From the
Department of Neuropathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the
Department of Life Science, Graduate School of Arts and Science, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan, the ¶Biomolecular Characterization Division, Characterization Center, The Institute of Physical and Chemical Research, Wako, Saitama 351-0198, Japan, the ||Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, and **Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Ludwig-Maximilians-University, D-80336 Munich, Germany
Received for publication, October 31, 2002 , and in revised form, April 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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One of the A species, A
42, has a much higher aggregation potential (8, 9) and is believed to be initially deposited in senile plaques (10). It is reasonable to postulate that A
42 accumulation in the brain is the very initial event in the development of AD including sporadic AD. Indeed, all mutations of PS1/2 and some mutations of APP that cause familial AD result in increased A
42 production (11).
Recently, we and other groups found that APP is cleaved by PS-dependent -secretase, not only in the middle of the transmembrane domain (
-cleavage) but also near the cytoplasmic membrane boundary (
-cleavage) (1215). The major product of the latter process is a CTF
of APP that begins at Val-50. This cleavage site is a few residues inside the membrane from the cytoplasmic/membrane boundary and is similar to site 3 cleavage of Notch (16). Since production of CTF
is inhibited by a dominant negative mutant of PS1 (17),
-cleavage is PS-dependent as well as
-cleavage (1315). Furthermore,
-cleavage is also inhibited with
-secretase inhibitors, which are known to selectively bind to PS1/2 (1215). However, it has remained unknown how
-cleavage relates to the generation of distinct A
species or whether this step is essential to generate A
. We therefore examined whether there is a link between CTF
and A
production using a cell-free system. Taking advantage of familial AD mutations of APP and PS1/2 and a
-secretase inhibitor, we show here that, when A
40 is predominantly produced, CTF
5099 is the major product of
-cleavage, and when a large amount of A
42 is produced, CTF
4999 is predominantly produced. Thus,
-cleavage may be linked to the specificity of
-cleavage.
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EXPERIMENTAL PROCEDURES |
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Cell-free Assay and Inhibitor TreatmentConfluent cells were harvested and homogenized in homogenization buffer (20 mM Pipes, pH 7.0, 140 mM KCl, 0.25 M sucrose, and 5 mM EGTA). Homogenized cells were centrifuged at 800 x g to remove nuclei and cell debris. The supernatant was further ultracentrifuged at 100,000 x g for 1 h. The resultant pellet, total membrane fraction, was suspended in homogenization buffer. The membrane fraction at a protein concentration of 2.5 mg/ml in homogenization buffer containing protease inhibitor mixture was incubated at 37 °C for the indicated time, and the reaction was stopped by placing the reaction mixture on ice. After extraction of lipids twice with chloroform/methanol (2:1) and chloroform/methanol/water (1:2:0.8), the residue was extracted with 70% formic acid, and the extract was dried. The remaining proteins were dissolved with the SDS sample buffer containing 9 M urea and subjected to 16.5% SDS-PAGE, followed by Western blotting using antibodies BA27 (specific for A40), BC05 (specific for A
42), 6E10 (raised against A
116, and appropriate for assessing total A
; Senetek PLC, Maryland Heights, MO), and C4 (raised against the 30 C-terminal residues of APP). A
40/42 and CTF
were quantified by a LAS-1000plus luminescent image analyzer (Fuji Film, Tokyo, Japan) using defined amounts of authentic A
(Bachem, Bubendorf, Switzerland) as a standard. DFK-167 was purchased from Enzyme Systems Products (Livermore, CA) and dissolved in Me2SO.
Mass Spectrometric and Amino Acid Sequence Analyses of CTF Membrane fractions prepared from 80 dishes of cultured cells were incubated at 37 °C for 30 min and then ultracentrifuged at 100,000 x g for 1 h to separate the soluble and membrane-bound CTF
. The membrane-bound fraction was suspended and homogenized in 1% Triton X-100, which completely extracted CTF
(data not shown). Solubilized CTF
was immunoprecipitated once with 4G8 (epitope: A
1724; Senetek PLC, Maryland Heights, MO) to remove the other C-terminal fragments of APP (CTF
and CTF
), and then CTF
was immunoprecipitated with C4, extracted with 70% formic acid, and dried by vacuum centrifugation. The partially purified samples were subjected to gel filtration on two tandemly arrayed TSK-gel Super SW2000 columns (Tosoh, Tokyo, Japan), which were developed with 6 M guanidine hydrochloride in 10 mM phosphate buffer (pH 6.0). Pooled putative CTF
fractions were further purified by reverse-phase high performance liquid chromatography (RP-HPLC) on CAPCELL PAK Phenyl SG300 (Shiseido, Tokyo, Japan).
