Identification of a beta -Secretase Activity, Which Truncates Amyloid beta -Peptide after Its Presenilin-dependent Generation*

Regina Fluhrerab, Gerd Multhaupbcd, Andrea Schlicksuppc, Masayasu Okochie, Masatoshi Takedae, Sven Lammicha, Michael Willema, Gil Westmeyera, Wolfram Bodef, Jochen Walteraghi, and Christian Haassahj

From the a Adolf-Butenandt Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Schillerstrasse 44, Ludwig-Maximilians University, 80336 Munich, Germany, the c ZMBH-Center for Molecular Biology, University of Heidelberg, Im Neuenheimer Feld 282, Germany, the e Department of Postgenomics and Diseases, Division of Psychiatry and Behavioral Proteomics, Osaka University Graduate School of Medicine, 565-0871 Osaka, Japan, and the f Max-Planck Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany

Received for publication, November 11, 2002, and in revised form, December 4, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The beta -amyloid precursor protein (beta APP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid beta -peptide (Abeta ). N-terminal cleavage is mediated by beta -secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) gamma -secretase complex. The Abeta -generating gamma -secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of Abeta -(1-40) or Abeta -(1-42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted Abeta -(1-40/42). However, mass spectrometry revealed a truncated Abeta species, which terminates at amino acid 34 (Abeta -(1-34)) suggesting an alternative gamma -secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of Abeta -(1-40) and Abeta -(1-42) but also that of Abeta -(1-34). However, expression levels of BACE correlate with the amount of Abeta -(1-34), and Abeta -(1-34) is produced at the expense of Abeta -(1-40) and Abeta -(1-42). Since this suggested that BACE is involved in a C-terminal truncation of Abeta , we incubated purified BACE with Abeta -(1-40) in vitro. Under these conditions Abeta -(1-34) was generated. Moreover, when conditioned media containing Abeta -(1-40) and Abeta -(1-42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, Abeta -(1-34) was efficiently produced in vivo. These data demonstrate that an apparently gamma -secretase-dependent Abeta derivative is produced after the generation of the non-truncated Abeta via an additional and unexpected activity of BACE.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because of the increasing mean life expectancy, there is considerable interest in the understanding of the molecular and biochemical mechanisms of age-related diseases. By far the most frequent age-related neurological disorder is Alzheimer's disease (AD).1 During the aging process the patients accumulate insoluble amyloid beta -peptide (Abeta ), which is deposited in senile plaques and microvessels in the brain. Abeta is generated by endoproteolytic processing of the beta -amyloid precursor protein (beta APP), involving beta - and gamma -secretase (1).

beta -secretase (also called BACE; beta -site APP-cleaving enzyme) was identified as a membrane-associated aspartyl protease (2-6). BACE mediates the primary amyloidogenic cleavage of beta APP and generates a membrane-bound beta APP C-terminal fragment (APP CTFbeta ), which is the immediate precursor for the intramembraneous gamma -secretase cleavage (1). BACE also generates N-terminally truncated Abeta species starting with amino acid 11 of the Abeta domain (2, 7-9). A close homologue (BACE-2) (6, 10-12) can also mediate the typical beta -secretase cut although with much lower efficiency (13). BACE-2 rather exhibits an alpha -secretase-like activity, which cleaves in the middle of the Abeta domain at amino acid 19 and 20 (9, 13, 14). Apparently BACE-2 does not contribute to the amyloidogenic processing of Abeta , since the deletion of BACE fully abrogates Abeta generation (15-17).

gamma -Secretase activity is associated with a protein complex, composed of presenilins (PS1 or PS2), Nicastrin (Nct), PEN-2, APH-1a, and APH-1b (18-25). The expression of these complex components is coordinately regulated, and gamma -secretase activity is only detected in the presence of all subunits (21, 23-25). Removing a single subunit results in the destabilization or reduced maturation of the remaining components (23-26). The catalytic activity is most likely contributed by the PSs (1, 27). PSs are polytopic transmembrane proteins, which together with the signal peptide peptidases and the type-4 prepilin peptidases may belong to a novel family of aspartyl proteases of the GXGD type (for review see Ref. 1). The cleavage of BACE-generated CTFbeta by gamma -secretase results in the secretion of Abeta into biological fluids (1). This cleavage principally occurs after amino acid 40 and 42, the latter being enhanced by numerous familial AD-associated mutations in the PS genes and beta APP itself (28). Beside the predominant cleavage after amino acid 40 and 42 slightly shorter peptides have been observed as well, suggesting that the gamma -secretase has loose sequence specificity (29). This includes peptides terminating after amino acid 34, 37, 38, and 39 (29). In addition or in parallel to these cleavages, gamma -secretase also cleaves within the transmembrane domain shortly before the cytoplasmic border after amino acid 49 to liberate the beta APP intracellular domain (AICD) (30-33), which may be involved in nuclear signaling (34, 35). The biological function of gamma -secretase is related to the very similar intramembraneous processing of Notch. Indeed, a depletion of PS1 leads to a very severe Notch phenotype (summarized in Ref. 36).

