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The Journal of Neuroscience, January 12, 2005, 25(2):436-445; doi:10.1523/JNEUROSCI.1575-04.2005
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Neurobiology of Disease
Longer Forms of Amyloid  Protein: Implications for the Mechanism of Intramembrane Cleavage by  -Secretase
Yue Qi-Takahara,1
Maho Morishima-Kawashima,1
Yu Tanimura,1
Georgia Dolios,2
Naoko Hirotani,3
Yuko Horikoshi,5
Fuyuki Kametani,6
Masahiro Maeda,5
Takaomi C. Saido,4
Rong Wang,2 and
Yasuo Ihara1
1Department of Neuropathology, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan, 2Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029-6574, 3Resources Center and 4Laboratory for Proteolytic Neuroscience, Brain Science Institute, RIKEN, Saitama 351-0198, Japan, 5Immuno-Biological Laboratories Company, Ltd., Gunma 375-0005, Japan, and 6Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo 156-8585, Japan
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Abstract
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 -Cleavage of  -amyloid precursor protein (APP) in the middle of the cell membrane generates amyloid protein (A), and  -cleavage,  10 residues downstream of the -cleavage site, releases the APP intracellular domain (AICD). A significant link between generation of A and AICD and failure to detect AICD41-99 led us to hypothesize that -cleavage generates longer As, which are then processed to A40/42. Using newly developed gel systems and an N-end-specific monoclonal antibody, we have identified the longer As (A1-43, A1-45, A1-46, and A1-48) within the cells and in brain tissues. The production of these longer As as well as A40/42 is presenilin dependent and is suppressed by {1S-benzyl-4R-[1S-carbamoyl-2-phenylethylcarbamoyl-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester, a transition state analog inhibitor for aspartyl protease. In contrast, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a potent dipeptide -secretase inhibitor, builds up A1-43 and A1-46 intracellularly, which was also confirmed by mass spectrometry. Notably, suppression of A40 appeared to lead to an increase in A43, which in turn brings an increase in A46, in a dose-dependent manner. We therefore propose an  -helical model in which longer A species generated by -cleavage is cleaved at every three residues in its carboxyl portion.
Key words: amyloid -protein; APP; -secretase; intramembrane cleavage; presenilin; Alzheimer's disease
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Introduction
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Senile plaques, one of the neuropathological hallmarks of Alzheimer's disease (AD), are composed of a small, 40-residue protein called amyloid protein (A). A is produced from -amyloid precursor protein (APP), through sequential cleavage by membrane proteases referred to as - and -secretases (Selkoe, 2001 ). -Secretase was identified as a membrane-bound aspartyl protease, -site APP-cleaving enzyme (Vassar et al., 1999), that generates CTF, an immediate substrate for -secretase, but the nature of -secretase has remained an enigma. Accumulating evidence strongly suggests that -secretase is also an aspartyl protease with its catalytic site(s) sitting within the membrane (for review, see Haass, 2004). Several A species consisting of 36-43 residues are finally generated and constitutively secreted. Among the secreted species, A40 is the most predominant species, and a longer species, A42, is a minor one (<10%). However, this minor form is indeed initially deposited in the brain and predominates in diffuse and mature plaques (Iwatsubo et al., 1994). This is probably because A42 has a much higher aggregation potential than A40. Most importantly, all of the mutations of presenilin (PS) 1/2 and APP that lead to familial AD (FAD) cause increased A42 production (for review, see Selkoe, 2001). Thus, the most important issue is how A, especially A42, is generated from APP through the action of PS1/2.
APP is cleaved by -secretase, not only in the middle of the transmembrane domain (-cleavage), but also near the membrane-cytoplasm boundary (referred to as -cleavage) (Gu et al., 2001; Sastre et al., 2001; Yu et al., 2001; Weidemann et al., 2002). This -cleavage site is located a few residues inside the membrane from the boundary and is very close to site 3 for cleavage of Notch (for review, see Selkoe and Kopan, 2003). The major product of -cleavage is an APP intracellular domain (AICD) that begins at Val-50, whereas the minor one is AICD49-99 (Gu et al., 2001; Sastre et al., 2001; Yu et al., 2001; Weidemann et al., 2002).
We have recently found that there is a link between AICD50-99 and A40 production and a link between AICD49-99 and A42 production (Sato et al., 2003). This potential link raises additional questions. Which cleavage, - or -cleavage, comes first, and how is one related to the other? Because we failed to detect a particular AICD longer than AICD49-99 (Gu et al., 2001), one possibility is that CTF is first cleaved at the -sites, and the products generated (A1-48 and 1-49) undergo -cleavage, generating A40/42. Another possibility is that - and -cleavage occur simultaneously or nearly so along the CTF molecule, leaving a small, 10-residue hydrophobic membrane peptide.
