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J. Biol. Chem., Vol. 281, Issue 21, 14776-14786, May 26, 2006
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-Protein and Amyloid Precursor Protein Intracellular Domain from -Carboxyl-terminal Fragment by 
-Secretase*
121
3
From the
Department of Neuropathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the Department of Life Science, Graduate School of Arts and Science, University of Tokyo, Tokyo 153-8902, and ¶Biomolecular Characterization Division, Characterization Center, RIKEN (The Institute of Physical and Chemical Research), Wako 351-0198, Japan
Received for publication, December 19, 2005 , and in revised form, March 30, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid -protein (A) 49 in cells resulted in predominant production of A40 and that of A48 leads to preferential production of A42. These observations suggest that 
-cleavage and 
-cleavage are interrelated. To determine the stoichiometry between A and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized -secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of A and AICD are produced from -carboxyl-terminal fragment (C99) by -secretase, irrespective of WT or MTAPP and PS1/2. Although various A species, including A40, A42, A43, A45, A48, and A49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, A48, in contrast to WT and other MTPS1s. These strongly suggest that-cleavage is the primary event, and the produced A48 and A49 rapidly undergo -cleavage, resulting in generation of various A species. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-protein (A)4 is the major component of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (AD). Current data indicate that A forms large fibrous aggregates that may not be toxic to the cells, but its intermediates, diffusible oligomers, exhibit neuronal toxicity, supporting the view that A is a real culprit for AD. This 38-43-amino acid residue, a small protein, is derived from -amyloid precursor protein (APP) through successive cleavages by - and -secretases (1). -Secretase is a membrane-bound aspartyl protease and produces a carboxyl-terminal fragment (CTF or C99) of APP by cleaving its luminal portion (2). Cumulative evidence indicates that -secretase is also an aspartyl protease, a high molecular mass protein complex composed of four different membrane proteins, Aph-1, nicastrin, Pen-2, and presenilin (PS) 1/2 (3-5). -Secretase cleaves C99 in the middle of its transmembrane domain (-cleavage), leading to release of A. The mechanism of A production is controversial, mainly because the hydrolytic event is postulated to occur in the very hydrophobic environment of the lipid bilayer.
Besides -cleavage sites, we and other groups identified novel cleavage sites close to the membrane/cytoplasmic boundary of APP (-cleavage) (6-8). -Cleavage sites of C99 are analogous to the -secretase-mediated Notch S3 cleavage site (9). As in Notch signaling, -cleavage results in release of the APP intracellular domains (AICD) 49-99 and 50-99, which translocate to the nucleus and influence transcriptional regulation (10-13). We also found that PS1/2 and APP carrying familial AD (FAD) mutations increase the proportion of AICD-(49-99) (14). As FAD mutations cause an increase in the A42/A40 ratio (1), we assumed that relationships exist between - and -cleavage sites. In fact, A48 and A49, the products that should have been generated by -cleavage, are converted to A40 and A42 within the cells (15). More importantly, the expression of A49 results in predominant release of A40 into media, whereas that of A48 preferentially produced A42. This indicates that the preceding -cleavage determines the preference for the final product, A40 or A42. Failure to identify AICD-(41-99) and AICD-(43-99), possible counterparts of A40/42, led us to assume that -cleavage precedes -cleavage.
