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J. Biol. Chem., Vol. 279, Issue 49, 50647-50650, December 3, 2004

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Identification of a New Presenilin-dependent -Cleavage Site within the Transmembrane Domain of Amyloid Precursor Protein^*

Guojun Zhao, Guozhang Mao, Jianxin Tan, Yunzhou Dong, Mei-Zhen Cui, Seong-Hun Kim, and Xuemin Xu¶

From the Department of Pathobiology, College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee 37996 and the Department of Neurobiology, Pharmacology and Physiology, the University of Chicago, Chicago, Illinois 60637

Received for publication, October 6, 2004 , and in revised form, October 12, 2004.

	ABSTRACT

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-Secretase cleavage of -amyloid precursor protein (APP) is crucial in the pathogenesis of Alzheimer disease, because it is the decisive step in the formation of the C terminus of -amyloid protein (A). To better understand the molecular events involved in -secretase cleavage of APP, in this study we report the identification of a new intracellular long A species containing residues 1–46 (A₄₆), which led to the identification of a novel -cleavage site between the known - and -cleavage sites within the transmembrane domain of APP. Our data clearly demonstrate that the new -cleavage is a presenilin-dependent event. It is also noted that the new -cleavage site at A46 is the APP717 mutation site. Furthermore, we show that the new -cleavage is inhibited by -secretase inhibitors known as transition state analogs but less affected by inhibitors known as non-transition state -secretase inhibitors. Thus, the identification of A₄₆ establishes a system to determine the specificity or the preference of the known -secretase inhibitors by examining their effects on the formation or turnover of A₄₆.

	INTRODUCTION

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The amyloid deposits in the brain of Alzheimer disease (AD)¹ patients are principally composed of the 39–43-amino acid residue amyloid -peptide (A), which is derived from a large -amyloid precursor protein (APP). In the amyloidogenic pathway, APP is first cleaved at the N terminus of A sequence by -secretase, to produce a soluble ectodomain, sAPP, and a membrane-anchored C-terminal fragment, CTF. CTF is then subsequently cleaved within the transmembrane domain by -secretase to produce the full-length A and the intracellular domain (AICD) (1). -Secretase has been identified as a type I membrane aspartyl protease (2, 3). The findings that knockout of presenilin 1 (PS1) and PS2 results in the abolishment of the -secretase cleavage of APP and that two aspartate residues in two transmembrane domains of presenilin have been identified as critical for the -secretase activity suggest that presenilin may be the -secretase (4–7). Recently, several other molecules, namely nicastrin, Aph-1, and Pen-2, have been identified as essential components of the -secretase complex of which presenilin may function as the catalytic subunit (8).

Most of the A species contain 40 or 42 amino acids. Recently, sequence analysis revealed that the N terminus of AICD starts at residue 50 of the A sequence, which is 7–9 amino acids away from the C termini of A₄₀ and A₄₂. This led to the finding of the -cleavage site between A49 and A50 (9–12). Now the cleavage at A40/42 has been specifically referred to as -cleavage site (12). However, neither the intermediate A peptide, which ends at the -cleavage site, nor the C-terminal fragment, which starts with an N terminus generated by -cleavage, has ever been detected. One possibility is that - and -cleavages occur simultaneously. The other possibility is that there may be additional cleavages(s) between - and -cleavages. Here we report that, in our effort to determine these possibilities, we identified a new cleavage site at A46, which we designated as -cleavage site.

	MATERIALS AND METHODS

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-Secretase inhibitors, DAPT, compound E, -secretase inhibitor XIX (XIX), L-685,458, and WPE-III-31C (31C), were from Calbiochem and dissolved in dimethyl sulfoxide (Me₂SO). A₄₀ was from Biopeptide. A₄₂ and A₄₃ were from American Peptide.

Cell Lines—Human neuroblastoma M17 cells, glioma HS683 cells, and embryonic kidney 293 cells were purchased from American Type Cell Collection. All stably transfected N2a cell lines were established as described previously (13). All cells were maintained in Dulbecco's modified Eagle's medium supplied with 10% fetal bovine serum.