To identify the A species longer than A1-42, we have modified an SDS/urea gel system and developed a monoclonal antibody highly specific for the N terminus of A. These two tools have now clearly shown the presence of distinct A species longer than A1-42 within the cells and brain. This has important implications for understanding the mechanism of intramembrane cleavage for APP.
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Materials and Methods
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Cell culture. Chinese hamster ovary (CHO) cells stably expressing wild-type (wt) APP751 (7WD10 cells) or V717F mutant (mt) APP and 7WD10 cells stably overexpressing M146L mtPS1 were cultured as described previously (Koo and Squazzo, 1994; Podlisny et al., 1995; Xia et al., 1997). Human embryonic kidney 293 (HEK 293) cells stably expressing wtAPP695 and mouse neuroblastoma N2a cells expressing "Swedish" mtAPP695, kindly provided by Drs. C. Haass (Ludwig-Maximilians University, Munich, Germany) and S. S. Sisodia (University of Chicago, Chicago, IL), respectively, were cultured as described previously (Citron et al., 1992; Thinakaran et al., 1996). The 7WD10 cells were transfected stably with wt or various mtPS1/2 cDNAs (Qi et al., 2003).
Generation of cell lines. The pcDNA4/TO vector (Invitrogen, Carlsbad, CA) with SP-DA-CTF1-99 cDNA insert (Lichtenthaler et al., 1999a) was transfected into T-Rex-CHO cells (Invitrogen) using Lipofectamine2000 (Invitrogen), and the stable cell lines were selected using 500 µg/ml Zeocin (Invitrogen). Expression of CTF was induced by the addition of 1 µg/ml tetracycline (Invitrogen) to the culture media. Dominant-negative (DN) mtPS1 (D257A/D385A) (Wolfe et al., 1999) cDNA was generated using the Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). The pIRESneo3 vectors (BD Biosciences, Palo Alto, CA) with wt and DN mtPS1 cDNA inserts were transfected into T-Rex-CHO cells stably expressing CTF, and stable cell lines were selected with 500 µg/ml G418 sulfate. The plasmids of wtAPP695, APP695 bearing a di-lysine endoplasmic reticulum (ER) retention signal (Jackson et al., 1990), and APP695 carrying the trans-Golgi network (TGN) sorting signal of TGN38 (SDYQRL) (Ponnambalam et al., 1994) at its C terminus were kindly provided by Dr. H. Komano (National Institute for Longevity Sciences, Aichi, Japan) (Sudoh et al., 2000). Each plasmid was transfected to CHO cells, and stable cell lines were selected with G418.
Treatment with -secretase inhibitors. CHO cells inducibly expressing CTF were incubated with a -secretase inhibitor, either {1S-benzyl-4R-[1-(1S-carbamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester (L-685,458) (Calbiochem, San Diego, CA) (Shearman et al., 2000) or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Calbiochem) (Dovey et al., 2001), at indicated concentrations for 2 hr and then cultured in the presence of 1 µg/ml tetracycline and each -secretase inhibitor for 4 hr to induce CTF production. The treatment with Compound E (Calbiochem) (Seiffert et al., 2000) was performed similarly.
Antibodies. Synthetic A peptides (DAEFRHDSGYEVHHQK and DAEFR) were conjugated to thyroglobulin through Cys at the C termini. The former was used for primary immunization with an adjuvant, whereas the latter was used for booster injections. Hybridomas were produced by polyethylene glycol-mediated fusion between immunized splenocytes and X63-Ag8-653 (Kinebuchi et al., 1991), and the clone 82E1 was selected using peptide-coated immunoplates.
Other monoclonal antibodies against A that were used were 6E10 (raised against A1-17), 4G8 (epitope: A17-24; Signet Laboratories, Dedham, MA), and BAN50 (raised against A1-16) (Suzuki et al., 1994). The polyclonal antibodies against the cytoplasmic domain of APP were UT421 (Tomita et al., 1998) and C4 (Takio et al., 1989). The polyclonal antibodies against PS1 (anti-G1L3) were described previously (Tomita et al., 1999).
Immunoprecipitation of A from the conditioned media and cell lysates. The conditioned media for 6-8 hr culture were incubated with BAN50 at 4°C for 6 hr. Harvested cells were lysed with Tris-buffered saline (TBS) (in mM: 50 Tris-HCl, pH 7.6, 150 NaCI, 1 EGTA, and 1 EDTA) containing 1% Triton X-100 and various protease inhibitors (0.1 mM diisopropyl fluorophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml N-ptosyl-L-lysine chloromethyl ketone, 1 µg/ml antipain, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml bestain, 1 µg/ml amerstain, 5 mM 1,10-phenanthroline monohydrate, and 10 mM thiorphan). The homogenates were cleared by centrifugation at 540,000 x g for 20 min. The cell lysates were first immunoprecipitated with C4-bound protein G-Sepharose at 4°C for 1 hr to remove full-length APP and CTF, and the resultant supernatants were incubated further with BAN50 at 4°C for 6 hr. The immune complexes were collected with protein G-Sepharose and eluted with the Laemmli SDS sample buffer. The immunoprecipitated proteins were separated on Tris/Tricine/8 M urea gels, followed by Western blotting with 82E1.