The fractions corresponding to peaks 1 and 2 were subjected to an Applied Biosystems model 494 cLC or model 492 protein sequencer. Both CTF 5099 and CTF
4999 were quantified from the yields of Val-50 at the first and second cycles, respectively.
Mass spectrometric analysis was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on the Voyager Biospectrometry Workstation (Applied Biosystems). Immunoprecipitated CTF was extracted with 1% trifluoroacetic acid, 30% acetonitrile. Masses of the peptides were determined in a linear mode, using sinapinic acid as a matrix. For calibration, bovine insulin (Sigma) was used as an external standard.
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RESULTS |
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We wondered whether production of A40 and A
42 is correlated with particular species of CTF
. The generated CTF
was separated into soluble and membrane-bound forms. A larger proportion of CTF
was released into soluble fraction during incubation, and the remaining proportion of CTF
was left membrane-bound (see Table I). As a first step to purification of CTF
, highly efficient immunoprecipitation with C4 (see "Experimental Procedures") was employed. CTF
immunoprecipitated from the soluble and membrane-bound fractions was subjected to mass spectrometric analysis; several species of CTF
were identified in each of the membranes (Fig. 1E), the results of which were consistent with our previous report (12). The largest peak in wtAPP and wtPS2 membranes represented CTF
5099. Notably, whereas CTF
4999 was a minor signal in wtAPP and wtPS2 membranes, it became a major signal in mtAPP (V717F) and mtPS2 (N141I) membranes. This raises the possibility that the extent of A
40 production is related to that of CTF
5099 production, and that of A
42 production is related to that of CTF
4999 production.
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Production of A42 and CTF
4999 Is Increased in Membranes from CHO Cells Expressing mtPS1To further confirm such increased production of CTF
4999, we examined membrane fractions prepared from wt or mtPS1 (M146L, M233T, and G384A)-overexpressing cells. By incubation for 20 min, wtPS1 membranes produced A
40 predominantly, a situation that is similar to that of wtAPP and wtPS2 membranes (Fig. 2, A and B). On the other hand, mtPS1 membranes produced larger amounts of A
42, although the ratios of A
42 to A
40 differed among mutations (Fig. 2A). Concomitantly produced CTF
was immunoprecipitated and subjected to mass spectrometric analysis (Fig. 2C). In the wtPS1 membranes, the largest signal was CTF
5099. In contrast, in the mtPS1 membranes, the peaks for CTF
4999 were found to be significantly larger. In M233T and G384A membranes in which A
42 was produced predominantly (Fig. 2A), the peaks for CTF
4999 were much higher (Fig. 2C). In contrast, in M146L membranes, the peak for CTF
4999 was almost at the same level as that in wtPS1 (Fig. 2C), and the A
42 production was only slightly increased (Fig. 2A).
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Membranes from HEK293 Cells Expressing mt APP Show an Increased Production of A42 and CTF
4999 To determine whether these differences were due to an idiosyncrasy of CHO cells, we examined the membrane fractions from wt, V717G, and V717F APP-overexpressing HEK293 cells. As shown in Fig. 3, A and B, whereas A
40 was predominantly produced in the membranes from wtAPP-overexpressing cells, the proportion of A
42 production was significantly increased in membranes from V717G and V717F APP-overexpressing cells. The CTF
immunoprecipitated from membranes of HEK293 cells was similarly analyzed by mass spectrometry. The major species was CTF
5099 in the membrane of wtAPP-overexpressing cells (Fig. 3C). On the other hand, the major peaks in the membranes from V717G- and V717F-overexpressing HEK293 cells were CTF
4999, which is similar to mtAPP-expressing CHO cells (see Fig. 1E). Interestingly, all cell lines of HEK293 cells appeared to produce significantly larger amounts of CTF
5299 as compared with CHO cells, an observation that agrees with previous reports (14, 24). From these results, it is likely that, although
-cleavage may show some variability among cell lines, CTF
5099 is a predominant species in wtAPP and wtPS1/2 membranes in which A
40 was predominantly produced, and CTF
4999 production is increased in mtAPP and mtPS1/2 membranes, in which A
42 production was increased.