Immunoprecipitation and Western Blotting—Eight hours after treatment with inhibitors, cells were harvested and lysed in Western blot lysis buffer (50 mM Tris-HCl, pH 6.8, 8 M urea, 5% -mercaptoethanol, 2% SDS, and protease inhibitors). Secreted A was immunoprecipitated from conditioned medium (CM) using a A-specific antibody 6E10 (Senetek). Both cell lysates and the immunoprecipitates were analyzed by 10% Bicine/urea SDS-PAGE or 10–18% regular SDS-PAGE. After being transferred to a polyvinylidene fluoride membrane (Millipore) and probed with specific antibodies, the immunoreactivity bands were visualized using ECL-Plus (Amersham Biosciences).

Mass Spectrometric Analyses—N2a cells cultured in the presence of DAPT were lysed with 1% Nonidet P-40 in IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 5 mM EDTA, protease inhibitor mixture). After centrifugation at 20,000 x g at 4 °C for 15 min, the supernatant was diluted with equal amounts of IP buffer to bring down the concentration of Nonidet P-40 to 0.5%. The intracellular A species were immunoprecipitated using 6E10. The immunoprecipitate was eluted from beads with a buffer of 1% trifluoroacetic acid and 45% acetonitrile and then subjected to matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis performed on a Voyager-DE STR mass spectrometer as described previously (14).

	RESULTS

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Identification of a New Intracellular A Species—N2a cells stably expressing wild type PS1 (PS1wt) and the myc-tagged Swedish mutant APP (APPsw), which have been used in previous studies (13, 15), were treated with or without -secretase inhibitors: DAPT, DAPM, compound E, and XIX. The cell lysates were analyzed by 10–18% Tris/glycine SDS-PAGE followed by Western blotting, using either 6E10, a monoclonal antibody specific to residues 1–17 of human A, or C15, an antibody raised against the C-terminal 15 amino acids of APP. As shown in Fig. 1A, treatment with these inhibitors resulted in the accumulation of the C-terminal fragments of APP, namely CTF and CTF, generated by - and -secretases, respectively, as detected by C15 (compare lanes 2–5 with lane 1, top panel). Surprisingly, when the same samples were probed by 6E10, a band, which is tentatively labeled as A₄₆ (lane 1, second panel) and has a migration rate much faster than that of CTF but slightly slower than those of synthetic A₄₀, A₄₂, and A₄₃ (compare lane 1 with lanes 6 and 7, second panel), was detected in untreated cells. More interestingly, this band was markedly increased in cells treated with -secretase inhibitors (compare lanes 2–5 with lane 1, second panel). The fact that this band was not detected by C15 but was detected by 6E10 suggests that it is more likely a novel intracellular A-containing peptide with a higher molecular mass than A₄₃. The same samples were also analyzed by 10% Bicine/urea SDS-PAGE as described previously (16). As shown in the third panel, this new A species (lanes 1–5) migrated much faster than A_40/42 and A₄₃ (lanes 6 and 7), indicating that this new A species is more hydrophobic than A₄₃. Next, we examined the effects of these inhibitors on the formation of secreted A. As shown in the bottom panel of Fig. 1A, secreted A₄₀ and A₄₂ were detected in CM of untreated cells (lane 1) but not detected in CM of cells treated with inhibitors (lanes 2–5), a result consistent with many previous studies.

