From the
Karolinska Institutet and Sumitomo Pharmaceuticals Alzheimer Center, Neurotec, Novum, Huddinge, SE-141 57 Sweden, ¶Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, SE-171 77 Sweden, and **Department of Neurotec, Karolinska Institutet, Section for Experimental Geriatrics, Novum, SE-141 86 Huddinge, Sweden
Received for publication, November 25, 2002 , and in revised form, April 11, 2003.
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ABSTRACT |
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INTRODUCTION |
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The majority of early-onset familial AD cases are associated with mutations in the presenilin (PS) genes, and all PS mutations cause an increase in the production of the amyloidogenic A42 (5). PS plays a central role in
-secretase activity (for review, see Refs. 6 and 7), but the precise molecular nature of this function in Alzheimer's disease is not fully understood. In the absence of PS,
-secretase-mediated cleavage of
-APP is fully abolished (8), and some studies suggest that PS is a novel aspartyl protease with an activity profile identical to
-secretase (913). Other studies implicate PS as an indirect regulator of
-secretase and hypothesized roles for PS in trafficking of the
-APP substrate to the site of cleavage or orientation of substrate within the membrane (1416).
-Secretase displays an evolutionarily conserved mechanism of regulated intramembrane proteolysis that is involved in the generation of signaling molecules from type I integral membrane proteins (17). As well as
-APP, substrates for
-secretase-mediated regulated intramembrane proteolysis include Notch, the receptor tyrosine kinase ErbB4, and E-cadherin (1820). In cells,
-secretase activity is associated with a PS-dependent high molecular weight integral membrane multiprotein complex (2124). Nicastrin, a transmembrane glycoprotein, was recently identified by co-immunoprecipitation with PS as part of this complex and was shown to be involved in Notch signaling in Caenorhabditis elegans and A
generation in human cells (25). The absence of nicastrin in Drosophila results in the destabilization and perturbation of both PS function and cellular localization (26, 27). The nicastrin ectodomain undergoes a major structural alteration during assembly with presenilin, and this alteration is necessary for
-secretase activity, suggesting that the nicastrin molecule is mechanistically central to the function of the
-secretase complex (28).
In addition to presenilin and nicastrin, the -secretase complex also contains two recently identified transmembrane proteins, namely Aph-1 and Pen-2. These proteins were initially identified in genetic screens in C. elegans (29, 30). Aph-1 is required for surface localization of nicastrin (30), and Pen-2 is required for both the expression of PS and the maturation of nicastrin (31). Similarly, Aph-1 plays an intimate role in both the function and stabilization of the
-secretase complex (32). More recent data in Drosophila and mammalian cells suggests that nicastrin, Aph-1, and Pen-2 stabilize presenilin and increase the formation of presenilin fragments and
-secretase activity. It is proposed, therefore, that Aph-1 stabilizes the presenilin holoprotein within the complex and Pen-2 is required for endoproteolysis of presenilin and activation of
-secretase (33, 34). The components described above are sufficient and necessary for
-secretase activity (35), but given the intricacies of the
-secretase complex and the broad diversity of substrate- and cleavage-site specificity, it is possible that there are other functional or regulatory components of
-secretase-mediated regulated intramembrane proteolysis.
A first step in the characterization of -secretase has been to identify conditions for the solubilization of the complex and the development of activity assays. Several approaches have been applied to solubilize the
-secretase complex. These have generally involved the use of membranes derived from cell lines, mild detergent treatments, and measurement of A
formation by enzyme-linked immunosorbent assay or Western blotting (23, 24, 36, 37). However, these approaches may not fully reflect
-secretase-mediated processing of
-APP that occurs in the human brain. It is possible that the
-secretase complex in brain contains brain- or neuronal-specific co-factors that modulate activity. The biochemical analysis of membrane-bound enzyme complexes from post-mortem brain is well established and has been applied to the investigation of enzyme activity in neurological disorders, including AD and schizophrenia (38, 39). An attractive novel approach, therefore, is the analysis of
-secretase in post-mortem non-AD and AD human brain isolates.