According to this model, -secretase should produce equal amounts of A and AICD from C99. However, it is difficult to determine accurately the stoichiometry between A and AICD in cells, because A is only derived from C99, whereas AICD is generated from both C99 and C83 (1), the latter of which is usually severalfold more abundant in cells than C99. In addition, AICD in the cells is more susceptible to proteases than A (16-19). For these reasons, the stoichiometry of A relative to AICD has remained unclear. However, its elucidation is important for understanding the relationship between - and -cleavages and the mechanism of A production by the enigmatic -secretase. In this study, we have examined the stoichiometry between A and AICD using a CHAPSO-solubilized -secretase assay system (20, 21).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Preparation of C99-FLAG Substrate—A carboxyl-terminal fragment of APP (C99) was carboxyl-terminally fused with FLAG tag (C99-FLAG) and amino-terminally with signal peptide (MQLRNPELHLGCALALRFLALVSWDIPGARA) of human 
-galactosidase A. Resultant fragment was inserted into pFASTBACTM1 (Invitrogen). Sf9 cells were infected with recombinant baculovirus according to the manufacturer's instructions. Infected cells (60-ml culture) were harvested after 36 h and resuspended in 0.3-0.5 ml of Tris-buffered saline (50 mM Tris-HCl, pH 7.6, 150 mM NaCl). The suspension was mixed with equal amount of lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2% Nonidet P-40 and 2x protease inhibitor mixture (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 245,000 x g for 20 min, the supernatant was agitated with 0.2 ml of ANTI-FLAG® M2-agarose beads (Sigma) overnight. C99-FLAG was eluted from the beads by incubation with 0.2 ml of 100 mM glycine HCl, pH 2.7, for 10 min at room temperature, and the eluate was immediately neutralized by addition of 1/25 volume of 1 M Tris-HCl, pH 8.0. Concentrations of residual Nonidet P-40 in purified C99-FLAG were estimated to be 0.3-0.4% from elution volume. The eluted C99-FLAG was confirmed for its purity and quantified by Coomassie Brilliant Blue staining after gel electrophoresis.
-Secretase Assay and Detection of A and AICD—Microsomal fractions of CHO cells were obtained as described previously (15). Briefly, the harvested cells were homogenized in buffer A (20 mM PIPES, pH 7.0, 140 mM KCl, 0.25 M sucrose, 5 mM EGTA) using a glass/Teflon homogenizer. The homogenates were centrifuged at 800 x g for 10 min to remove nuclei and cell debris. The postnuclear supernatants were recentrifuged at 100,000 x g for 1 h. The resulting pellets representing the microsomal fractions were suspended in a buffer (50 mM PIPES, pH 7.0, 0.25 M sucrose, 1 mM EGTA). Their protein concentrations were adjusted at 10 mg/ml. The membranes were solubilized by the addition of equal volume of 2x NK buffer (50 mM PIPES, pH 7.0, 0.25 M sucrose, 1 mM EGTA, 2% CHAPSO (Sigma; catalogue number C3649; lot numbers 013K5314 and 015K5313), 2 mM diisopropyl fluorophosphate, 20 µg/ml antipain, 20 µg/ml leupeptin, 20 µg/ml TLCK, 10 mM phenanthroline, and 2 mM thiorphan) and incubated on ice for 1 h (20, 21). After centrifugation at 100,000 x g for 1 h, the supernatants were saved (1% CHAPSO lysate). 1% CHAPSO lysate was diluted with 3 volumes of CHAPSO-free buffer (50 mM PIPES, pH 7.0, 0.25 M sucrose, 1 mM EGTA, 1 mM diisopropyl fluorophosphate, 10 µg/ml antipain, 10 µg/ml leupeptin, 10 µg/ml TLCK, 5 mM phenanthroline, and 1 mM thiorphan) containing defined amounts of C99-FLAG. Furthermore, 0.1% phosphatidylcholine (catalogue number P3556, lot number 034K5218; Sigma) was added into the diluted lysate, which significantly enhanced the activity of -secretase but did not alter the proportion of A40/42 produced. Residual Nonidet P-40 in the substrate should be estimated carefully before starting the reaction. The Nonidet P-40 concentrations at 0.2% and above in the reaction mixture abolished -secretase activity. Its final concentrations in all reactions in this study were kept less than 0.06%, which exhibited no noticeable suppression in A and AICD productions. After incubation at 37 °C for 3 h, lipids were extracted with chloroform/methanol (2:1), and protein residues were subjected to quantitative Western blotting with defined amounts of synthetic A as a control. For AICD standard, we used CTF50 synthetic peptide (AICD-(50-99); Calbiochem) or AICD-(50-99)-FLAG expressed in Escherichia coli as described below. For detection of A, 82E1, a monoclonal antibody end-specific for the amino terminus of human A (IBL, Gunma, Japan) was used (23). To visualize AICD, UT-421 (gift of Dr. T. Suzuki, Hokkaido University) raised against carboxyl-terminal sequence of APP was used (24). All blots in this study were immersed in boiled phosphate-buffered saline for 5 min before blocking in 5% skim milk, which significantly enhanced the detectability of the antigens.