View larger version (46K): [in this window] [in a new window]   FIG. 1. Detection of a new intracellular A species. A, APP derivatives in lysates or media of cells treated with (lanes 2–5) or without (lane 1) various inhibitors (500 nM DAPT, 100 nM DAPM, 3 nM compound E (CPDE), and 5 nM XIX, respectively) for 8 h were analyzed by 10–18% Tris/glycine-SDS-PAGE (top and second panels) or by Bicine/urea SDS-PAGE (third and bottom panels). Lane 6 is the mix of synthetic A40 and A42. Lane 7 is the synthetic A43. The top panel is the blot probed with C15. The second, third, and bottom panels are the blots probed with 6E10. Since the CTF and CTF derived from recombinant APPsw are tagged with a myc epitope, they were detected as bands with slower migration rates than the endogenous CTF (CTF(end), top panel) (15). The bottom panel is the A immunoprecipitated from CM. B, cell lysates of human glioma HS683 (lanes 1 and 2) and human neuroblastoma M17 (lanes 3 and 4) cells treated with (3 nM) or without (–) compound E (CPDE) as indicated for 8 h were analyzed for the presence of the new A species. As controls, cell lysates of N2a cells stably expressing APPsw and PS1wt treated with (lane 6) or without (lane 5) compound E were included. Lane 7 is the mix of synthetic A40 and A42.

Identification of a New -Cleavage Site at A46 —To determine the identity of the novel A-containing peptide, mass spectrometric analysis was carried out. The new A species was immunoprecipitated with 6E10 from the lysates of cells treated with DAPT and analyzed by MALDI-MS. As shown in Fig. 2, a major spectral peak with a mass of 4927.53 Da, which is in agreement with the expected mass of 4926 for A₄₆, was observed. Thus, we determined the new A species as A₄₆ and identified a new cleavage site at A46 within the transmembrane domain of APP between the known -cleavage site at A40/42 and the -cleavage site at A49. We named this new cleavage site as "-cleavage" site, following the tradition by which other APP processing sites were named. It is noted that in addition to A₄₆, a weak spectral peak corresponding to A₄₃ (measured mass = 4614.86 Da; expected mass = 4615 Da) was detected in the immunoprecipitate.

View larger version (10K): [in this window] [in a new window]   FIG. 2. MALDI-MS spectra for A species immunoprecipitated from lysate of cells treated with DAPT. Mass spectrometric analysis was performed as described previously (14) except that after applying the sample matrix solution to the sample plate, formic acid and isopropanol were added to a final concentration of 30% each.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Requirement of presenilin for A46 formation. Cell lines are labeled at the top. Lysates of cells cultured in the presence (+) or absence (–) of 0.5 µM DAPT were analyzed by Western blot for the expression of PS1 (top panel) using anti-PS1N, which is specific for the N-terminal domain of PS1 and was raised against a peptide of 24 amino acids corresponding to residues 27–50 of human PS1. Full-length APP (fAPP), CTF, and A46 in the cell lysate were detected by 6E10 (middle panel). Secreted A and soluble APP (sAPP) immunoprecipitated from CM were also detected by 6E10 (bottom panel).

View larger version (44K): [in this window] [in a new window]   FIG. 4. Deferential inhibitions of A46 formation by transition state analogs and non-transition state inhibitors. Accumulation of CTF and the new intracellular A46 (upper panel) and the secreted A40 and A42 immunoprecipitated from CM with 6E10 (lower panel) were analyzed by 10% Bicine/urea PAGE followed by Western blotting using 6E10. Inhibitors and concentrations used are indicated on the top of the gels. Lanes 1, 9, and 19 are controls of samples from untreated cells. Lanes 18 and 28 are the mix of synthetic A40 and A42 used as standards.

	DISCUSSION

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The identification of this new A₄₆ offers several novel insights into the mechanism of the normal and pathogenic intramembranous processing of APP and the formation of A.

First, the identification of A₄₆ reveals a novel cleavage site, -cleavage site between the two known - and -cleavage sites. Our data also clearly reveal that this new -cleavage occurs as a presenilin-dependent event, indicating that it is a new intramembranous cleavage of APP by -secretase or related protease activity. Moreover, the facts that A₄₆ can be detected in the absence of inhibitors and it is the predominant intracellular A species as determined by both Western blot and mass spectrometry analyses strongly indicate that the cleavage site at A46 is another major cleavage site in APP, besides the known -cleavage site at A40/42 and the -cleavage site at A49. It is also noted that the new -cleavage site at A46 is the site at which the AD-linked APP mutation, APP717 mutation also known as London mutation, occurs (19, 20). Thus, our finding reveals an interesting fact that the well characterized AD-linked APP717 mutation actually occurs at a major APP processing site, the -site near the C terminus of A. Also the other well characterized AD-linked APP mutation, the Swedish mutation occurs at another major cleavage site, the -site at the N terminus of A.