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Quantification of CTF Species Using an Amino Acid SequencerPeak heights for several CTF
species on mass spectrometric profiles may not be quantitative. Thus, we sought to quantify precisely each molecular species of CTF
produced using an amino acid sequencer. The CTF
species produced in membranes from four cell lines as shown in Fig. 1 were carefully analyzed. C4-immunoprecipitated CTF
was fractionated by gel filtration, followed by RP-HPLC (Fig. 4A). The fractions corresponding to peaks 1 and 2 (see Fig. 4A) were subjected to amino acid sequence analysis. Five species of CTF
were identified (see Fig. 1E); peak 1 contained four molecular species (CTF
5299, 5199, and 5099 and a trace amount of 4999), whereas peak 2 contained two molecular species (CTF
4999 and 4899). Among them, CTF
5099 and 4999 accounted for >90% of CTF
in each cell line, and the remaining species (CTF
5299, 5199, and 4899) were at detectable but negligible levels. Other species (e.g. CTF
5399, 5499, etc.) were undetectable by sequence analysis. The accurate proportion of CTF
4999/5099 for each cell line is provided in Table I.
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The proportions of CTF 4999 were 28.7 and 19.3% for wtAPP and wtPS2 membranes, respectively (Table I and Fig. 4B). Both membranes produced A
40 predominantly (Fig. 1A). In contrast, the proportions of CTF
4999 were increased to 72.2 and 50.3% for the membranes of mtAPP (V717F) and mtPS2 (N141I) cells, respectively, both of which produced increased amounts of A
42 (Fig. 1A). These data were consistent with the results of mass spectrometric analysis, indicating that the evaluation by mass spectrometry is very informative in the present study.
Proportion of CTF 5099 Is Decreased at a Low Concentration of DFK-167To further verify that the increase in the proportion of CTF
4999 is independent of the cell type, we next examined the effect of DFK-167, an inhibitor that at low concentrations specifically inhibited the production of A
40 and paradoxically increased A
42 production (Fig. 1D), on the proportion of CTF
species produced by the membranes (Table I). The proportions of CTF
5099 and CTF
4999 in the soluble fraction of 10 µM inhibitor-treated wtPS2 membrane were significantly (p < 0.05) changed to 74.8 ± 0.9% from 81.5 ± 3.7% and to 25.2 ± 0.9% from 18.5 ± 3.7%, respectively, concomitantly with the proportion of A
40 produced decreasing to
60% from more than 80% and A
42 produced increasing to
40% from less than 20% (Figs. 1D and 4B). These results strongly suggest that A
40 and A
42 production is related to that of CTF
5099 and CTF
4999, respectively (Fig. 4C).
Additive Effects of mtAPP and mtPS1 on A and CTF
SpeciesAn increase in CTF
4999 production accompanying a decrease in CTF
5099 production was observed in mtAPP-, mtPS1-, and mtPS2-overexpressing cells. To examine whether the combination of mtAPP and mtPS1 has an additional effect on the produced CTF
species, we established stable transfectants co-expressing mtAPP (V717F; hereafter VF) and wt or mtPS1 (M233T or G384A). The ratio of A
40/42 produced in wtPS1 membranes (VF/wtPS1) was almost the same as that observed in V717F-only membranes (Fig. 5, A and B). On the other hand, the proportion of A
42 production was significantly increased in mtPS1 membranes (VF/M233T and VF/G384A) as compared with VF/wtPS1. This additive effect of mtAPP and mtPS1 on A
42 production was consistent with the previous report (25). Interestingly, the membranes from VF/G384A cells produced much smaller amounts of A
and CTF
and accumulated CTF
/
(Fig. 5, A and B). It is possible that this mtPS1-associated
-secretase has a lower affinity and/or inefficient cleavage for V717F substrate.