Quantification of Authentic AICD-(50-99)-FLAG—AICD-(50-99)-FLAG was expressed in E. coli and affinity-purified with ANTI-FLAG M2 beads®. The protein concentrations of authentic AICD-(50-99)-FLAG were determined using both amino acid compositional analysis that determines the total protein amount, and sequence analysis that assesses the specific amount of AICD-(50-99)-FLAG. The appropriate amounts of AICD-(50-99)-FLAG were purified by reverse-phase liquid chromatography on super-Phenyl column (Tosoh, Tokyo, Japan) with 0-48% gradient of acetonitrile in 0.1% trifluoroacetic acid. Peak fractions were collected and rechromatographed under the same condition. Compared between the peak areas of the first and second chromatogram, chromatographic recovery was estimated (about 58%). Pooled fractions from the second chromatography were dried and hydrolyzed in 6 N HCl vapor at 110 °C for 20 h. The acid hydrolysate was derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and quantified as described by Shindo et al. (25). For amino acid sequence analysis, an appropriate amount of AICD-FLAG was subjected to Edman degradation using a Procise cLC protein sequencing system (Applied Biosystems, Foster City, CA) to obtain the amino-terminal 10-residue sequence. Repetitive yield and initial yield were calculated from the sequence.
Detection of Longer As Produced by -Secretase—Various longer A species were separated on a Tris, Tricine, 8 M urea gel with minor modifications (22, 27). A 10% T/3% C separation gel, pH 8.45, containing 8 M urea (gel system I) was used to separate A37 through A45. A 12% T/3% C separation gel, pH 8.90, containing 8 M urea (gel system II) was used to separate A46 through A49. The spacer and stacking gels did not contain urea. Following transfer, the blots were probed with 82E1 to detect only As that begin at Asp-1 and developed using an ECL system. Intensities of the bands were quantified using a LAS-1000 plus luminescent image analyzer (Fuji Film, Tokyo, Japan).
Immunoprecipitation of -Secretase—1% CHAPSO lysate of mouse embryonic fibroblasts (MEF) or WT or MTPS1-transfected CHO cells was diluted in 3 volumes of CHAPSO-free buffer (26). -Secretase complex was immunoprecipitated with anti-nicastrin antibody (Sigma) and protein G-Sepharose (Amersham Biosciences). After sufficient washing with 0.25% CHAPSO buffer (50 mM PIPES, pH 7.0, 250 mM sucrose, 1 mM EGTA, 0.25% CHAPSO, 1 mM diisopropyl fluorophosphate, 10 µg/ml antipain, 10 µg/ml leupeptin, 10 µg/ml TLCK, 5 mM phenanthroline, and 1 mM thiorphan), -secretase complex bound to protein G-Sepharose was allowed to react with 500 nM C99-FLAG in 0.25% CHAPSO buffer containing 0.1% phosphatidylcholine at 37 °C for 4 h with gentle agitation. The reaction mixtures were subjected to quantitative Western blotting for A and AICD (22, 27).
Mass Spectrometric Analysis of AICDs—After incubating 0.25% CHAPSO lysate of CHO cells with C99-FLAG in the presence of 0.1 mM bestatin, 10 µM amastatin, 0.1 µM arphamenine A, the uncleaved excess C99-FLAG was largely removed by immunoprecipitation with 4G8, a monoclonal antibody raised against 17-28 residues of A (epitope, 17-24 residues) (Signet Laboratories, Dedham, MA). The produced AICD was immunoprecipitated with Anti-FLAG® M2-agarose beads and extracted with 30% acetonitrile in 1% trifluoroacetate. Masses of the peptides were determined with a matrix-assisted laser desorption ionization-TOF-mass spectrometer, Autoflex® (Bruker Daltonics, Ibaraki, Japan). Samples were prepared by the thin layer method using sinapinic acid as a matrix (14).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Secretase Assay System—The relationship between -cleavage and AICD production has been a matter of debate. Studies by multiple research groups implied that -cleavage was largely independent of -cleavage (28-31). On the other hand, Pinnix et al. (32) noted a potential link between A and AICD productions. In their paper, they identified and considered AICD as a counterpart of A. Recently, we and others (14, 15, 33) identified novel -cleavage that liberates AICD and assumed that -cleavage was the primary event. We also assumed that equal amounts of total A and AICD are produced by -secretase (14, 15, 33). Most of quantitative studies on A and AICD productions were performed in the presence of large amounts of CTF, which is processed to p3 and AICD but not to A. In addition, levels of A and AICD were assessed by methods based on different principles, such as enzyme-linked immunosorbent assay for A released into media and indirect luciferase transactivation assay for AICD. To our knowledge, rigorous quantitative determination by conventional Western blotting of the produced A and AICD has never been performed.