As an initial step toward this aim, we describe the development of a sensitive in vitro -secretase assay based on a fluorogenic peptide probe. This assay system is dependent on the presence of PS, as described previously (8). We report the solubilization and characterization of
-secretase from human brain and show that
-secretase-mediated cleavage occurs at the putative
-secretase site(s) within the
-APP amino acid sequence that results in the generation of A
. This activity is reduced by compounds previously shown to inhibit
-secretase. In addition, we show that
-secretase activity in human brain co-localizes with a high molecular weight protein complex that comprises PS1, nicastrin, Aph-1, and Pen-2. These findings provide the basis for the further characterization of
-secretase and may facilitate the elucidation of essential factors in human brain involved in the pathobiology of AD.
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EXPERIMENTAL PROCEDURES |
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Presenilin 1 Stable Cell LinesThe PS-deficient blastocyst derived mouse embryonic stem cells, BD8 (40), were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2.4 mM L-glutamine, 0.1 mM -mercaptoethanol, and nonessential amino acids. Site-specific recombination was achieved by mixing the gateway-compatible pCAG-IRESpuro vector (a gift from Dr. Stephen Wood and Poon-Yu Khut, Child Health Research Institute, Adelaide, Australia) and pEntr-human PS1 in the presence of LR clonase as per the manufacturer's instructions, creating the plasmid pCAG-PS1-IRESpuro. Transformants were selected on LB-ampicillin plates. BD8 cells were stably transfected with pCAG-PS1-IRESpuro and selected for 10 days in 1 µg/ml puromycin (Sigma). Surviving colonies were expanded and screened for PS1 overexpression by Western blotting.
Membrane Isolation from Cells and Human BrainHuman brain material (2 g of temporal cortex, non-AD, 2 h post-mortem interval) was obtained from Huddinge Brain Bank (Huddinge, Sweden). Human embryonic kidney 293 cells and BD8 cells (
2 x 108 cells/experiment) were propagated under standard conditions. All procedures were carried out at 4 °C. Brain pieces were kept on ice, dissected to remove blood vessels and white matter, and resuspended in buffer A (1 ml/0.2 g) containing 20 mM Hepes, pH 7.5, 50 mM KCl, 2 mM EGTA, and CompleteTM protease inhibitor mixture (Roche Applied Science). Tissue was homogenized using a mechanical pestle-homogenizer (IKALaborteknik RW20; 1 500 rpm/25 strokes). Cells were harvested with a cell scraper, pelleted, and washed once in ice-cold phosphate-buffered saline. Cells were resuspended in 9 volumes of buffer A and homogenized using 15 strokes of a Dounce homogenizer with a B pestle. After homogenization, brain and cell lysates were processed under identical conditions. Lysates were centrifuged at 800 x g for 10 min to remove nuclei and large cell debris, and the pellet was rehomogenized as above. The resulting supernatants were pooled and centrifuged at 100,000 x g for 1 h. The membrane pellet was washed once in buffer A and recollected by centrifugation at 100,000 x g for 30 min. All centrifugations were carried out using a fixed-angle rotor. The membranes were resuspended in buffer A plus 10% glycerol, flash-frozen in liquid N2, and stored at 70 °C before use.
Solubilization of Membrane PreparationsProtein concentration in membrane preparations was determined using BCA reagents (Pierce). Membranes were resuspended (0.5 mg/ml) in 20 mM Hepes, pH 7.0, 150 mM KCl, 2 mM EGTA, 1% (w/v) CHAPSO (Calbiochem), and protease inhibitor mixture and solubilized at 4 °C for 1 h with end-over-end rotation. The solubilized membranes were centrifuged at 100,000 x g for 1 h, and the supernatants were collected.
-Secretase InhibitorsThe highly specific
-secretase inhibitors L-685,458 (Bachem) and compound 1 (Dr. Mark Shearman, Merck Sharp and Dohme) and pepstatin A (Sigma), a more general aspartyl protease inhibitor, were dissolved in Me2SO, separated into aliquots, and stored at 70 °C until use. An inactive deshydroxy derivative of L-658,458 (synthesized by Dr. Alan Nadin, Merck Sharp and Dohme) was also used in this study.