BACE and gamma -secretase are obvious targets for therapeutic strategies aimed to inhibit Abeta generation. Unfortunately gamma -secretase inhibitors not only block Abeta generation but also interfere with Notch signaling (37-39). Therefore treatment of patients with such inhibitors remains problematic. On the other side it has been shown that the gene encoding BACE can be removed without any deleterious effects (15-17). Therefore inhibition of BACE with small chemical compounds seems to be a safer approach for long term treatment. However, a detailed understanding of the cleavage specificity and the substrate specificity of BACE is required for the generation of selective drugs. Surprisingly, overexpression of BACE in cell culture models leads to reduced Abeta secretion (2). In order to investigate this paradox we analyzed Abeta peptides secreted from cells stably expressing various levels of BACE and made the surprising observation that BACE can also cleave 34 amino acids C-terminal from its primary cleavage site, thus mimicking a PS-like cleavage specificity.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture, cDNAs, and Transfection-- HEK293 were cultured as described (9). The cell lines stably overexpressing wild type beta APP695 (40) or beta APP695 containing the Swedish double mutation (beta APPsw) (41) and cell lines co-expressing either PS1 wt or PS1 D385N have been described (42). To transfect BACE into these cell lines, the BACE cDNA was cloned into the EcoRI/XhoI sites of pcDNA3.1 hygro (+) expression vector (Invitrogen). Transfection was carried out using FuGENE 6 reagent (Roche Molecular Biochemicals). Pooled stable cell clones were selected in 150 µg/ml hygromycin (Invitrogen). The cell lines expressing BACE-2 have been described previously (9).

Antibodies, Metabolic Labeling, Immunoprecipitation, and Immunoblotting-- Antibodies 7520 (43, 44) and 7524 (9) directed against the respective C termini of BACE or BACE-2 and antibody 3926 (45) against the Abeta domain of beta APP, as well as the antibodies 6687, against the C terminus of beta APP, and 5313, against the N terminus of beta APP (46), have been described previously. The monoclonal antibody 6E10 directed against amino acids 1-17 of the Abeta domain was obtained from Senetek Inc. For immunodetection of PS1 the polyclonal and monoclonal antibodies against the large hydrophilic loop of PS1 (3027 and BI.3D7) were used (47, 48). Metabolic labeling, immunoprecipitations, and Western blotting were carried out as described previously (9).

BACE Activity Assay-- The fluorometric BACE activity assay was carried out as described previously (46). To selectively inhibit BACE activity GL189 was used as described previously (46). Soluble BACE (sBACE) was isolated and incubated with synthetic Abeta as follows: HEK 293 cells expressing sBACE were incubated with Optimem 1 containing Glutamax (Invitrogen) for 24 h. 400 ml of the conditioned medium were purified using a mono Q-Sepharose column (Amersham Biosciences). 20 µl of the fraction derived from cells expressing sBACE or from control fractions not containing sBACE were incubated with 50 µg of synthetic Abeta -(1-40) for MALDI-TOF MS and with 6 µg of synthetic Abeta -(1-40) for gel analysis at 37 °C for the indicated time points. The pH was adjusted to 4.5 with acetic acid. Samples were dried in a Speed Vac and resuspended in acetic acid. Subsequently samples were purified by using a Zip-Tip column and were then subjected to MALDI-TOF MS.