To examine the stoichiometry of A relative to AICD, we took advantage of the CHAPSO-solubilized -secretase assay system (20, 21). C99 was fused at the carboxyl terminus with FLAG tag (C99-FLAG) and expressed in Sf9 cells. After C99-FLAG was purified by Anti-FLAG M2 (see "Experimental Procedures"), and its purity was confirmed, and its amounts were quantified by Coomassie Brilliant Blue staining after gel electrophoresis (see Fig. 2B). Purified 0.5 µM C99-FLAG was incubated in 0.25% CHAPSO-lysate of CHO cells. A species produced by our assay system were separated on gel system I (22, 27) (Fig. 1A). The amount of each A species was estimated from densitometric scanning of the Western blot and increased in a time-dependent linear fashion (data not shown). Each A species appeared to be generated by random cleavage depending on the interaction between susceptible sites and catalytic sites. Robust signals for A42 and A43 were detected as well as for A40, which contrasts with the major A species in cell-based and cell-free assays (1, 14, 34, 35) (Fig. 1A). In addition to A45, A species longer than A45 were also detected and stuck at the bottom of gel (Fig. 1A, top panel). Gel system II (22, 27) revealed that these longer species were A48 and A49 (Fig. 1A, bottom panel, and see also Fig. 4B). Against increasing concentrations of C99-FLAG, band intensities of all A species were plotted as the percentage of intensity of synthetic A45 (50 pg) on each blot (Fig. 1, B and C). The production rates of all A species appeared to follow the Michaelis-Menten type curve (Fig. 1C). The apparent Km (Kapp) and Vmax values for various A species were summarized in Table 1. As can be seen in Fig. 1, A and B, this solubilized -secretase assay system was characterized by abundant A40, A42, and A43 and small or trace amounts of A45, A48, and A49. The majority of A species produced by the assay system consisted of A40, A42, and A43 (see below).
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production in a dose-dependent manner (Fig. 1, D and E). As expected, L-685,485, a transition state analogue inhibitor, uniformly suppressed production of all A species (Fig. 1D). Most unexpectedly, DAPT, a nontransition state analogue inhibitor, did not result in a build up of A43 and A46 (22, 27, 33, 36) but uniformly suppressed the production of all A species (Fig. 1E). This may indicate that membrane integrity is a prerequisite for DAPT-induced accumulation of A43 and A46, as this phenomenon was observed in cell-based assay (27) and cell-free assay using a microsomal fraction.5
Stoichiometric Relationship between A and AICD—Purified C99-FLAG was incubated with CHAPSO-solubilized microsomal fraction from CHO cells. The A at about 4 kDa on the SDS gel was quantified by Western blotting as reported previously (15). Total AICD was quantified using synthetic CTF50 (AICD-(50-99), eight residues smaller than the substrate AICD-FLAG) as a control. The highest molecular mass band at about 15 kDa and the lowest molecular mass band at about 9 kDa before incubation represent presumably amino-terminally extended C99-FLAG with residual signal peptide and truncated C99-FLAG, respectively (Fig. 2A, bottom panel). Coomassie Brilliant Blue staining cannot detect those fragments, and thus only trace amounts of the fragments contaminate the reaction mixture (Fig. 2B). The A band at about 4 kDa on the SDS gel most likely represents A40, A42, and A43. As longer than A43 have slower mobilities (data not shown and see Ref. 22) and can be separated from the major A band at about 4 kDa. The longer As appear to be present in very small amounts, as they constituted around 10% of the total amount estimated from densitometric scanning of the Western blot (gel I; Fig. 1A and Table 1), assuming that the retention efficiency for each A species is equal. Thus, A here indicates virtually the sum of A40, A42, and A43. Time course analysis showed that equal amounts of A and AICD increased similarly in a time-dependent manner (Fig. 2, A and C); the kinetics of A and AICD production was statistically indistinguishable (Fig. 2D). These observations indicate that cleavage of C99 provides equal amounts of A (mostly A40, -42, and -43) and AICD. The rates of A and AICD production apparently followed Michaelis-Menten type curve (Fig. 2D). The apparent Km (Kapp) values of C99 for A and AICD production were 507.93 ± 26.45 and 468.72 ± 129.26 nM, respectively (Table 2). Those values are roughly consistent with Km values reported previously for crude and purified -secretase (20, 21, 37). The apparent Vmax values for A and AICD were 433.53 ± 51.94 and 419.62 ± 19.39 pM/min, respectively (Fig. 2D and Table 2). Those values were more than 30-fold higher than reported previously (21, 35). Such high activity of -secretase in our system made it possible to use conventional Western blotting to accurately quantify the amounts of A and AICD produced.