DeglycosylationCHAPSO-solubilized membrane protein samples (50 µg) containing protease inhibitor mixture (as above) were denatured by boiling for 10 min at 100 °C in the presence of 0.5% (v/v) SDS and 1% (v/v) -mercaptoethanol. After cooling on ice, samples were adjusted to 50 mM sodium citrate, pH 5.5. For Endo H treatment, 100 milliunits of Endo H (Roche Applied Science) was added. For PNGase F treatment, Nonidet P-40 was added to a final concentration of 1%, and this was followed by the addition of 15.4 milliunits of PNGase F (Roche Applied Science). Samples were incubated overnight at 37 °C, and reactions were stopped by the addition of 2x SDS-PAGE sample buffer. Glycosylation status was analyzed by SDS-PAGE/Western blotting and immunolabeling with nicastrin antibody.
ImmunoprecipitationCHAPSO-solubilized membrane fractions were diluted with immunoprecipitation buffer (20 mM Hepes, pH 7.0, 150 mM KCl, 2 mM EDTA, 2 mM EGTA, and 0.5% CHAPSO). Samples were pre-cleared by the addition of 20 µl of a 1:1 slurry of protein A/G-Sepharose (Amersham Biosciences) and end-over-end rotation at 4 °C for 30 min. After incubation, samples were subjected to centrifugation at 16,100 x g for 2 min, and the supernatant was removed to a fresh tube. Antibodies were added to each tube, and incubation was continued overnight at 4 °C with end-over-end rotation. Protein A/G-Sepharose was added, and incubation was continued for 12 h. The beads were then isolated and washed three times with immunoprecipitation buffer containing 0.25% (w/v) CHAPSO. SDS-PAGE sample buffer was added to the beads, and samples were subjected to SDS-PAGE as described below.
SDS-PAGE and Western BlotMembrane preparations were boiled in Laemmli sample buffer and separated by SDS-PAGE (1020% Tricine gels, Novex). After electrophoresis proteins were transferred to PVDF membranes (Bio-Rad) and probed with specific antibodies. Immune complexes were visualized by SuperSignal West Pico enhanced chemiluminescence reagents (Pierce). Hyperfilm ECL (Amersham Biosciences) was used for exposure, and films were scanned using an AGFA Duoscan. Figures were produced for publication with Photoshop (Adobe) or Canvas v.8 (Deneba Systems, Inc.) software.
-Secretase-mediated Peptide Cleavage AssayTo measure
-secretase activity, solubilized membranes (treatments, samples, and protein concentrations are indicated in individual experiments) were incubated at 37 °C in 150 µl of assay buffer containing 50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 0.25% CHAPSO (w/v). Peptide substrate (Fig. 1A) was synthesized and purified by high performance liquid chromatography by Peptide Inc., Osaka, Japan. Solubilized membrane preparations were incubated with 8 µM peptide at 37 °C overnight, unless otherwise specified. After incubation, reactions were centrifuged at 16,100 x g for 15 min and placed on ice. Supernatants were transferred to a 96-well plate (Nunclon, Nunc), and fluorescence was measured using a plate reader (Fluorstar Galaxy) with excitation wavelength at 355 nm and emission wavelength at 440 nm. Analysis and presentation of results was carried out using Microsoft Excel software.
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Liquid Chromatography-Mass SpectrometrySamples from the -secretase assay containing solubilized membranes and peptide probe were injected onto a PLRP-S 300-Å (150 x 2.1 mm) reverse phase column (Polymer Laboratories). Water with 0.2% formic acid and acetonitrile with 0.2% formic acid was used as the mobile phase. The samples were eluted with a gradient from 10 to 40% acetonitrile over 30 min. The column was coupled on-line to an electrospray ion-trap mass spectrometer (Agilent 1100 series, Agilent Technologies). As a control, samples were also prepared in the absence of membranes.
