarly, reduction in the yield of the 6-kDa CTFgamma was also observed when brain membranes were spiked with purified caspase, which was restored in the presence of Z-VAD-FMK (Fig. 6).

gamma -Secretase Inhibitors Reduce CTFgamma -- A number of protease inhibitors were tested to determine the class of proteases involved in the generation of the putative CTFgamma and to understand the role of gamma -secretase in generating this fragment. Previous reports have already identified several inhibitors that lower gamma -secretase activity as defined by a reduction in the production of Abeta and by the increase in CTFalpha and CTFbeta levels in cell lysates (2). Three of these inhibitors are pepstatin-A, MG132, and Wolfe-1 (a gamma -secretase substrate-based difluoroketone inhibitor). F2P membranes treated with the inhibitors pepstatin-A (Fig. 7A, lanes 5-8), Wolfe-1 ((42) Fig. 7A, lanes 9-12), and MG132 (Fig. 7B, lanes 7-12) showed a dose-dependent reduction in the yield of the 6-kDa fragment generated from brain membranes in vitro. These data provide additional evidence, indicating that the putative CTFgamma fragment is a product of gamma -secretase cleavage.



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Fig. 7.   Known gamma -secretase inhibitors reduce the yield of CTFgamma . F2P from guinea pig brain was incubated in the presence of 5 mM PNT and the indicated protease inhibitors and supernatants were analyzed on Western blots with the O443 antibody as described under "Experimental Procedures." A compares CTFgamma in PNT alone for 0 (C-0) and 2 h (C-2) with PNT + 10 µM (P-1) and 100 µM (P-2) pepstatin-A, or 0.2 mM (W-1) and 1 mM (W-2) Wolfe-1. B compares samples incubated in the presence of PNT alone for 0 (C-0) and 2 h (C-2), and PNT + 50 µM (M-1) or 200 µM (M-2) MG132 for 2 h. The relative yield of CTFgamma from the various conditions is summarized in C, with each bar representing the mean and S.E. of 8-10 independent incubations from three separate experiments.

CTFgamma Is Generated in a Fraction Enriched in CTFalpha , CTFbeta , and Presenilin-- CTFalpha and CTFbeta are the postulated substrates for gamma -secretase activity, and PS1/PS2 are reported to be an integral part of purified gamma -secretase (39). Furthermore, reports show that PS1 binds APP and its C-terminal fragments, consistent with its role in gamma -secretase activity (50-52). To examine the role of PS1 in the generation of CTFgamma , we analyzed brain membrane fractions. As described earlier, we initially obtained three membrane pellet fractions F1P, F2P, and F3P generated after sequential centrifugation. The fractions were probed with antibodies against APP and PS1 and marker proteins as described under "Experimental Procedures". The cell-surface GPI-anchored proteins (53) and the Golgi-marker syntaxin-6 (43) were preferentially found in F3P, which also had the highest concentration of protein (Fig. 8). In contrast, most of the gamma -secretase activity together with CTFalpha and CTFbeta was in F2P, suggesting that gamma -secretase activity was not enriched in the plasma membrane or in Golgi vesicles. Although the N-terminal fragment of PS1 was observed in all three fractions, its mobility was somewhat retarded in F3P. The reason for this shift in mobility is not known but may be due to post-translational modification such as phosphorylation of PS1 (54).



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Fig. 8.   CTFgamma is generated in a fraction distinct from the cell surface and the Golgi apparatus. Guinea pig brain fractions F1P, F2P, and F3P were analyzed on Western blots stained using reagents shown on the right side of each panel. Note that full-length APP is enriched in F3P, whereas CTFalpha , CTFgamma , and some of the CTFbeta are enriched in F2P. The N-terminal fragment of PS1 at ~30 kDa was seen in all fractions. However, the fragment was slightly shifted up in F3P. The anti-CRD antibody against PI-PLC-cleaved GPI-anchored proteins detected two major groups of bands in the 38- and 62-kDa range primarily in F3P. Each of these groups presumably represents several GPI-anchored proteins in these size ranges. The Golgi marker, syntaxin-6 at 31 kDa, was also primarily in F3P. The synaptic vesicle marker, synaptophysin, was primarily detected in F1P and F2P indicating that most of it is pelleted at 10,000 × g, although a sizable quantity is also seen in F3P. A total protein stain (Ponceau S) detected the strongest signal in F3P, although Fig. 2 shows that most of the CTFgamma is generated in F2P.