For each set of the experiments, each supernatant was appropriately diluted to the same protein concentrations, and an equal volume of the supernatant was used for immunoprecipitation.
Fractionation of Tg2576 mouse brain homogenates. The cerebra of 2.5-month-old male Tg2576 mice (IBL, Fujioka, Japan) (Hsiao et al., 1996) were homogenized in 5 vol of TBS buffer containing protease inhibitors. The homogenate was centrifuged at 540,000 x g for 20 min to obtain a TBS-soluble fraction. After washing with the same buffer, the resultant pellet was homogenized in 5 vol of TBS buffer containing 1% Triton X-100 and protease inhibitors, and the homogenate was centrifuged at 540,000 x g for 20 min to obtain a Triton-soluble fraction. The resulting pellet was suspended in 1 vol of guanidine hydrochloride (GuHCI) by sonication. The suspension was then centrifuged at 265,000 x g for 20 min to obtain a GuHCI-soluble fraction, which was diluted 12-fold. Each fraction was subjected to immunoprecipitation with BAN50, as described above, and then the immunoprecipitates were separated on Tris/Tricine/8 M urea gels.
Modified Tris/Tricine/8 M urea gels and Western blotting. To separate A37 through A49, the protocol for Tris/Tricine/8 M urea gel described previously (Klafki et al., 1996) has been modified here. An 11% T plus 3% C separation gel, pH 8.45, containing 8 M urea was used to separate A37 through A45 (referred to as gel I). The dimensions of this slab gel were as follows: length of separation gel, 16 cm; length of spacer gel, 0.5 cm; length of stacking gel, 1.5 cm; width, 8.5 cm; thickness, 0.1 cm. To separate A46 through A49, a 10% T plus 3% C separation gel, pH 8.95, containing 8 M urea was used (referred to as gel II). The dimensions of this slab gel were as follows: length of separation gel, 20 cm; length of spacer gel, 1.0 cm; length of stacking gel, 1.5 cm; width, 8.5 cm; thickness, 0.1 cm. The compositions of the spacer gel and stacking gel were as described previously (Klafki et al., 1996).
Development of the blots was performed using an ECL system, and intensities of the bands were quantified with a LAS-1000plus luminescent image analyzer (Fuji Film, Tokyo, Japan) (Qi et al., 2003).
Peptide synthesis, purification, and characterization. All A peptides, A1-45 through A1-49, were synthesized using an automated peptide synthesizer (ABI 433A; Applied Biosystems, Foster City, CA). To avoid the formation of deleted derivatives, the amino-terminal extremities without fluorenylmethoxycarbonyl were capped with 0.5 M acetic anhydride, 0.125 M N,N'diisopropylethylamine, 0.015 M 1-hydroxybenzotriazole in 1-methyl-2-pyrrolidinone. For the final cleavage from the resin and removal of trifluoroacetic acid (TFA)-labile protecting groups, a mixture of 90% TFA, 6% phenol, 4% thioanisole, 2% ethanedithiol, and 4% H2O (v/v) was used for a total volume of 5 ml, and the mixture was kept at room temperature for 3 hr. Crude peptides were partially purified by size exclusion chromatography on Superdex 75 10/300 GL (10 x 300 mm) and Superdex peptide 10/300 GL (10 x 300 mm; Amersham Biosciences, Piscataway, NJ) columns, equilibrated with 20% 2-propanol and 80% formic acid (v/v) at a flow rate of 0.4 ml/min.
Sequences of peptides were confirmed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (Biflex, Bruker, Germany) and by amino acid analysis.
Immunoprecipitation/mass spectrometry assay. A peptide profiles were analyzed by immunoprecipitation/mass spectrometry (Wang et al., 1996). Aliquots of conditioned media containing 1% FCS and lysates from cells inducibly expressing CTF that were treated with and without DAPT were immunoprecipitated by monoclonal antibodies 4G8/6E10 and protein G/A agarose. CTF in the cell lysates was preabsorbed with C4-bound protein G-Sepharose. The molecular masses of immunoprecipitated A species were measured using a Voyager-DE STR MALDI TOF mass spectrometer (Applied Biosystems). Peptide samples were prepared by the thin-layer method using -cyano-4-hydroxycinnamic acid as matrix. Each spectrum was collected from 1000 laser irradiations. Mass spectra were calibrated using bovine insulin as the internal mass calibrant. Peaks corresponding to A peptides were identified using the measured molecular masses searching against A peptide sequence with a mass error tolerance of 200 ppm.