Blue Native (BN) PAGEBN PAGE was performed by modification of methods described in Schülke et al. (41) and Brookes et al. (42). All buffers and solutions were pH 7.0 at 4 °C. Membranes (0.5 mg of protein) were prepared as described above and resuspended in 100 µlof extraction buffer composed of aminocaproic acid (0.75 M) and BisTris (50 mM). 12.5 µl of n-dodecyl
-D-maltoside (10% w/v, Calbiochem) was added to the suspension. After incubation on ice for 20 min with vortexing every 5 min, samples were cleared by centrifugation at 100,000 x g for 10 min, and protein concentration was quantified. To 100 µl of supernatant, 6.3 µl of a 5% suspension of Coomassie Brilliant Blue G-250 in aminocaproic acid (0.5 M) was added. Samples (30-µl aliquots containing
50 µg of protein) were loaded onto a 412% BisTris NuPAGE gel (Invitrogen). Molecular weight standards (high molecular weight calibration kit, Amersham Biosciences) were resuspended in extraction buffer (200 µl per 250-µg vial) plus 25 µl of 10% n-dodecyl
-D-maltoside and 12 µl of 5% Coomassie Blue, as above. Gel electrophoresis was performed overnight at 75 V using 50 mM Tricine, 15 mM BisTris, 0.02% Coomassie Blue G-250 as cathode buffer, and 50 mM BisTris as anode buffer. After electrophoresis, gels were equilibrated for 30 min in Western blot transfer buffer containing 0.05% (w/v) SDS. The samples were transferred to PVDF membrane as described above, except that transfer buffer contained 0.05% SDS.
Size Exclusion ChromatographyThe solubilized membrane preparation (300 µg of protein) was injected onto a Superose 6 HR 10/30 column (Amersham Biosciences). Solubilization buffer with 0.25% CHAPSO was used as mobile phase at a flow rate of 0.4 ml/min, and 0.4-ml fractions were collected and assayed for activity. The protein content of selected fractions was concentrated using StratacleanTM resin (Stratagene), according to the manufacturer's instructions, and subjected to Western blot analysis as described above.
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RESULTS |
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Solubilized Membranes Isolated from Post-mortem Human Brain Can Cleave the Peptide ProbeAfter the development of a sensitive in vitro assay we first sought to establish whether cleavage of the peptide probe was comparable with that seen using solubilized membranes from freshly isolated cultured human cells. Initially, cell membranes were prepared from human embryonic kidney 293 cells and temporal cortex to compare activities. After solubilization and quantification of protein content, samples with equal amounts of protein were incubated overnight with the peptide probe. The activities from the different membrane preparations were compared (Fig. 2A). Maximal activity was obtained from the human embryonic kidney 293 cell membranes, whereas -secretase activity from brain membranes was
20% lower. Activity increased in a time-(Fig. 2B) and protein concentration-dependent (Fig. 2C) manner. Control samples without membranes showed a slight increase in signal in the presence of peptide, probably due to hydrolysis of the probe (Fig. 2, A and B). The membrane fraction has some autofluorescence (Fig. 2A), and this signal is subtracted from subsequent readings.
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The Peptide Probe Is Cleaved at the A40- and A
42-generating SitesThe peptide cleavage products generated during the incubation were analyzed by electrospray ionization mass spectrometry (shown in Table I). N- and C-terminal peptide cleavage products were observed corresponding to the A
42 cleavage site (Nma-GGVVIA and TVK(Dnp)rrr-NH2), whereas only N-terminal products were detected corresponding to the A
40 cleavage (Nma-GGVV). This suggests that a specific protease was active in the crude membrane fraction, generating cleavage products corresponding to both A
40 and A
42.