The active F2P was further fractionated by centrifugation to equilibrium in a sucrose step gradient ranging from 20 to 40% sucrose as described above. The eight fractions were examined for gamma -secretase activity by incubating membrane pellets as described earlier. In addition, the membranes were analyzed on Western blots for APP, CTFalpha , CTFbeta , and PS1 (Fig. 9). The peak of gamma -secretase activity as determined by the relative intensity of the CTFgamma fragment generated was in fraction 5. Interestingly, PS1, CTFalpha , and CTFbeta are all enriched in this fraction. However, it is important to note that PS1 was distributed in several fractions with low gamma -secretase activity (Figs. 8 and 9). Recent findings show that most of the PS1 is in the endoplasmic reticulum and intermediate compartments with small amounts in the Golgi and cell surface (55). PS1 was also detected in detergent-resistant glycosphingolipid-enriched membranes (DIGs (56)), which was also enriched in cell-associated Abeta (57). Our preliminary observations2 show that PS1, BACE, and the ADAM 10 protease (Kuzbanian) are all enriched in detergent-insoluble glycosphingolipid-enriched membranes isolated from guinea pig brain.3 Since we did not detect gamma -secretase activity in detergent-insoluble glycosphingolipid-enriched membranes prepared by flotation of Triton X-100-extracted membranes, we homogenized guinea pig brain in carbonate buffer to strip peripheral proteins, and generated a membrane fraction enriched in caveolin as described by Lisanti and coworkers (58). The gamma -secretase activity was enriched in the caveolin-rich fractions suggesting that it may be present in DIG-related membranes.3



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Fig. 9.   The peak of PS1, CTFalpha , and CTFbeta coincide with the peak of CTFgamma generation. F2P from guinea pig brain was further fractionated on sucrose density gradients, and eight fractions were collected from the top of the tube as described under "Experimental Procedures," and equal volumes of each fraction were analyzed by Western blotting with O443 (top three panels) and anti-PS1 N terminus (bottom panel). Fractions 1 and 2 were mixed and loaded in the 1st lane, and the remaining fractions were loaded in individual lanes. The top panel shows full-length APP, the 2nd panel CTFalpha , and the 3rd panel CTFgamma generated after incubation for 2 h. CTFbeta can be detected as a faint band above CTFalpha in the 2nd panel. The bottom panel shows the ~30-kDa PS1-NTF. Note that PS1-NTF, APP, CTFalpha , CTFbeta , and generation of CTFgamma peak in fraction 5.

Solubilization of the Active gamma -Secretase-- The in vitro gamma -secretase activity is lost when either F2P or membrane fraction 5 purified by sucrose density gradients is extracted with several detergents such as Triton X-100, methyl beta -cyclodextrin, digitonin, Nonidet P-40, CHAPS, and octyl beta -glucoside (data not shown). The data presented compare gamma -secretase activity in fraction 5 in the presence of Brij 35, Big CHAP, and CHAPSO (Fig. 10A). gamma -Secretase activity, determined as an increase in CTFgamma production, appeared equivalent to the detergent-free control in CHAPSO, reduced in Brij 35, and almost absent in Big CHAP. Thus, the detergent extraction profile of this in vitro activity is similar to the in vitro assay recently reported by Li et al. (39). Since dissolving the activity is essential for its further purification and characterization, we removed the CHAPSO-insoluble membranes by centrifugation at 100,000 × g for 60 min and compared gamma -secretase activity in the supernatant with activity in the mixture (Fig. 10B). Our data indicate that most of the activity was extracted from the sucrose gradient purified fraction 5 in this detergent, as judged by the approximately similar intensities of the CTFgamma band obtained by incubation of equivalent amounts of the CHAPSO-solubilized supernatant alone and the CHAPSO-treated membrane mix. Solubilization of the activity is necessary for further biochemical analysis of gamma -secretase in vitro.