Other methods. Protein concentrations were determined in the presence of 1% SDS using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).
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Results
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Modified Tris/Tricine/8 M urea gels and a monoclonal antibody, 82E1
Because we were unable to detect longer AICD forms, including AICD41-99 and AICD43-99, in the cell lysates, we postulated that there might be a particular A species longer than A40/42. Thus, we modified the Tris/Tricine/8 M urea gel system (Klafki et al., 1996) and developed two gel systems to separate the A species, A1-37 through A1-45 and A1-46 through A1-49.
Synthetic A1-37 through A1-45 peptides were well separated by modifying the length of the gel and reducing the concentrations of acrylamide (see Materials and Methods). Because this gel system (termed gel I) cannot separate A1-46 through A1-49, which are stuck at the gel front (Fig. 1A), we developed a second gel system (termed gel II). Gel II has three modifications: extended gel length, reduced acrylamide concentrations, and slight alkalization of the separation gel (see Materials and Methods). In contrast to gel I, gel II is unable to separate A species shorter than A1-45, which are now stuck at the upper region of the gel, but can separate well A1-45 through A1-49 (Fig. 1B). The striking characteristic of these gel systems is that longer As migrate faster than shorter ones.
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Figure 1. Modified Tris/Tricine/8 M urea gels and specificity of 82E1. A, B, Synthetic A1-37 through A1-49 were separated using gel I (A) and gel II (B). In gel I (A), A1-37 through A1-45 are clearly separated, whereas A1-46 through A1-49 are stuck at the gel front. In gel II (B), A1-45 through A1-49 are well separated, whereas A1-37 through A1-44 are stuck at the upper region of the gel. C, Synthetic A1-40, A2-40, and A3-40 (500 and 50 pg of each) were subjected to Western blotting using 6E10 (top) or 82E1 (bottom). Whereas 6E10 labeled all of these As to similar extents, 82E1 recognized only A1-40. D, The cell lysates from CHO cells overexpressing CTF (lanes 1, 3) or APP (lanes 2, 4) were subjected to Western blotting with 82E1 (left) or 6E10 (right). 82E1 specifically labeled CTF but not full-length APP or several APP-derived products that are strongly labeled with 6E10.
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Because our primary concern was the C-terminal processing of A, we sought to focus on A1-Xs, A peptides that start from Asp-1. Otherwise, results must have been confounded by the presence of numerous N-terminally truncated As that extend similarly to various C termini (Haass et al., 1994; Wang et al., 1996). If we could collect only A1-Xs, identification of longer As would become possible by comparing their electrophoretic mobilities with those of authentic synthetic A1-Xs. We thus developed a new monoclonal antibody, 82E1. Whereas 6E10 labeled A1-40, A2-40, and A3-40 to the same extent, 82E1 labeled only A1-40, but never those truncated As (Fig. 1C). The full-length APP and its several derivatives, CTF and its N-terminally extended forms, were strongly labeled with 6E10 but never with 82E1, except for CTF (Fig. 1D). Thus, 82E1 is highly specific for the N terminus of A or CTF and is virtually end specific. Furthermore, 82E1 showed similar affinities for A1-40, A1-42, and A1-43 (data not shown), suggesting that this would also be the case with other shorter and longer As that start from Asp-1. Thus, the intensity of the 82E1 immunoreactivity should reflect the concentrations of various A1-Xs in the cell lysates. Hereafter, AX represents A1-X.
Longer As in various cell lysates and APP-transgenic mouse brain
We first sought to detect longer As in the cultured media and in the cell lysates of 7WD10 cells, CHO cells overexpressing wtAPP. The A species in the culture media and lysates were immunoprecipitated with BAN50 and subjected to electrophoresis on the above two gels, followed by Western blotting with 82E1.
Various A species, including A37, A38, A39, A40, and A42, were present in the conditioned media, an observation consistent with previous reports (Wang et al., 1996; Clarke et al., 1998; Beher et al., 2002) (Fig. 2A). A43 was virtually undetectable in the media using this protocol. In contrast, A species longer than A42, including A43, A45, A46, and A48, were reproducibly detectable in the cell lysates (Fig. 2A). It was difficult to see A37 (and sometimes A38) clearly in the lysate, because of superimposition with CTF on gel I (Fig. 2A). In the 7WD10 cells secreting predominantly A40, three species (A40, A43, and A46) were the major ones in the lysates, which contrasts with trace amounts of A42, A45, and A48 (Fig. 2A). We were consistently unable to detect A49, the longest A that we postulated exists (Fig. 2A).