Mass Spectrometry/MALDI-TOF-- Cells were grown on 10-cm dishes and incubated with 4 ml of Dulbecco's modified Eagle's medium high glucose (DMEM; PAA Laboratories) supplemented with 10% fetal calf serum (PAA Laboratories) and penicillin/streptomycin for 24 h. Subsequently the samples were prepared for mass spectrometry as described previously (49-51). Samples were analyzed on MALDI-target plates by matrix-assisted laser desorption ionization (Brucker Reflex III).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In order to analyze the Abeta species secreted by BACE-expressing cells we collected conditioned media from HEK 293 cells stably transfected with Swedish mutant beta APP695 (beta APPsw) and BACE. Cells expressing either endogenous levels of BACE or moderate or high levels of transfected BACE were investigated (Fig. 1A). To prove the catalytic activity of BACE in these cell lines we performed in vitro activity assays using solubilized membranes of the respective cell lines (46). As expected we found substantially increased beta -secretase activity in the cell line expressing high levels of BACE as compared with non-transfected cells or cells expressing low levels of BACE (data not shown). These cell lines were labeled with [35S]methionine and conditioned media were immunoprecipitated with the anti-Abeta antibody 3926. Surprisingly, increasing BACE expression negatively correlated with Abeta production (Fig. 1B). This is consistent with previous findings by Vassar et al. (2) who observed reduced Abeta production despite increased BACE activity in cells transfected with beta APPsw (2). This paradoxical finding raised the possibility that Abeta species produced under these conditions could either not be metabolically labeled or not detected by conventional gel electrophoresis. We thus used an independent method and attempted to identify secreted Abeta species by a combined immunoprecipitation/MALDI-TOF MS method. As expected cells expressing endogenous BACE secreted predominantly Abeta -(1-40) (Fig. 1C). In addition we also obtained small amounts of Abeta -(1-42) and Abeta -(1-37/38/39) (Fig. 1C). Upon expression of moderate levels of BACE we observed an additional Abeta species (Abeta -(1-34); Fig. 1C). In order to analyze if the production of this truncated species is related to BACE expression levels, we next investigated Abeta species secreted from cells expressing higher levels of BACE (Fig. 1A). This revealed robust amounts of Abeta -(1-34), which was accompanied by reduced levels of Abeta -(1-40), Abeta -(1-42), and Abeta -(1-37/38/39) (Fig. 1C). Similar results were obtained using cell lines co-expressing wtAPP and BACE (data not shown). The detection of robust levels of Abeta -(1-34) upon expression of BACE explains the lack of its detection upon metabolic labeling, since the single radioactively labeled Met residue at position 35 of the Abeta peptide has been removed by the additional cleavage.


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Fig. 1.   Production of Abeta -(1-34) correlates with BACE expression. A, HEK 293 cells stably overexpressing beta APP695 sw (control; lane 1) or beta APP695 sw and BACE (lanes 2 and 3) were labeled with [35S]methionine. BACE was immunoprecipitated from cell lysates with antibody 7520. B, conditioned media were analyzed for Abeta accumulation by immunoprecipitation with antibody 3926. C, MALDI-TOF MS of Abeta peptides immunoprecipitated from conditioned media of the three cell lines with antibody 3926. Arbitrary intensities are given on the y-axis (a.i.). The tables below the spectra indicate the peak masses obtained by mass spectrometry (mass) and the respective calculated masses (mass calc.). Note that the relative levels of Abeta -(1-34) correlate with increasing amounts of BACE expression while other Abeta species are reduced.

Although the above described results suggest that BACE is directly involved in the enhanced production of Abeta -(1-34), previous observations indicated that C-terminally truncated Abeta species including Abeta -(1-34) are generated by the gamma -secretase complex in a PS-dependent manner (52, 53). In order to analyze if a PS-dependent gamma -secretase activity is required for Abeta -(1-34) generation, we co-expressed BACE with either PS1 wt or the non-functional PS1 D385N mutant (Fig. 2A). As shown previously (42), PS1 wt undergoes endoproteolysis whereas no endoproteolysis was obtained in cells expressing PS1 D385N (Fig. 2A, right panel; Ref. 27). The non-functional PS1 D385N fully replaced biologically active endogenous PS (Fig. 2A, right panel). Whereas robust levels of Abeta -(1-34) and Abeta -(1-40) (and all other minor Abeta species) were produced from cells co-expressing PS1 wt and BACE, Abeta -(1-34) generation as well as generation of all other Abeta species was almost completely inhibited in the presence of the non-functional PS1 D385N (Fig. 2B, right panel). This clearly demonstrates that a PS-dependent gamma -secretase activity is involved directly or indirectly in the production of Abeta -(1-34). However, the results described in Fig. 1, demonstrated that upon BACE expression Abeta -(1-34) generation occurs to the expense of the production of all other Abeta variants and thus suggests a direct involvement of BACE in the cleavage of Abeta at position 34. This apparent paradox may indicate that gamma -secretase activity is required first to produce secreted Abeta species, which are then trimmed at their C termini by a so far unknown BACE activity.