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Fig. 10.   The in vitro gamma -secretase activity can be solubilized in select detergents. Membranes from fraction 5 shown in Fig. 9 were diluted and collected by centrifugation as described under "Experimental Procedures." A, the fraction was incubated in pairs in the absence (lanes 1 and 2) and presence (lanes 3-14) of the indicated detergents for 0 and 2 h. Activity was recognized as an increase in the CTFgamma level at 2 h (even lanes) over 0 h (odd lanes) for each condition. Note that CTFgamma levels did not increase in the presence of Big CHAP (lanes 7-10). B shows that CTFgamma is generated in the CHAPSO-soluble fraction. CHAPSO-resistant membranes were removed by spinning at 100,000 × g for 1 h, and the supernatants were incubated for 0 (lanes 1 and 4) and 2 h (lanes 2 and 5). The CHAPSO-treated membranes were incubated for 2 h (lanes 3 and 6) as in A. Note that removal of CHAPSO-resistant membranes did not reduce the yield of CTFgamma . The CHAPSO-resistant membrane pellet did not generate any CTFgamma (data not shown).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The current study reports a novel gamma -secretase assay from brain membranes and is the initial description of CTFgamma , a previously undescribed fragment predicted as a product of gamma -secretase cleavage of APP. The CTFgamma fragment is normally not detected in cell lysates and brain homogenates, probably due to its instability in vivo. A possible exception is a report of a faint 5.8-kDa CTF in human brain homogenates (22). However, CTFgamma is readily detected in an in vitro gamma -secretase assay based on fractionated brain membranes or cell homogenates. The in vitro detection of CTFgamma strongly suggests that gamma -secretase cleavage is due to a specific endoproteolytic cleavage and not random degradation of the cytoplasmic tail domain of APP as suggested previously (59).

An in vitro assay for gamma -secretase has been recently reported by looking for Abeta as a product using a purified substrate consisting of the C-terminal 99 residues of APP (39). In the presence of thiorphan, an inhibitor of Abeta degradation in the brain (60), we were also able to detect the generation of Abeta 40 (Fig. 3) as well as Abeta 42 in our in vitro assay. Since a pool of CTFgamma is not already present in brain or cell lysates at detectable levels, the low background allows the characterization of gamma -secretase activity from fractionated membranes more easily than the measurement of Abeta . Based on the reports that beta -secretase accounts for <10% of the secretory processing of APP (22), using CTFgamma as an end point for gamma -secretase activity may be 10 times more sensitive than the measurement of Abeta . In addition, the measurement of CTFgamma is not complicated by aggregation like Abeta or by the presence of alternative fragments such as P3 (Fig. 1).

Subcellular fractionation studies based on the in vitro generation of CTFgamma suggest that gamma -secretase activity is localized in fractions enriched in CTFalpha and CTFbeta . PS1 is apparently enriched in these fractions, consistent with the currently favored hypothesis that presenilins include the active site of gamma  secretase. However, high levels of PS1 are still seen in fractions showing little in vitro gamma -secretase activity. Thus, if PS1 is indeed the active subunit of gamma -secretase as suggested, only a small subset of it, presumably in a special compartment, is involved in gamma -secretase activity. The restriction of the activity may be due to either the observed enrichment of the substrate CTFalpha and CTFbeta in this compartment, limitations in essential components of the gamma -secretase complex other than presenilins, or unfavorable conditions in the isolated organelle.

Although high levels of Abeta are secreted by several cell lines in culture, CTFgamma is not readily detected in cell lysates, indicating that it is either rapidly degraded in the cytoplasm or sequestered in a manner that prevents its detection. Similar fragments from other membrane proteins such as Notch and SREBP are transported into the nucleus where they act as transcription factors, are rapidly degraded, and are difficult to detect (61, 62). The C-terminal tail of APP includes a sequence (KFFEQ) that resembles a motif present in soluble proteins that are rapidly degraded in lysosomes (63). It will be useful to determine whether this sequence is responsible for rapid CTFgamma turnover in cells and to identify alternative cellular pathways for its degradation, if any.

Recent reports show that PS1 specifically binds a gamma -secretase inhibitor indicating that the active site of gamma -secretase lies within the transmembrane domains of PS1 and PS2 (40, 41). It was proposed that presenilins are unusual aspartyl proteases, but this is not definitive as the aspartyl protease inhibitor, pepstatin A, can inhibit other classes of proteases at high concentrations (64). In addition, other gamma -secretase inhibitors are known to inhibit several classes of proteases including serine proteases, cysteine proteases, and the proteasome (2). The data showing gamma -secretase activity in fractions enriched in PS1 agree with studies using knockout mice deficient in PS1/PS2 that fail to generate Abeta and accumulate CTFalpha and CTFbeta (37, 65, 66). The detection of CTFgamma provides another powerful tool for the analysis of gamma -secretase activity in vitro and for identifying mechanisms involved in its regulation and for developing drugs that block this activity.