Second, our dose-curve experiments clearly demonstrate that, at the lower range of concentrations, DAPT, DAPM, compound E, and XIX cause a dose-dependent decrease in secreted A_40/42 and a concomitant increase in A₄₆, suggesting a possible precursor-product relationship between A₄₆ and A_40/42. However, the observation that -cleavage can be differentially inhibited by -secretase inhibitors known as transition state analogs but less affected by the inhibitors known as non-transition state inhibitors also raises a possibility that -cleavage may be catalyzed by a -secretase-like activity. According to this model, the dose-dependent decrease in secreted A_40/42 and the concomitant increase in A₄₆ caused by non-transition state inhibitors at the lower range of concentrations may be related to the substrate availability, i.e. when the true -secretase, which cleaves APP at A40/42, is inhibited, it makes more CTF available for the putative -secretase-like activity, which produces A₄₆. At higher ranges of concentrations, these inhibitors also caused a dose-dependent increase of CTF, suggesting an allosteric mechanism of the inhibitory effects of these inhibitors on the turnover of A₄₆ and CTF.

Third and more importantly, the identification of A₄₆ made it possible to determine the specificity of the known -secretase inhibitors. Our experiment examining the effects of the known inhibitors on the formation and turnover of the newly identified A₄₆ led to a novel finding that the newly identified -cleavage is specifically inhibited by inhibitors known as transition state analogs and is less affected by inhibitors known as non-transition state inhibitors. According to the amyloid hypothesis (1), the longer and more hydrophobic and more amyloidogenic A is more toxic and pathogenic. In this regard, it is notable that DAPT, DAPM, compound E, and XIX, which were previously known to inhibit the formation of secreted A, cause an intracellular accumulation of an even longer A species, A₄₆. This finding provides information important for the strategy of prevention and treatment of AD aimed at the design of -secretase inhibitors. Therefore, the observation that -cleavage is differentially inhibited by so called transition state inhibitors and is less affected by non-transition state inhibitors is significant because it establishes, for the first time, a system to determine the specificity or the preference of the known -secretase inhibitors by examining their effects on the formation or turnover of A₄₆. In general, the identification of the intracellular A₄₆ and the new -cleavage site may provide a potential new therapeutic target for the treatment and prevention of AD.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant NS42314 (to X. X.) and by American Heart Association Grant 0355339B (to M.-Z. C). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article was selected as a Paper of the Week.

^¶ To whom correspondence should be addressed: Dept. of Pathobiology, College of Veterinary Medicine, The University of Tennessee, 2407 River Dr., Knoxville, TN 37996. E-mail: xmx{at}utk.edu.

¹ The abbreviations used are: AD, Alzheimer disease; A, amyloid -peptide; APP, -amyloid precursor protein; APPsw, Swedish mutant APP; AICD, APP intracellular domain; CTF, C-terminal fragment; CM, conditioned medium; PS, presenilin; Bicine, N,N-bis(2-hydroxyethyl)-glycine; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; DAPM, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine methyl ester; XIX, -secretase inhibitor XIX; 31C, WPE-III-31C.

	ACKNOWLEDGMENTS

 
We thank Dr. Ying Yang and Dr. Ling Xu (Mass Spectrometry Laboratory, University of Toronto) for the mass spectrometric analyses, Dr. M. Donald McGavin (Department of Pathology) and Jada R. Huskey (technical writer; College of Veterinary Medicine, The University of Tennessee) for their critical reading of the manuscript.

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