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Figure 2. Longer As in the various cell lysates and the APP-transgenic mouse brain. A, BAN50 immunoprecipitates from the conditioned medium (Medium) and Triton-soluble fraction (Lysate) of 7WD10 cells were separated on gel I (top and middle) and gel II (bottom) and subjected to Western blotting with 82E1. The middle panel represents an overexposed blot (the same as above). A species longer than A42 are undetectable in the conditioned medium but are detectable in the cell lysate. B, BAN50 immunoprecipitates of Triton-soluble fractions from CHO, N2a, and HEK cell lines stably expressing APP were separated on gel I (top and middle) and gel II (bottom) and subjected to Western blotting with 82E1. The middle panel represents an overexposed blot. Although the relative levels of A species differ, the same longer A species as found in CHO cells are detectable in the N2a and HEK cell lines. In N2a cell line, a band just above A42 presumably represents A41. The origins of an extra band below A43 in the N2a lane and a band located between A45 and A46 seen in all three lanes are unknown. C, A43, A45, A46, and A48 are found exclusively in the Triton-soluble fraction of (plaque-free) brain homogenates from 2.5-month-old APP-transgenic mice (Tg2576). Immunoprecipitates from TBS- (TS), Triton-, and GuHCl-soluble fractions of Tg2576 mouse brain homogenates were separated on gel I (top and middle) and gel II ((TS) bottom) and subjected to Western blotting with 82E1. The middle panel represents an overexposed blot. A weakly immunoreactive band above A42 in the Triton lane presumably represents A41. The asterisks in A and B are presumably C-terminally truncated CTFs. When different gel conditions are used, these bands exhibit the various mobilities relative to those of synthetic As, which contrasts with the observation that longer As identified here always comigrate with corresponding synthetic As.
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To exclude the possibility that these longer As are inherent to CHO cells, we searched for longer As in N2a cells stably expressing Swedish mtAPP and HEK 293 cells stably expressing wtAPP. Those longer As, including A43, A45, A46, and A48, were detectable in N2a and HEK 293 cells as in CHO cells, although the proportions and levels of each longer A differed among these three cell lines (Fig. 2B). The levels of longer As relative to those of A40/42 were slightly higher in CHO and HEK cells (Fig. 2B). In contrast, N2a cells showed higher levels of A39/40/42 and relatively lower levels of longer As (Fig. 2B). One more characteristic of N2a cells may be the presence of A41 (Fig. 2B). Most interestingly, there were no differences in the levels of secreted A species among those cell lines (data not shown). These results clearly show that various A species longer than A42 exist in the lysates of various cell lines but are undetectable in their culture media.
We next asked whether those longer As exist in the brain of the APP-transgenic mouse strain Tg2576 (Hsiao et al., 1996). The homogenates of brains from 2.5-month-old Tg2576 mice were fractionated into three fractions (TBS-soluble, Tritonsoluble, and GuHCl-soluble fractions), each of which was subjected to immunoprecipitation and Western blotting. Longer As, including A43, A45, A46, and A48, were identified mostly in the Triton-soluble fraction but not in the TBS-soluble fraction of the homogenates (Fig. 2C), strongly suggesting that these longer As are associated with the membrane in the brain. Interestingly, the Triton-soluble fraction contained A41, which was detected in N2a cells (Fig. 2B,C). The same longer A species were also detected in the brains of 1.3-month-old PDAPP mice (Games et al., 1995) (data not shown).
Generation of longer As depends on PS
Because the generation of A40/42 requires PSs (De Strooper et al., 1998), we next investigated whether the generation of longer As also depends on PSs. To readily detect longer As, a stable CHO cell line that inducibly expresses CTF using the T-Rex system was generated, and the vectors carrying wtPS1 or DN (D257A/D385A) mtPS1 construct were stably transfected into this CTF cell line. After induction of CTF for 4 hr, the wtPS1 and DN mtPS1 cells were examined for the levels of AICD and A.
The expression of DN mtPS1 caused a marked reduction in the levels of both AICD and intracellular As (Fig. 3B). Note that trace amounts of AICD and A were still generated in DN mtPS1 cells. This is probably because endogenous PS1/2 were not completely replaced by the biologically inactive mtPS1 (Fig. 3A) and maintained some -cleavage activities. Gel I clearly showed that DN mtPS1 greatly reduced the levels not only of A40/42 but also of longer As, including A43, A45, and A46 (Fig. 3C). Several 82E1-immunoreactive bands that do not correspond to any of the authentic AXs were discernible in the lysates of CTF cells (Fig. 3B,C). They are, presumably, C-terminally truncated products derived from CTF. Their levels apparently increased by suppression of -cleavage, presumably because steady-state levels of CTF become elevated and it is more susceptible to proteolysis (Fig. 3B,C).
In addition, the cells that inducibly express CTF were treated with a -secretase inhibitor, L-685,458 (Shearman et al., 2000; Li et al., 2000). The cells were first treated with various concentrations of L-685,458 for 2 hr, and then CTF production was induced for 4 hr in the presence of the inhibitor. The levels of AICD and intracellular and secreted A were suppressed in dose-dependent manners (Fig. 4A). Gels I and II clearly showed that the intracellular levels of A40/42 and longer As were also uniformly suppressed in dose-dependent manners (Fig. 4B,C). Thus, the generation of longer As, including A43, A45, A46, and A48, is mediated by -secretase.