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Fig. 2.   Production of Abeta -(1-34) is PS1-dependent. A, left panel, BACE expression in cells co-expressing PS1 wt or PS1 D385N. Membrane fractions were probed with antibody 7520. Immature BACE can only be detected upon longer exposure (data not shown). Right panel, PS1 derivatives were identified by a combined immunoprecipitation/immunoblotting protocol using antibody 3027 and BI.3D7 (47). Note full replacement of endogenous PS1 by the uncleaved non-functional PS1 D385N mutant. B, MALDI-TOF MS of Abeta peptides immunocaptured from conditioned media of cell lines shown in A using antibody 3926. The tables below the spectra indicate the peak masses measured (mass) and the respective masses calculated (mass calc.). Note that in the presence of a non-functional PS1 mutant (PS1 D385N) and BACE almost no Abeta species is generated, whereas in the presence of PS1 wt and BACE Abeta -(1-34) is the predominant species.

In order to prove this hypothesis, synthetic Abeta -(1-40) was incubated with purified BACE isolated from conditioned media of cells secreting a soluble version of BACE lacking the transmembrane domain and the C terminus (43). To prove the catalytic activity of secreted BACE we carried out in vitro assays (46). Soluble BACE was fully active in the in vitro assay, whereas no activity was obtained in control media (Fig. 3A). This activity was fully blocked by the BACE-specific inhibitor GL189 (Fig. 3A). Upon incubation of synthetic Abeta -(1-40) with soluble BACE an additional peptide, which co-migrated with synthetic Abeta -(1-34) was detected on a gel system capable to separate low molecular weight peptides (Fig. 3B; Ref. 54). In vitro generation of Abeta -(1-34) was completely inhibited upon addition of the specific BACE inhibitor GL189 (Fig. 3B). Using MALDI-TOF MS we confirmed a time-dependent generation of Abeta -(1-34) during incubation of soluble BACE with Abeta -(1-40) (Fig. 3C). In contrast incubation of synthetic Abeta -(1-40) with conditioned media, not containing soluble BACE does not reveal truncated Abeta species (Fig. 3C). This finding demonstrates that BACE has the ability to cleave Abeta after amino acid 34 and, together with the results shown in Fig. 1, excludes artificial trimming by exopeptidases.


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Fig. 3.   BACE cleaves synthetic Abeta -(1-40) after amino acid 34 in vitro. A, conditioned media from cells expressing sBACE were analyzed in vitro for beta -secretase activity. Note that medium containing sBACE (filled squares) shows significant activity upon incubation with the fluorogenic substrate, while the control medium without sBACE (open circles) and sBACE in the presence of the specific BACE inhibitor GL189 (filled triangles) do not display activity. B, sBACE was incubated with 6 µg of synthetic Abeta -(1-40) overnight at 37 °C in the presence or absence of the inhibitor GL189. As a control the synthetic peptides Abeta -(1-40) and Abeta -(1-34) were co-migrated. The proteins were visualized by Coomassie Blue staining. Note the aberrant migration of Abeta -(1-34). Significant in vitro production of an Abeta species co-migrating with synthetic Abeta -(1-34) was obtained. The production of this peptide was fully inhibited by the specific BACE inhibitor GL189. C, analysis of in vitro produced Abeta -(1-34) by MALDI-TOF MS. 50 µg of synthetic Abeta -(1-40) were incubated at 37 °C for 1, 5, or 8 h with purified sBACE or a control fraction not containing sBACE. The tables below the spectra indicate the peak masses measured (mass) and the respective masses calculated (mass calc.). Note that BACE is capable of producing Abeta -(1-34) under in vitro conditions.

Because in vivo, only very minor amounts of BACE are secreted (data not shown and Ref. 55) we next investigated if membrane-bound BACE can convert secreted Abeta -(1-40/42) to Abeta -(1-34) in living cells. To do so we collected conditioned media (Fig. 4A) from cells expressing beta APPsw and endogenous BACE. These media were then added either to cells expressing PS1 D385N alone or to cells co-expressing PS1 D385N and BACE. Because of the lack of gamma -secretase activity in the latter cell line almost no de novo synthesis of any Abeta species occurs (Ref. 42; compare also Fig. 2B). Therefore any truncation of Abeta should occur independent of gamma -secretase activity. In conditioned media incubated with cells expressing PS1 D385N no detectable conversion of Abeta -(1-40) to Abeta -(1-34) was observed (Fig. 4B). However, upon addition of the conditioned media to cells expressing both PS1 D385N and BACE, Abeta -(1-34) was readily produced (Fig. 4C). Taken together these results demonstrate that BACE can proteolytically modify Abeta species, which were originally produced by a gamma -secretase-dependent pathway.