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and AICD in reaction mixtures containing MTAPP and MTPS1/2. The CHAPSO lysates were reacted with C99-FLAG containing FAD mutations T714I, V717F, and L723P, and an artificial mutation I716F (I45F, according to A numbering) (38). As shown in Fig. 3A, the MTAPPs tested in this study caused substantial to profound reductions in A and AICD productions (Fig. 3A). Nevertheless, these MTAPPs did not alter the stoichiometry between A and AICD (Fig. 3B). Similarly, MTPSs led to significantly reduced production of A and AICD but maintained the one-to-one stoichiometry indicated for WTPS1/2 (Fig. 3, C and D). In G384A MTPS1, an additional signal was detected above the A band (Fig. 3C) and was identified as a mixture of A48 and A49 on gel II (data not shown). Densitometric analysis showed that the longer As produced by G384A MTPS1 amount to more than 40% of total As, as compared with less than 10% in WTPS1. Signals from longer As were included in the quantification of A for G384A MTPS1. Weak additional signals for longer As were also observed in WTPS2 and N141I MTPS2 and similarly included in quantification of A (Fig. 3, C and D).
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s, A48 and A49, Produced by -Secretase—Our previous observations on -cleavage itself led to predict the two counterparts of AICDs, namely A48 and A49 (14). Consistent with this assumption, the cells expressing A48 or A49 secreted A40 and A42 into media in a PS-dependent manner (15).
By using this solubilized system, we sought to confirm -secretase-dependent production of A48 and A49. -Secretase complex that has been known to contain mature (highly glycosylated) nicastrin (39) was immunoprecipitated with anti-nicastrin antibody from 0.25% CHAPSO-solubilized MEF lysate. This partially purified -secretase complex was incubated with C99-FLAG, and the reaction mixtures were subjected to gel system II to detect A48 and A49. The immunoprecipitate from WTMEF by anti-nicastrin antibody produced A and AICD from C99-FLAG, whereas that by preimmune serum generated negligible levels of A and AICD (Fig. 4A). Precipitate from PS1/2-deficient MEF contained only an immature form of nicastrin and generated no products (Fig. 4A). Gel II clearly shows the two distinct bands for A48 and A49 in the immunoprecipitate from WTMEF but none in that from PS1/2-deficient MEF (Fig. 4B). Furthermore, A48 and A49 were profoundly reduced by the addition of L-685,458, in a dose-dependent manner (data not shown). These data indicate that -secretase does produce A48 and A49 as well as other As. A46 was undetectable in this CHAPSO-solubilized system, for which currently we cannot offer an appropriate explanation (22, 27, 33, 36).
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-Secretase—In the cell-free system, two -cleavage sites along C99, which released AICD-(49-99) and AICD-(50-99), were detected. Despite intensive efforts, we failed to identify any longer AICDs, for example AICD-(41-99) and AICD-(43-99), as potential counterparts of A40 and A42, respectively (6, 14). Because our solubilized system allows free collision of enzyme and substrate in the solution, it is quite possible that various -cleavage sites could emerge, generating various lengths of AICDs. If C99-FLAG is cleaved once along its transmembrane domain, various AICDs can be expected as counterparts of various A species. Because A40, A42, and A43 are abundant in the assay system, their counterparts, AICD-(41-99), AICD-(43-99), and AICD44-99, should have been abundant.