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Figure 4. L-685,458 suppressed the levels of longer As. A, The cells that inducibly express CTF were first treated with indicated concentrations of L-685,458 for 2 hr, and CTF was induced for 4 hr in the presence of L-685,458. Equal amounts of protein from whole-cell lysates were subjected to Western blotting with UT421 (top) and 82E1 (middle). The BAN50 immunoprecipitates from the medium were separated on gel I and subjected to Western blotting with 82E1 (bottom). B, Triton-soluble fractions of those treated cells were immunoprecipitated with BAN50, and the collected proteins were separated on gel I (top and middle) and gel II (bottom) and subjected to Western blotting with 82E1. The middle panel represents an overexposed blot. The asterisks in A and B indicate C-terminally truncated CTFs. C, The amounts of intracellular A were quantified using LAS-1000plus luminescent image analyzer. The levels of each A species were normalized to those in the nontreated cells. The data shown are the means of the values from three (for A40, A42, A43, and A45) or two (for A46 and A48) independent experiments.
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Longer As are produced at the same subcellular location as A40/42
To investigate where the longer As are produced, stable CHO cells overexpressing wtAPP, APP bearing a di-lysine motif (ER retention signal) (APP/ER) (Jackson et al., 1990), or APP carrying the sorting signal SDYQRL of TGN38 (APP/TGN) (Ponnambalam et al., 1994) were established and examined for the intracellular levels of various A species.
In APP/ER cells, the level of AICD was similar to that in nontransfected CHO cells, whereas in wtAPP and APP/TGN cells, the level of AICD was increased (data not shown). No intracellular A species were detectable in the APP/ER cell lysates, whereas in APP/TGN cells the intracellular levels of As were increased compared with those in wtAPP cells (Fig. 5). Moreover, the A species found in APP/TGN cells were the same as detected in the lysates of wtAPP cells and other cell lines (Fig. 2B, 5B): A40, A43, and A46 were abundant, whereas A42, A45, and A48 were minor species. These data suggest that both A40/42 and longer A species are produced mainly in the TGN but not in the ER.
MtPS1/2 and mtAPP affect the intracellular levels of longer As
We next asked whether and how the mutations of PS1/2 affect the intracellular levels of longer As. WtPS1 and wtPS2 had varying effects on the levels of longer As. Overproduction of wtPS2 caused increases in the intracellular levels of A40/42 and concomitant decreases in the levels of A43, A45, and A46 compared with 7WD10 cells (Fig. 6A). In contrast, overproduction of wtPS1 caused small increases in the levels of A43 and A46 but did not alter the levels of A40/42 (Fig. 6B).
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Figure 6. MtPS1/2 altered the intracellular levels of longer As. A, Immunoprecipitates from the lysates of 7WD10 (7W) cells, wt (WT), N141I (NI), and T122P (TP) mtPS2 transfectants were separated on gel I (top and middle) and gel II (bottom). The middle panel represents an overexposed blot. Striking increases in the A42 levels and concomitant decreases in the A40 levels in N141I and T122P mtPS2 cells were noted. B, Similarly, immunoprecipitates from 7WD10 (7W) cells, wt (WT), N135D (ND), and M233T (MT) mtPS1 transfectants were separated on gel I (top and middle) and gel II (bottom). The middle panel represents an overexposed blot. N135D PS1 is homologous to N141I PS2 and showed similar alterations in the intracellular A levels. A remarkable increase in the A48 level was a characteristic of M233T mtPS1. C, Immunoprecipitates from wt (WT), M146L (ML), H163R (HR), and G384A (GA) mtPS1 transfectants were separated on gel I (top and middle) and gel II (bottom). A middle panel represents an overexposed blot. All of these blots were probed with 82E1.
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In contrast, mtPS2s caused distinct changes in the intracellular levels of A40/42 and longer As. N141I and T122P mtPS2s showed similar alterations in the intracellular A levels: a marked increase in A42 and a concomitant decrease in A40 (Fig. 6A) (Qi et al., 2003). Both also caused an increase in the level of A45 and decreases in the levels of A43 and A46 (Fig. 6A). N135D mtPS1 is homologous to N141I mtPS2, and both showed similar alterations in the intracellular levels of As (Fig. 6B).