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-Secretase Is Active in Brain-derived Solubilized MembranesThe peptide probe is cleaved in the presence of the solubilized membrane fraction, and peptide fragments corresponding the C termini of A
are generated. To verify the specificity of the isolated activity, the membrane fraction was treated with known
-secretase inhibitors and subjected to the
-secretase activity assay. Three independent inhibitors were used, L-685,458 (described above), pepstatin A (a potent inhibitor of aspartyl proteases (44, 45)), and compound 1 (prototype
-secretase inhibitor shown recently to promote the accumulation of C83/C99 and to potently inhibit A
40 peptide production (46)). After overnight incubation in the presence of 10 µM L-685,458, activity was reduced by
55%. Similarly, 100 µM pepstatin A reduced activity by
70%. Treatment with 20 µM compound 1 resulted in
80% reduction in activity (Fig. 3A). A dose-dependent inhibition of activity was seen in the presence of increasing concentrations of L-685,458 (Fig. 3B). To further examine the specificity of the observed
-secretase activity, an additional parallel control experiment was carried out using an inactive deshydroxy derivative of L-685,458. This compound did not inhibit
-secretase activity using concentrations up to 5 µM. The proteolysis of the peptide probe by proteinase K was not affected by treatment with L-685,458, pepstatin A, or compound 1 (not shown).
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Known Components of -Secretase Can Be Isolated from Human Brain as a Protein ComplexIn human cells,
-secretase activity is dependent on several essential protein co-factors, including presenilin. PS1 N- and C-terminal fragments are generated by the endoproteolysis of PS1 holoprotein and remain heterodimerically associated at tightly regulated levels in the cell (for review, see Ref. 47). In addition to PS, the
-secretase complex also contains the recently identified components nicastrin, Aph-1, and Pen-2 (25, 32, 48). To characterize the molecular composition of the
-secretase complex in human brain, we performed co-immunoprecipitation studies.
-Secretase complexes were immunoprecipitated from CHAPSO-solubilized membranes using antibodies directed against PS1-NTF, PS1-CTF, nicastrin, and Aph-1. Nicastrin was found to co-immunoprecipitate with PS1-NTF and -CTF and Aph-1. PS1-NTF co-immunoprecipitated together with PS1-CTF, nicastrin, and Aph-1, and similarly, Pen-2 co-immunoprecipitated together with PS1-NTF, PS1-CTF, nicastrin, and Aph-1 (Fig. 4A). The specificity of interactions between PS1-NTF, PS1-CTF, nicastrin, Aph-1, and Pen-2 was verified by the absence of co-immunoprecipitation of these proteins with pre-immune serum (not shown) or the co-immunoprecipitation of the unrelated integral membrane protein calnexin with the
-secretase complex components (Fig. 4B). These results confirm that an intact protein complex comprising PS1 N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2 exists in human brain membranes.
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Mature Nicastrin Is Associated with the CHAPSO-solubilized Membrane FractionNicastrin is a type I integral membrane glycoprotein (25, 49). We investigated the maturation status of nicastrin in the CHAPSO-solubilized membrane fraction by carrying out deglycosylation with Endo H and PNGase F. Western blotting of untreated, control membranes with antibodies against the C terminus of nicastrin revealed a predominant protein species of 130 kDa (Fig. 5, lane 1), which is in agreement with the molecular weight of mature nicastrin seen in cells (49, 50). Treatment with Endo H increased the mobility of this fully mature glycosylated nicastrin species by
20 kDa, resulting in the loss of the
130-kDa species and formation of an
110-kDa species (Fig. 5, lane 3). This limited sensitivity to Endo H suggests that the higher molecular weight nicastrin species comprises a mixture of both ER-derived high mannose residues and Golgi-derived complex oligosaccharides. Complex glycosylation of nicastrin was confirmed by treatment with PNGase F, which completely removes all oligosaccharide side chains. A single protein species of
80 kDa, corresponding to the deglycosylated protein core of nicastrin, was generated by treatment with PNGase F (Fig. 5, lane 5). A less prominent, higher molecular weight band was also observed that also appeared to be partially Endo H-sensitive (Fig. 5, lane 3). This "hyper"-mature form of human brain-associated nicastrin does not appear to be related to
-secretase activity, as discussed later.