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Fig. 4.   Membrane-bound BACE generates Abeta -(1-34) in living cells. A, HEK 293 cells stably expressing beta APP695 sw were incubated with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum for 48 h. One-third of the conditioned media of HEK 293 cells was immunoprecipitated with antibody 3926. Immunopreciptated Abeta was analyzed by MALDI-TOF MS. The remaining conditioned media (see A) was added to cells expressing either beta APP695 sw and PS1 D385N alone (B) or beta APP695 sw, PS1 D385N, and BACE (C). Abeta species were immunoprecipitated from the media after an incubation period of 24 h using antibody 3926. Subsequently samples were subjected to MALDI-TOF MS. The tables below the spectra indicate the peak masses measured (mass) and the respective masses calculated (mass calc.). Note that although no Abeta species are secreted in cells expressing PS1 D385N (compare Fig. 2), Abeta -(1-34) is generated in conditioned media when BACE is overexpressed.

While BACE is the protease with the major beta -secretase activity (15-17), the homologous BACE-2 can also proteolytically process beta APP to some extend (9, 13, 14, 56). To investigate if the BACE homologue BACE-2 may also be able to convert Abeta -(1-40/42) into Abeta -(1-34) we co-expressed wild type beta APP together with either moderate or high levels of BACE-2 (Fig. 5A). Low level expression of BACE-2 allowed the recovery of very small amounts of Abeta -(1-34), which were not detectable in cells expressing no exogenous BACE-2 (Fig. 5B). However, high level expression of BACE-2 allowed the generation of increased amounts of Abeta -(1-34) thus demonstrating a BACE-2-dependent generation of Abeta -(1-34). These data demonstrate that both BACE and BACE-2 have the unexpected ability to generate C-terminally truncated Abeta species.


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Fig. 5.   Abeta -(1-34) generation correlates with BACE-2 expression. A, membranes of HEK 293 cells stably overexpressing either beta APP695 wt (control; lane 1) or additionally BACE-2 (lanes 2 and 3) were probed with antibody 7524. Note the increasing amounts of BACE-2 expression in the three cell lines. Longer exposure reveals endogenous BACE-2 (data not shown). B, MALDI-TOF MS of Abeta peptides immunocaptured directly from conditioned media of the cell lines shown in A, using antibody 3926. Arbitrary intensities are given on the y-axis (a.i.). The tables below the spectra indicate the peak masses measured (mass) and the respective masses calculated (mass calc.). Note that Abeta -(1-34) levels correlate with BACE-2 expression.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BACE plays a central role in the pathogenesis of AD and appears to be the sole beta -secretase, since its knock-out in mice fully abolishes Abeta generation (15-17). In addition and in contrast to the loss of gamma -secretase activity the knock out of BACE has no obvious phenotype (15-17). Thus BACE became a primary target for the development of therapeutic strategies. However, very little is known about the biological function of BACE. In addition we do not yet know precisely where the major proteolytic acitivity of BACE is localized within the cell. BACE is co-translated into the endoplasmic reticulum (ER) as a pro-enzyme (43, 57). During its trafficking through the secretory pathway the pro-domain is removed, and complex glycosylation occurs (43, 57-61). Upon reaching the plasma membrane BACE is reinternalized and targeted to endosomes (44, 62). From endosomes BACE is retrieved in a phosphorylation-dependent manner and transported back to the trans-Golgi network (44). Although BACE has an acidic pH optimum it is apparently active in early compartments such as the ER, since small amounts of Abeta can accumulate in pre-Golgi compartments (45). Work on the Swedish beta APP mutation also demonstrated beta -secretase activity within the trans-Golgi network (64). In contrast wild type beta APP is apparently processed by BACE within early endosomes after reinternalization from the plasma membrane (65). So far no proteolytic activity of BACE was demonstrated on the cell surface. Here we show that Abeta can be truncated by BACE at its C terminus after its generation by gamma -secretase (Fig. 6). Thus it appears likely that secreted Abeta is further processed by BACE at or close to the plasma membrane although we cannot exclude uptake of Abeta prior to its processing by BACE.