The CHAPSO lysate of CHO cells was incubated with C99-FLAG at 37 °C. Uncleaved C99-FLAG was removed by the pretreatment with 4G8, and AICD-FLAG was subsequently precipitated with Anti-FLAG® M2-agarose. The immunoprecipitate was subjected TOF-mass spectrometry. Most surprisingly, even under this solubilized condition, only two species of AICD, AICD-(49-99)-FLAG and AICD-(50-99)-FLAG, were detected (6, 14) (Fig. 4C), which contrasts with the presence of various A species generated, as shown by the gel system I (Fig. 1A).
To confirm further the stoichiometry between A and AICD produced by -secretase, immunoprecipitated -secretase was used to avoid possible contamination with proteases. In addition, instead of AICD-(50-99), AICD-(50-99)-FLAG expressed in E. coli was used as a strict control. AICD-(50-99)-FLAG purified from E. coli was quantified by amino acid compositional analysis and sequence analysis (see "Experimental Procedures"). Compared with that from WT and M146L MTPS1, the purified -secretase from G384A MTPS1 exhibited profoundly reduced enzymatic activity, as observed in the CHAPSO-solubilized membrane (Fig. 4D, left panel). More importantly, purified -secretases from three cell lines similarly exhibited one-to-one stoichiometry between A and AICD (Fig. 4D, right panel). Again, only two AICDs were detectable (6, 14) (Fig. 4C).
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-cleavage sites using the CHAPSO-solubilized assay system. The CHAPSO lysate was incubated with WT or MTC99-FLAG and subjected to the gel system I (Fig. 5A). T714I MTC99-FLAG remarkably increased the proportion of A42, decreased the proportions of A40 and A43 (Fig. 5A), and released only AICD-(49-99)-FLAG (Fig. 5B). This contrasts with WTC99-FLAG, which produced both AICD-(49-99)-FLAG and AICD-(50-99)-FLAG. With V717F MTC99-FLAG, the abundance of individual A species on gel I was similar to that with WTC99-FLAG, but only AICD-(49-99)-FLAG was detected. An artificial mutant, I716F MTC99-FLAG (I45F, according to A numbering), predominantly produced A42, but both AICD-(49-99)-FLAG and AICD-(50-99)-FLAG were detected. No detectable amounts of A and AICD were produced from L723P MTC99-FLAG.
Similarly, the effects of MTPS1/2 on the A and AICD species generated were investigated. As was the case with WTC99-FLAG, WTPS1/2 showed robust production of A42 and A43 (Fig. 6A) and generated AICD-(49-99)-FLAG and AICD-(50-99)-FLAG, with the latter giving a stronger signal (Fig. 6B). Although M146L MTPS1 produced A40 as did WTPS1, M233T and G384A MTPS1 and N141I MTPS2 similarly exhibited an increase in the A42/A40 ratio on gel I (Fig. 6A). In addition, it is of note that each MTPS1/2 invariably provides higher signal intensity for AICD-(49-99)-FLAG relative to that for AICD-(50-99)-FLAG (Fig. 6B). Most interestingly, M233T MTPS1 produced only AICD-(49-99)-FLAG, as did T714I and V717F MTC99-FLAG (see Fig. 5B). After incubation, the reaction mixture was subjected to the gel system II. Although both A48 and A49 were detectable in the WTPS1-containing reaction mixture, only A48 was identified in the M233T MTPS1-containing reaction mixture (Fig. 6C). These observations strongly suggest that A48 is a counterpart of AICD-(49-99)-FLAG exclusively generated by M233T MTPS1. These also warrant the accuracy of identification of longer A by the present gel systems.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Secretase cleaves type I membrane proteins within their transmembrane domains (40). The type I membrane proteins as the substrates of -secretase are characterized by ectodomain shedding, which is mediated by a wide variety of membrane-bound proteases (shed-dases) (41). The relationship between ectodomain shedding and intramembrane proteolysis is well known for the processing of APP, in which ectodomain shedding occurs through - or -cleavage (1). The cleavages at the - and -sites are necessary and sufficient for the execution of -cleavage, which results in A production. However, the relationship between - and -cleavages has remained unclear (42). Although several reports implied that - and -cleavages are independent processes (28-31), we found previously that there is a significant correlation between - and -cleavages (14, 15). Here we have further examined the relationship by taking advantage of a solubilized -secretase assay system with high specific activity, in which use of recombinant C99-FLAG substrate allowed us to exclude contamination by C83-derived AICD. Our major finding using this CHAPSO-solubilized assay system is that equal amounts of A and AICD are produced from C99 and that only two AICD-(49-99)-FLAG and AICD-(50-99)-FLAG are detectable. Even in MTAPP and MTPS1/2, the stoichiometry between A and AICD is almost identical.