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-Secretase in Human Brain Can Be Partially Purified as an Active High Molecular Weight Protein ComplexSeveral studies using a variety of biochemical techniques have shown previously that
-secretase activity/PS1 is associated with a protein complex ranging from
100 to 2000 kDa (2123, 51). We investigated the molecular weight of PS1-dependent-
-secretase complex in the brain membrane fraction by subjecting samples to blue native gel electrophoresis and immunoblotting with antibodies specific to PS1-NTF, PS1-CTF, nicastrin, Aph-1, and Pen-2 (Fig. 6A). The same predominant immunoreactive band, with a molecular mass of
500 kDa, was observed with all antibodies. In a parallel experiment, PAGE under denaturing conditions revealed the monomeric protein species of the known
-secretase complex components (Fig. 6B).
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To verify that -secretase activity was associated with a high molecular weight protein complex, we separated total brain CHAPSO-solubilized membrane extracts by size exclusion chromatography and measured
-secretase activity in the eluted fractions (Fig. 7A). Two distinct peak activities were observed, peak 1 (fractions 610) and peak 2 (fractions 2935) (Fig. 7A), with apparent approximate molecular masses of >1000 kDa (peak 1) and <66 kDa (peak 2). When the eluted fractions were treated with 10 µM L-685,458 (Fig. 7A) or 10 µM compound 1 (not shown) only the activity in peak 1 was inhibited.
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The peak fractions were also analyzed by Western blot using PS1-NTF, PS1-CTF, nicastrin, Aph-1, and Pen-2 antibodies, which showed that the -secretase complex components could only be detected together in peak 1 (Fig. 7B). This indicates that these proteins are associated as a high molecular weight complex with
-secretase activity. The CHAPSO-soluble membrane fraction does not contain unassembled forms of PS1 fragments, Aph-1, and Pen-2 because these were not detected in fractions corresponding to their monomeric molecular weights, supporting investigations in cells which suggest that assembly is required for stabilization of the individual members of the
-secretase complex (31, 32, 34).
Interestingly, the hyper-mature form of nicastrin that is observed in the crude solubilized membrane extract (Fig. 5) does not appear to be associated with the -secretase complex (Fig. 7B). Instead, this form of the protein remains unassociated and monomeric (not shown). PS is required for the trafficking of nicastrin through the secretory pathway but not its complex glycosylation, and this association is also required for
-secretase activity (52). It is conceivable, therefore, that the hyper-mature form of nicastrin observed in human brain extracts belongs to a pool of unassembled nicastrin not directly involved in
-secretase activity. Other functions for nicastrin have been suggested, such as interacting with
-site APP cleaving enzyme and activating
-secretase (53), but the significance of this observation in human brain remains to be elucidated.
We conclude that active -secretase can be isolated from post-mortem human brain. The
-secretase-/PS1-dependent nature of this activity is supported by the specificity and inhibition profile of the fluorogenic peptide cleavage assay and the detection of
-secretase complex components in the active peak after size exclusion chromatography. This is extended by BN PAGE data, which confirms that a high molecular weight complex in the crude solubilized membrane fraction contains PS1-NTF, PS1-CTF, nicastrin, Aph-1, and Pen-2. The apparent molecular weight of the PS1-
-secretase complex in brain as resolved by BN PAGE and size exclusion chromatography are in agreement with those published previously. Using size exclusion chromatography, Li et al. (23) resolved a PS1 complex that associates with
-secretase activity, with a molecular mass of
2000 kDa (23), whereas BN PAGE data by Steiner et al. (31) suggests a PS1 complex of
500 kDa. The molecular characteristics of the
-secretase complex are also suggested by co-immunoprecipitation data, indicating that a complex comprising PS1-NTF and -CTF, nicastrin, Aph-1, and Pen-2 can be isolated. Our data support a functional link between this protein complex and
-secretase activity.
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DISCUSSION |
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To develop an in vitro -secretase assay we adapted a recently published method to solubilize membrane preparations from BD8 cells and BD8 cells transfected with PS1 (23) using the mild detergent CHAPSO. Incubation of solubilized membranes with a novel fluorogenic
-APP-mimicking probe confirmed that
-secretase activity in mouse BD8 cells is PS1-dependent. The availability of endogenous substrates in cortical extracts from post-mortem human brain is highly variable.2 To control substrate availability, the fluorogenic peptide cleavage assay was developed and used to investigate
-secretase further in brain. Using this approach, activity is comparable with that seen in freshly isolated membranes obtained from cells grown in culture.