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Fig. 6.   beta APP processing by BACE, BACE-2, and gamma -secretase. A, BACE cleaves beta APP at Asp-1 of the Abeta domain (black and light gray) to liberate a soluble part of the ectodomain (APPs beta ; large white box). The C-terminal stub remains within the membrane (APPCTFbeta ). Subsequently PS-dependent gamma -secretase cleavages occur, which then release AICD (small white box) or Abeta . Only upon gamma -secretase cleavage BACE further processes Abeta at position 34 of the Abeta domain, which results in the generation of Abeta -(1-34) (black box) and a small hydrophobic peptide (light gray box), which may be quickly degraded. B, enlargement of the Abeta domain. The known BACE cleavage sites are indicated, and the respective BACE-2 cleavage sites are indicated. The Abeta domain is marked by the black and light gray, with the blue box indicating Abeta -(1-34).

Our data demonstrate a novel cleavage site at position 34 of the Abeta domain. This was unexpected since full-length beta APP is cleaved by BACE in a highly sequence-specific manner (66). Moreover, previous work demonstrated that in vivo a membrane bound substrate is required for recognition by BACE (66). Obviously, soluble Abeta escapes these requirements and is cleaved by BACE after amino acid 34 of the Abeta domain. This suggests that the initial cleavage of BACE at the Met-Asp bond of the Abeta domain occurs in a different structural context than the secondary cut at position 34. This latter cleavage occurs only after Abeta -(1-40/42) generation, whereas the first cleavage requires the membrane bound precursor with a specific recognition sequence at the Met-Asp bond. Furthermore, BACE-2, which differs in its cleavage specificity of beta APP (Fig. 6) by predominantly generating an alpha -secretase-like cleavage after amino acid 19 of the Abeta domain (9, 13, 14) is also able to generate Abeta -(1-34) from Abeta -(1-40/42). Both enzymes apparently share similar sequence requirements for recognition and cleavage of soluble Abeta but different preferences for cleavage of full-length beta APP. This may also have implications for the search for physiological substrates of BACE. So far only a sialyltransferase (ST6 Gal 1) has been identified as a putative BACE substrate beside beta APP (67). Our results suggest that substrate-mimicking peptides have to be considered as natural BACE substrates in vivo.

Abeta -(1-34) has been shown to exist in vivo (29). The novel cleavage activity of BACE and BACE-2 removes the most hydrophobic sequences of the Abeta domain. This may inhibit aggregation and thus facilitate proteolytic clearance by the insulin-degrading enzyme (68) or neprilysin (63, 69). Thus BACE may play an unexpected role in Abeta clearance.

Finally, our data also demonstrate that an apparently PS- and gamma -secretase-dependent cut is in fact mediated by BACE. Since the cleavage after amino acid 34 is fully dependent on the previous PS/gamma -secretase cleavage, the involvement of proteases in the generation of C-terminally truncated Abeta peptides may have been misinterpreted (52, 53). Moreover, our results also solve the apparent paradox that increased BACE expression results in reduced Abeta production (2), since the truncated Abeta -(1-34) species cannot be detected by autoradiography due to the loss of the Met residue at position 35 of the Abeta domain. Furthermore, under the electrophoretic conditions used (54), the peptide aberrantly migrates at a higher molecular weight as expected, again confusing the analysis of Abeta produced in BACE-expressing cells. Finally our findings may also suggest that substrate-mimetic peptides could represent a novel therapeutic approach in vivo.

    ACKNOWLEDGEMENTS

We thank H. Steiner, P. Kahle, and A. Capell for critical discussion and Klaus Maskos and Luis Moroder for the generation of the BACE inhibitor GL189.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 596-Project B1 (to C. H.) and the Priority Program (to J. W., C. H., and G. M.)) and the DIADEM Project (to C. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

b These authors contributed equally to this work.

d Present address: Dept. of Chemistry/Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.

g Present address: Dept. of Neurology, University of Bonn; Sigmund-Freud-Str. 25, 53105 Bonn, Germany.

h These authors contributed equally to this work.

i To whom correspondence may be addressed. Tel.: 49-89-5996-471/472; Fax: 49-89-5996-415; E-mail: chaass@pbm.med.uni-muenchen.de.

j To whom correspondence may be addressed. Tel.: 49-228-2879782; Fax: 49-228-2874387; E-mail: jochen.walter@ukb.uni-bonn.de.

Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M211485200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta -peptide; beta APP, beta -amyloid precursor protein; PS, presenilin; BACE, beta -site APP-cleaving enzyme; sBACE, soluble BACE; wt, wild type; HEK, human embryonic kidney; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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