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We next analyzed CTF species produced in these membranes by mass spectrometry. The major species was CTF
4999 in the VF/wtPS1 membranes, similarly to V717F membranes (Fig. 5C). On the other hand, the peaks for CTF
5099 were found to be smaller in both VF/M233T and VF/G384A membranes. In these membranes, the peaks of CTF
5099 were also remarkably smaller as compared with mtPS1-only membranes (Figs. 2C and 5C). Because mass spectrometric analyses are not so quantitative, we could not accurately assess whether or how much CTF
4999 in these membranes was increased as compared with that in VF/wt-PS1 membrane. However, these results strongly suggest that mtAPP and mtPS1 have an additive effect on CTF
species produced.
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DISCUSSION |
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In contrast to CHO cells, HEK293 cell membranes produced a significant amount of another CTF, CTF
5299 (Fig. 3C). Moreover, CTF
5299 was significantly increased in mtAPP membranes. Even in CHO cells, CTF
5299 appears to be very slightly increased in the membranes from mtAPP and mtPS1/2 cells (Figs. 1E, 2C, and 5C). Thus, the production of CTF
5299 may also be related to that of A
42, although further study is required to confirm this view.
A number of recent reports have argued for a reciprocal relationship between -cleavage and Notch site 3 cleavage. Several PS1 mutations that increase the production of A
42 were found to reduce Notch site 3 cleavage (24, 2629) and production of CTF
as well (24, 29). These results are consistent with the present quantitative data based on sequencing, showing that the total amounts of CTF
produced in the mtPS2 membranes are reduced to
50% of those of wtPS2 membranes (Table I). Thus, mtPS1/2 causes not only a reduction in the extent of
-cleavage but also an alteration in the proportion of CTF
species (i.e. an increase of long CTF
). This contrasts with mtAPP, which does not affect the extent of
-cleavage (24) but produces an unexpectedly large amount of CTF
4999 (Fig. 1E, 3C, and 4C). It is therefore reasonable to speculate that this particular characteristic underlies the mechanisms for increased A
42 production by each mtAPP and mtPS1/2 membranes. The differing characteristics of mtPS1/2 and mtAPP might further complicate the relationship between
- and
-cleavage.
Thus far identified substrates of -secretase are cleaved at or near the cytoplasmic membrane boundary, and some are also in the middle of the transmembrane domain (16, 3033). This suggests that
-cleavage and
-cleavage are universal phenomena in a particular subset of type I membrane proteins. Further, a potential link between A
42 and CTF
4999 raises further questions. Which cleavage,
- or
-cleavage, comes first, and how does one cleavage affect the other? We failed to detect a particular CTF
longer than CTF
4899 by either mass spectrometric analysis or sequencing. One possible interpretation is that CTF
is first cleaved at the
-site, and thus cleaved products (A
148 and 149) undergo
-cleavage, and A
40/42 are secreted. Previous studies reported the existence of long A
146 by mass spectrometric analysis (34, 35). However, in our hands, longer A
species were undetectable, although we cannot rule out the possibility that the steady-state levels of such intermediates in the lysates are below the detection limit. It is also possible that
- and
-cleavage occur simultaneously or nearly so along the CTF
molecule, leaving a small hydrophobic membrane peptide that must be difficult to isolate and detect. Development of specific inhibitors for
- or
-cleavage might help us to identify a particular intermediate(s) and then lead us to a better understanding of the relationship between two kinds of cleavage.
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FOOTNOTES |
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Supported by a grant from the American Health Association Foundation.
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: 81-3-5800-6852; E-mail: yihara{at}m.u-tokyo.ac.jp.
1 The abbreviations used are: AD, Alzheimer's disease; A, amyloid
-protein; APP,
-amyloid precursor protein; PS, presenilin; CTF, carboxyl-terminal fragment; CHO, Chinese hamster ovary; HEK, human embryonic kidney; wt, wild type; mt, mutant; RP-HPLC, reverse-phase high performance liquid chromatography; Pipes, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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