The -secretase complex contains and is functionally dependent upon the N- and C-terminal fragments of PS1, the transmembrane glycoprotein nicastrin, and the recently identified transmembrane proteins Aph-1 and Pen-2. It has recently been shown that interactions between these proteins mediate
-secretase activity. Our results indicate that the solubilized crude membrane fraction contains PS1-NTF, PS1-CTF, mature nicastrin, Aph-1, and Pen-2 and that these proteins can be co-immunoprecipitated as a complex. The fact that we could detect only mature nicastrin in association with the detergent-soluble fraction supports recent data showing that maturation and endoglycosidase H-resistant glycosylation of nicastrin is required for interaction with PS1 (48). In addition, PS1-NTF, PS2-CTF, nicastrin, Aph-1, and Pen-2 in the solubilized human brain membrane fraction localize within an SDS-sensitive high molecular weight protein complex.
-Secretase activity is associated with the same high molecular weight complex, and this can be inhibited by treatment with the specific
-secretase inhibitor L-685,458, suggesting that the complex is functional.
How the C termini of A40 and A
42 are generated is an area of great attention. It has been suggested that several distinct proteases acting at the C terminus of C99 could be responsible for the generation of different A
species (54, 55). Other reports favor a single
-secretase as responsible for the generation of A
40 and A
42 (56). We observed a range of A
cleavage products, most of them corresponding to A
40- and A
42-specific cleavage after incubation of the fluorogenic peptide with the total solubilized membrane fraction. Taken together with our observations that
-secretase activity could not be fully inhibited by treatment with L-685,458, compound 1, and pepstatin A using physiologically active concentration ranges suggests nonspecific or presenilin/
-secretase-independent proteolytic activity could account for part of the peptide cleavage and generation of heterogeneous fragments that occurs in our system in the presence of total solubilized membranes. This notion is supported by the fact that cleavage of the peptide probe in lower molecular weight size exclusion chromatography fractions occurs in the absence of PS1-NTF, peak 2 (Fig. 7) and that activity is almost completely inhibited in the isolated high molecular weight fraction where
-secretase is present. Further experimentation will determine whether or not this is related to the normal metabolism of C99, a hypothesis supported by a recent study that describes the generation of intracellular A
in presenilin-deficient cells (57). Nevertheless, the fact that
-secretase activity can be inhibited by up to
80% using three independent inhibitory compounds provides compelling evidence that the
-secretase complex is functional in solubilized membrane fractions from post-mortem human brain.
The biochemistry of -secretase has been determined to a high degree of detail. However, experimentation has largely been restricted to the use of cell lines grown in tissue culture, and questions remain regarding the functional properties of the complex. Here we have shown that
-secretase is active in post-mortem human brain. These observations provide the basis for the further characterization of
-secretase such as the identification of other components of the complex and the elucidation of brain-specific or brain region-specific factors that may underlie pathogenic processes in Alzheimer's disease.
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FOOTNOTES |
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|| Supported by a stipend from David och Astrid Hageléns stiftelse.
To whom correspondence should be addressed: Karolinska Institutet, Neurotec, Novum-KASPAC, SE-141 57 Huddinge, Sweden. Tel.: 46-8-58583625; Fax: 46-8-58583610; E-mail: mark.farmery{at}neurotec.ki.se.
1 The abbreviations used are: A, amyloid
-peptide;
-APP, A
precursor protein; C83, C-terminal
-APP stub generated by
-secretase cleavage; C99, C-terminal
-APP stub generated by
-secretase cleavage; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; Nma, N-methyl-o-aminobenzoic acid; Dnp, 2,4-dinitrophenyl; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; BN PAGE, blue native PAGE; Endo H, endo-
-N-acetylglucosaminidase H; PNGase F, peptide N-glycosidase F; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; AD, Alzheimer's disease; Tricine, N-[2-hydroxy-1,1-/bis(hydroxymethyl)ethyl]glycine; PVDF, polyvinylidene difluoride.
2 M. Farmery, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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