M233T mtPS1 caused a remarkable increase in the level of A48 and substantial increases in the levels of A39 and A42 (Fig. 6B). At the same time, it accompanied decreases in the levels of A40, A43, and A46 (Fig. 6B). Other mtPS1s caused various changes in the levels of longer As, although they invariably resulted in a significant increase in the levels of A42 (Fig. 6C) (Qi et al., 2003). M146L and G384A mtPS1 caused decreases in the levels of A43 and A46, whereas H163R mtPS1 did not alter their levels compared with wtPS1 (Fig. 6C). M146L mtPS1 caused an increase in A48, whereas G384A and H163R mtPS1 caused decreases in A48 (Fig. 6C). None of these three mtPS1s caused an increase in the levels of A45 (Fig. 6C).
Therefore, we conclude that the FAD-associated PS1/2 mutations have distinct effects on the levels of each longer A and bring about increased intracellular levels of A42. This indicates that numerous, diverse effects of mtPS1/2 on the levels of each longer A could cause finally increased levels of intracellular A42.
FAD-associated mtAPPs also enhance A42 production (for review, see Selkoe, 2001). We thus examined the intracellular A species in CHO cells overexpressing wtAPP (7WD10 cells), V717F mtAPP, or L723P mtAPP. The levels of APP and CTF were similar among these three transfectant lines (Fig. 7A). V717F and L723P mtAPP caused increases in the intracellular levels of A42 and slight decreases in the levels of A40 compared with 7WD10 cells (Fig. 7B). L723P mtAPP caused a discernible increase in the level of A45, whereas V717F mtAPP did not alter the level of A45 compared with 7WD10 cells, but both caused subtle decreases in the levels of A48 (Fig. 7B,C). In addition, the levels of A43 and A46 in V717F mtAPP cells were increased, whereas their levels were decreased in L723P mtAPP cells (Fig. 7B,C). These results indicate that these two mtAPPs alter the intracellular levels not only of A42 but also of longer As.
DAPT causes differential accumulations of longer As within the cell
A dipeptide inhibitor, DAPT very efficiently inhibits the secretion of A40/42 (Dovey et al., 2001; Sastre et al., 2001) and is regarded as a potent -secretase inhibitor. As expected, DAPT caused a reduction in the levels of AICD and secreted A in a dose-dependent manner (Fig. 8A) (Dovey et al., 2001; Sastre et al., 2001). It also reduced the levels of total intracellular A (Fig. 8A). However, after careful inspection of the blots, it becomes evident that, with increasing concentrations of DAPT, the proportion of intracellular A with fast mobility decreased and that of the A with slow mobility even slightly increased on a Tris/Tricine gel without urea (Fig. 8A). Moreover, the intracellular A was more resistant to DAPT than the secreted A. The intracellular A was clearly visible even at 1000 nM DAPT, whereas at the same concentration, the secreted A was barely discernible (Fig. 8A). Regarding AICD, only small amounts were detected at 1000 nM DAPT (Fig. 8A), and thus its counterpart would be converted to intracellular A, although the decay response of AICD should be best reflected by the amounts of the secreted A which accounts for the majority of produced A. This contrast with the effects of L-685,458, in which the intracellular A, the secreted A, and AICD showed similar decay responses (Fig. 4A).
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Figure 8. Differential effects of DAPT on the levels of longer As. A, The cells that inducibly express CTF were treated with the indicated concentrations of DAPT for 2 hr, and CTF was induced for 4 hr in the presence of DAPT. Equal amounts of protein from the cell lysates were subjected to conventional Tris/Tricine gel electrophoresis, followed by Western blotting with UT421 to detect AICD (top) and with 82E1 to detect intracellular A (middle). The relatively broad band representing intracellular A apparently consists of two components that have slow and fast mobilities, as indicated by arrowheads. The fast-migrating A component declined at 5 nM DAPT and was not discernible at 50 nM, where as the slow-migrating A component appeared to increase at 50 nM and was discernible even at 1000 nM. The conditioned media were immunoprecipitated with BAN50, separated on gel I, and subjected to Western blotting with 82E1 (bottom). B, The lysates from those DAPT-treated cells were immunoprecipitated with BAN50, separated on gel I (top and middle) or gel II (bottom), and subjected to Western blotting with 82E1. The middle panel represents an overexposure of the top blot. A couple of bands indicated by asterisks are presumably C-terminally truncated CTFs. C, The amounts of intracellular A were quantified using LAS-1000plus luminescent image analyzer. The levels of each A species were normalized to those in the nontreated cells. The data shown are the means of the values from three (for A40, A42, A43, and A45) or two (for A46 and A48) independent experiments. D, Mass spectra of secreted A (top left) and intracellular A from cells treated without and with 250 nM DAPT (middle and bottom left, respectively) after immunoprecipitation using monoclonal antibodies 4G8 and 6E10. The calculated (c) and observed (m) masses are shown in the right panel. The suppression of secreted As by DAPT was very similar to that by L-685,458. However, the effects on the intracellular As were quite distinct between these two inhibitors (see Fig. 4).