In the this study, we identified nothing but AICD-(49-99)-FLAG and AICD-(50-99)-FLAG, despite detection of various A species, including A40, A42, A43, A45, A48, and A49. This is surprising because the present CHAPSO-solubilized system warrants free collision of enzyme and substrate. One may point out that very hydrophobic peptides often cannot be detected by TOF-mass spectrometry, and thus longer AICDs, even if they exist, may not be detected. However, we previously detected AICD-(46-99), which was produced by the membrane prepared from cells transfected with artificial MTAPP (Ew; -cleavage sites are replaced by a stretch of tryptophan) (43). This strongly suggests that no significant amounts of longer AICDs are produced in the assay system.
In our previous report, we detected A46 and A48 but failed to identify A49 in the cell lysates (27). But in the solubilized assay system, small or trace amounts of A48 and A49 were invariably detected. At present, we cannot explain the discrepancy between the present data and previous report. Possibly, the absence or presence of the membrane may have an effect on the processing of C99 by -secretase. However, this study has clearly shown that the immunoprecipitated -secretase generates A48 and A49 in a presenilin/nicastrin-dependent manner. In addition, production of A48 and A49 was prevented by L-685,458 in a dose-dependent manner (data not shown). It is most likely that A48 and A49 are counterparts of AICD-(49-99)-FLAG and AICD-(50-99)-FLAG, respectively, and potential intermediates for A40, A42, and A43. In this context, it is of note that, even if -cleaved AICD-(49-99)-FLAG and AICD-(50-99)-FLAG are predominant in the reaction mixture, only a trace or very small amounts of A48 and A49 can be detected. This indicates that, once generated, A48 and A49 promptly undergo -cleavage, which occurs at the carboxyl termini of Ile-45, Thr-43, Ala-42, and Val-40, presumably depending upon the affinity between the cleavage sites of the substrate and catalytic sites of -secretase (Fig. 1A). Thus, the abundance of various A species in the reaction mixture may reflect varying affinities between susceptible sites and catalytic sites of -secretase. The order of susceptibility to -secretase (in decreasing order) on the carboxyl terminus is Val-40, Ala-42, Thr-43 >> Ile-45 > Leu-49 > Thr-48. Most interestingly, A46 is undetectable, and the carboxyl terminus of Val-46 is least susceptible in this assay system, which contrasts sharply with the ready detectability of A46 in the cell-free system (22, 27). In addition, DAPT treatment of solubilized -secretase uniformly suppressed the levels of A40, A42, and A43 but failed to accumulate A43 and A46 (22, 27) (Fig. 1E). Thus, one should bear in mind that the present -secretase assay system can simulate only a part of cell-based or cell-free system. Nevertheless, MTC99 and MTPS1/2 produced a higher proportion of A42 and a higher proportion of AICD-(49-99), thus still maintaining the important characteristics of MTAPP and MTPS1/2.
The increased proportion of AICD-(49-99) produced by MTC99 and MTPS1/2 in the CHAPSO-solubilized -secretase assay system is consistent with our previous report on AICD production in the cell-free system (14). Interestingly, T714I and V717F MTC99 generated only AICD-(49-99). It would be of interest to identify longer As in these MTC99-containing reaction mixtures. Unfortunately, we were unable to identify corresponding longer As species because they have altered mobilities on the gel, because of amino acid substitutions. In our experience it is not possible to identify longer As by TOF-mass spectrometry, presumably because of their hydrophobicity.