Although presenilins have been identified as being essential for gamma -secretase activity and are shown to contain the active sites, it is probably not sufficient, as it has not been possible to reconstitute the activity with pure PS1/PS2 and purified gamma -secretase is a multisubunit complex (39). The mechanism of the endoproteolytic cleavage of the membrane-spanning domains of proteins may identify the role of each component. For example, it has been suggested that the cleavage is initiated by the sliding of the peptide out of the membrane into the cytoplasm (67). However, the energy required for this sliding reaction is likely to be very high. It is also likely that the reaction is mediated by a water molecule within the active site of the enzyme for the hydrolytic reaction. Thus, a water molecule may be held between the two transmembrane aspartate residues on PS1/PS2 that serve to hydrolyze within the transmembrane domain. It is possible that some of the unidentified subunits are involved in either transport of water or the APP for hydrolysis. The mechanisms for maintaining a supply of the water molecules in the membrane for the hydrolysis may provide useful insights into the mechanisms of gamma -secretase cleavage and identify additional therapeutic targets for inhibiting this activity. A similar intramembrane proteolytic cleavage has been described for the sterol regulatory element binding protein (SREBP). The protease that cleaves SREBP has been identified as S2P by genetic complementation of a mutant cell line. The predicted active site of this enzyme resembles that of a metalloprotease with the exception that hydrophobic residues flank the domain, suggesting its intramembranous location (62). The development of an in vitro assay for S2P may be useful for comparison with gamma -secretase and also provide useful insights into intramembrane proteolysis.

Since the generation of CTFgamma is closely tied to the generation of Abeta , the mutations that increase the production of Abeta 42 should also increase the 57-residue CTFgamma starting at residue 43 of the Abeta sequence. Since FAD mutations that increase Abeta by preventing its degradation have not been reported, the correlation between CTFgamma -57 and AD is as strong as Abeta 42. In addition, CTFgamma includes a peptide sequence that is reported to induce apoptosis in neurons as discussed in the Introduction. The localization of the enzyme, the biological activity of the fragment, the cleavage site involved in its production, the mechanism of its turnover, and its role in AD are important unanswered questions that will be facilitated by this initial description of CTFgamma .


    ACKNOWLEDGEMENTS

We thank Dr. Daniel Sevlever of the Mayo Clinic and Dr. Debomoy Lahiri for several antibodies used in this study and Pfizer Corp. for synthetic CTFgamma 57 and antibody PF998. We also thank Meera Parasuraman, Dr. Daniel Sevlever, Dr. Debomoy Lahiri, and Dr. John Hardy for critical reading of the manuscript.


    FOOTNOTES

* This study was supported by grants from ISOA, Bayer Corp., Axonyx Corp., and Smith fellowship (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Brandeis University, Waltham, MA 02454.

§ To whom correspondence should be addressed: Mayo Clinic, 4500 San Pablo Rd., Jacksonville, FL 32224. Tel.: 904-953-7383; Fax: 904-953-7370; E-mail: samba@mayo.edu.

Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005968200

2 I. Pinnix, U. Musunuru, H. Tun, A. Sridharan, T. Golde, C. Eckman, C. Ziani-Cherif, L. Onstead, and K. Sambamurti, unpublished observations.

3 H. Tun, I. Pinnix, and K. Sambamurti, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: Abeta , Alzheimer's amyloid beta  protein; AD, Alzheimer's disease; FAD, familial AD; Abeta 40, Abeta ending at residue 40; Abeta 42, Abeta ending at residue 42; APP, Abeta protein precursor; sAPP, secreted APP derived from APP by proteolytic cleavage; sAPPalpha , sAPP product of alpha  secretase cleavage; sAPPbeta , sAPP product of beta  secretase cleavage; CTFalpha , C-terminal membrane associated product of alpha  secretase cleavage; CTFbeta , C-terminal membrane associated product of beta  secretase cleavage; CTFgamma , C-terminal soluble product of gamma  secretase cleavage; ADAM, adamalysin, a new class of disintegrin metalloproteases; PS1, presenilin-1; PS2, presenilin-2; PNT, 1,10-phenanthroline; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonic acid; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; PLAP, placental alkaline phosphatase; CHO, Chinese hamster ovary; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2F; PI-PLC, phosphatidylinositol-specific phospholipase C; KLH, keyhole limpet hemocyanin; ELISA, enzyme-linked immunosorbent assay; SREBP, sterol regulatory element-binding protein; GPI, glycosylphosphatidylinositol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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