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The new gel systems demonstrated that the intracellular levels of A40 and A42 were increasingly suppressed by DAPT in dose-dependent manners: 5 nM DAPT had a negligible effect on their levels, but 50 nM DAPT substantially suppressed their levels (Fig. 8B,C). The levels of A45 appeared to be less affected by the DAPT treatment than those of A42 (Fig. 8B,C). Surprisingly, A43 and A46 accumulated in the cells and showed distinct patterns. The levels of A43 increased at 5 nM DAPT, reached a maximum at 50 nM, and gradually declined. The levels of A46 gradually increased to a maximum at 250 nM, followed by a slight decline at 1000 nM (Fig. 8B,C). These increases in the intracellular A43 and A46 levels after DAPT treatment were confirmed by immunoprecipitation/mass spectrometry analysis (Fig. 8D). The intracellular accumulations of longer As and "up and down" responses are a striking characteristic of the DAPT treatment. Notably, decreasing levels of A40 are followed by an increase in the levels of A43, and decreasing levels of this, in turn, are followed by increases in the levels of A46 (Fig. 8B,C). Thus, it appears that suppression of A40 leads to an increase in A43, which in turn brings an increase in A46. These data suggest that A40 is produced successively from A46 through A43 or, alternatively, that the substrate is competitive for the A40-, A43-, or A46-cleavage site of -secretase, and DAPT is the most potent for A40-cleavage.
Compound E (Seiffert et al., 2000), another nontransitionstate analog inhibitor for -secretase, provided very similar, but not identical, results. When the cells were treated with increasing concentrations of Compound E, the levels of A40 and A43 showed similar decay profiles, which accompanied a gradual increase and a subsequent decrease in the levels of A46 (data not shown).
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Discussion
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Multiple cleavage sites exist between the - and -cleavage sites
The A species that were detected in the cells and brain are not necessarily all of the possible A species of A43 through A49 differing from each other by one residue. For example, we did not consistently detect A41, A44, and A47, although it is likely that a trace amount of A41 exists in the N2a cell line and the Tg2576 brain. Despite intensive efforts, we were unable to detect A49 in the cell lysates; this is a counterpart of the major AICD50-99 that would have been generated by -cleavage. Thus, the major intracellular A species were A40, A43, and A46, whereas A42, A45, and A48 were minor ones in the CHO cells overexpressing wtAPP. In contrast, in mtPS2 cell lines, the levels of A42 and A45 were increased, whereas those of A40, A43, and A46 were decreased (Fig. 6A). In mtPS1 cell lines, when the levels of A40 were suppressed, those of A43 and A46 were also decreased (Fig. 6B) compared with 7WD10 or wtPS1 cells. In addition, according to our preliminary experiments, CHO cells expressing V721K APP produced mainly AICD47-99 and A40. We therefore categorize A40, A43, and A46 as one group and A42, A45, and A48 as the other. All of these indicate the presence of multiple cleavage sites between the - and -cleavage sites along the CTF molecule: the carboxyl sides of Thr-43, Val-46, and Ileu-45.
Cleavage of the substrate at every three residues fits well with an -helical model
The transmembrane domain of CTF is postulated to adopt an -helix that needs 3.6 residues for one complete turn (Lichtenthaler et al., 1999b). According to this model, the cleavage sites for A49, A46, A43, and A40 are aligned on the -helical surface of the CTF molecule, whereas those for A48, A45, and A42 are aligned on the other -helical surface (Fig. 9). We observed that the transient expression of A49, a postulated major counterpart generated by -cleavage, leads to predominant production of A40, whereas the expression of A48, a minor counterpart generated by -cleavage, leads to preferential production of A42 (Funamoto et al., 2004). Thus, it is possible that A40 is produced from A49 by cleaving at every three residues, whereas A42 is produced similarly from A48. Alternatively, once the substrate, possibly a counterpart of AICD, is bound to-secretase, it is preferentially cleaved at the A40, A43, or A46 site, all aligning at one particular -helical surface of CTF, or at the A42 or A45 site, both aligning at the opposite surface (Fig. 9). However, there is insufficient evidence in our hands for coordinated cleavages for A42, A45, and A48. We did not observe similar differential accumulations of A45 and A48 as for A43 and A46 (Fig. 8B,C), although differential susceptibility of A42 and A45 was observed: whereas A42 was already undetectable at 50 nM DAPT, A45 was still visible even at 250 nM DAPT (Fig. 8B).
Because three residues are insufficient for one complete -helical turn, the cleavage at every three residues cannot always occur and must be compensated by a cleavage at a four-residue distance. Presumably, this may explain our observation that the transient expression of A51 leads to predominant production of A42 and some A38 (Funamoto et al., 2004). This possible conversion of A42 to A38 might be promoted by treatment with certain nonsteroidal anti-inflammatory drugs (Weggen et al., 2001 |
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Abstract
Introduction