Alternative proof that -cleavage is followed by -cleavage could be provided by a reciprocal experiment. We attempted to generate substrates CTF-(41-99)-FLAG and CTF-(43-99)-FLAG, potential counterparts of A40 and A42, respectively, and we examined whether AICD was produced from those substrates. When those CTF-FLAG substrates, amino-terminally fused with signal peptide of-galactosidase A, were expressed in Sf9, most of purified CTF-FLAG fragments still had uncleaved signal peptide at the amino terminus, which were not appropriate for the reciprocal experiment. Although we failed to do this experiment, Shah et al. (44) recently proposed a very interesting model for the mechanism of -cleavage. They found that nicastrin acts as a gatekeeper in -secretase complex by binding to an amino terminus of the substrate (44). According to their model, the amino terminus of the substrate should protrude from membrane to the extracellular/luminal side. In view of this, it is unlikely that CTF-(41-99) and CTF-(43-99) act as -secretase substrates, because of absence of their ectodomains. Taken together, our data support the view that -cleavage precedes -cleavage and triggers subsequent -cleavage to produce various A species.
The most striking feature of our assay system is high specific activity of -secretase. Apparent Vmax values of A and AICD were 
400 pM/min. These values are roughly more than 30-fold higher than those reported previously (21, 35). This high activity of our assay system makes possible unambiguous detection of A and AICD by conventional Western blotting. In fact, we successfully determined the stoichiometry between A and AICD and detected minor A species, such as A45, A48, and A49, which are probably unable to be detected by enzyme-linked immunosorbent assay, by combination of new gel systems I and II. The reasons for higher activity of -secretase in our assay system would be as follows. First, 0.25 M sucrose was added to the reaction mixture. In the presence of such polyol, -secretase might be stabilized and active in cleaving substrates for a longer time. We examined the effects of 0.25 M sucrose on -secretase activity, and we observed 4-5-fold A production in the presence of sucrose, but such an increase is not enough to account for the present high activity.6
Second, the substrate C99-FLAG was expressed in and purified from Sf9 cells. We thought that protection of the transmembrane domain of substrate with native lipids would be important to take a right conformation with which the substrate readily interacts with -secretase. Even after solubilization with Nonidet P-40, C99-FLAG may still retain cellular lipids derived from Sf9 cell membranes. In this condition, the addition of phosphatidylcholine to the reaction mixture further enhanced -secretase activity. This cannot be realized by the bacterial expression system in which the expressed substrate was recovered as insoluble inclusion bodies probably caused by interaction through its exposed transmembrane domain. Once inclusion bodies were formed, it would be difficult for the substrate to become soluble and to take a right conformation, even if solubilized, by subsequent addition of lipid or detergent. Thus, we believe that the substrate expressed in and purified from Sf9 cells greatly contributes to the high specific activity of our solubilized assay system. Accordingly, C99-FLAG substrate from Sf9 cells was compared with that from E. coli in terms of A production. Roughly 7-fold more A was produced with C99-FLAG derived from Sf9 cells, compared with that from E. coli.6
Third, the absence of a formyl group at the amino terminus of the substrate may also contribute to high activity of the assay system. As the substrate fused with a signal peptide was expressed in Sf9 cells, the purified substrate should possess a free amino terminus after removal of signal peptide. When expressed in E. coli, a protein is usually N-formylated, and this modification leads to significantly reduced -secretase activity (44). It would be possible that the free amino terminus of the substrate is required for its efficient interaction with -secretase.
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FOOTNOTES |
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1 Both authors contributed equally to this work.
2 Support by IBL Co. Present address: Immuno-Biological Laboratories Co., Ltd., 1091-1 Naka, Fujioka, Gunma 375-0005, Japan.
3 To whom correspondence should be addressed: Dept. of Neuropathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-3541; Fax: 81-3-5800-6854; E-mail: yihara{at}m.u-tokyo.ac.jp.
4 Theabbreviationsusedare:A,amyloid-protein;AD,Alzheimer'sdisease;APP,-amyloid precursor protein; CTF, carboxyl-terminal fragment; AICD, APP intracellular domain; FAD, familial Alzheimer's disease; PS1, presenilin 1; PS2, presenilin 2; MEF, mouse embryonic fibroblast; WT, wild-type; MT, mutant; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; CHO, Chinese hamster ovary; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; TOF, time-of-flight; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester.
5 M. Takami, S. Funamoto, Y. Ihara, unpublished data.
6 N. Kakuda, S. Funamoto, and Y. Ihara, unpublished data.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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