Identification of a β-Secretase Activity, Which Truncates Amyloid β-Peptide after Its Presenilin-dependent Generation
2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês
10.1074/jbc.m211485200
ISSN1083-351X
AutoresRegina Fluhrer, Gerd Multhaup, Andrea Schlicksupp, Masayasu Okochi, Masatoshi Takeda, Sven Lammich, Michael Willem, Gil G. Westmeyer, Wolfram Bode, Jochen Walter, Christian Haass,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe β-amyloid precursor protein (βAPP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid β-peptide (Aβ). N-terminal cleavage is mediated by β-secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) γ-secretase complex. The Aβ-generating γ-secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of Aβ-(1–40) or Aβ-(1–42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted Aβ-(1–40/42). However, mass spectrometry revealed a truncated Aβ species, which terminates at amino acid 34 (Aβ-(1–34)) suggesting an alternative γ-secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of Aβ-(1–40) and Aβ-(1–42) but also that of Aβ-(1–34). However, expression levels of BACE correlate with the amount of Aβ-(1–34), and Aβ-(1–34) is produced at the expense of Aβ-(1–40) and Aβ-(1–42). Since this suggested that BACE is involved in a C-terminal truncation of Aβ, we incubated purified BACE with Aβ-(1–40) in vitro. Under these conditions Aβ-(1–34) was generated. Moreover, when conditioned media containing Aβ-(1–40) and Aβ-(1–42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, Aβ-(1–34) was efficiently produced in vivo. These data demonstrate that an apparently γ-secretase-dependent Aβ derivative is produced after the generation of the non-truncated Aβ via an additional and unexpected activity of BACE. The β-amyloid precursor protein (βAPP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid β-peptide (Aβ). N-terminal cleavage is mediated by β-secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) γ-secretase complex. The Aβ-generating γ-secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of Aβ-(1–40) or Aβ-(1–42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted Aβ-(1–40/42). However, mass spectrometry revealed a truncated Aβ species, which terminates at amino acid 34 (Aβ-(1–34)) suggesting an alternative γ-secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of Aβ-(1–40) and Aβ-(1–42) but also that of Aβ-(1–34). However, expression levels of BACE correlate with the amount of Aβ-(1–34), and Aβ-(1–34) is produced at the expense of Aβ-(1–40) and Aβ-(1–42). Since this suggested that BACE is involved in a C-terminal truncation of Aβ, we incubated purified BACE with Aβ-(1–40) in vitro. Under these conditions Aβ-(1–34) was generated. Moreover, when conditioned media containing Aβ-(1–40) and Aβ-(1–42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, Aβ-(1–34) was efficiently produced in vivo. These data demonstrate that an apparently γ-secretase-dependent Aβ derivative is produced after the generation of the non-truncated Aβ via an additional and unexpected activity of BACE. 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). 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-peptide; βAPP, β-amyloid precursor protein; PS, presenilin; BACE, β-site APP-cleaving enzyme; sBACE, soluble BACE; wt, wild type; HEK, human embryonic kidney; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-peptide; βAPP, β-amyloid precursor protein; PS, presenilin; BACE, β-site APP-cleaving enzyme; sBACE, soluble BACE; wt, wild type; HEK, human embryonic kidney; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight During the aging process the patients accumulate insoluble amyloid β-peptide (Aβ), which is deposited in senile plaques and microvessels in the brain. Aβ is generated by endoproteolytic processing of the β-amyloid precursor protein (βAPP), involving β- and γ-secretase (1Haass C. Steiner H. 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Trends Cell Biol. 2002; 12: 556-562Google Scholar, 27Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Google Scholar). 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. 1Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Google Scholar). The cleavage of BACE-generated CTFβ by γ-secretase results in the secretion of Aβ into biological fluids (1Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Google Scholar). 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 βAPP itself (28Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Google Scholar). Beside the predominant cleavage after amino acid 40 and 42 slightly shorter peptides have been observed as well, suggesting that the γ-secretase has loose sequence specificity (29Wang R. Sweeney D. Gandy S.E. Sisodia S.S. J. Biol. Chem. 1996; 271: 31894-31902Google Scholar). This includes peptides terminating after amino acid 34, 37, 38, and 39 (29). In addition or in parallel to these cleavages, γ-secretase also cleaves within the transmembrane domain shortly before the cytoplasmic border after amino acid 49 to liberate the βAPP intracellular domain (AICD) (30Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Google Scholar, 31Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Google Scholar, 32Yu C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Google Scholar, 33Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. 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Wong P.C. Nat. Neurosci. 2001; 4: 233-234Google Scholar). 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 Aβ secretion (2Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Google Scholar). In order to investigate this paradox we analyzed Aβ 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. HEK293 were cultured as described (9Fluhrer R. Capell A. Westmeyer G. Willem M. Hartung B. Condron M.M. Teplow D.B. Haass C. Walter J. J. Neurochem. 2002; 81: 1011-1020Google Scholar). The cell lines stably overexpressing wild type βAPP695 (40Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Google Scholar) or βAPP695 containing the Swedish double mutation (βAPPsw) (41Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberburg I. Selkoe D.J. Nature. 1992; 360: 672-674Google Scholar) and cell lines co-expressing either PS1 wt or PS1 D385N have been described (42Steiner H. Romig H. Pesold B. Philipp U. Baader M. Citron M. Loetscher H. Jacobsen H. Haass C. Biochemistry. 1999; 38: 14600-14605Google Scholar). To transfect BACE into these cell lines, the BACE cDNA was cloned into theEcoRI/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 (9Fluhrer R. Capell A. Westmeyer G. Willem M. Hartung B. Condron M.M. Teplow D.B. Haass C. Walter J. J. Neurochem. 2002; 81: 1011-1020Google Scholar). Antibodies 7520 (43Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Google Scholar, 44Walter J. Fluhrer R. Hartung B. Willem M. 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For immunodetection of PS1 the polyclonal and monoclonal antibodies against the large hydrophilic loop of PS1 (3027 and BI.3D7) were used (47Steiner H. Romig H. Grim M.G. Philipp U. Pesold B. Citron M. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 7615-7618Google Scholar, 48Walter J. Capell A. Grunberg J. Pesold B. Schindzielorz A. Prior R. Podlisny M.B. Fraser P. Hyslop P.S. Selkoe D.J. Haass C. Mol. Med. 1996; 2: 673-691Google Scholar). Metabolic labeling, immunoprecipitations, and Western blotting were carried out as described previously (9Fluhrer R. Capell A. Westmeyer G. Willem M. Hartung B. Condron M.M. Teplow D.B. Haass C. Walter J. J. Neurochem. 2002; 81: 1011-1020Google Scholar). The fluorometric BACE activity assay was carried out as described previously (46Capell A. Meyn L. Fluhrer R. Teplow D.B. Walter J. Haass C. J. Biol. Chem. 2002; 277: 5637-5643Google Scholar). To selectively inhibit BACE activity GL189 was used as described previously (46Capell A. Meyn L. Fluhrer R. Teplow D.B. Walter J. Haass C. J. Biol. Chem. 2002; 277: 5637-5643Google Scholar). Soluble BACE (sBACE) was isolated and incubated with synthetic Aβ 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 Aβ-(1–40) for MALDI-TOF MS and with 6 μg of synthetic Aβ-(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. 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 (49Kulic L. Walter J. Multhaup G. Teplow D.B. Baumeister R. Romig H. Capell A. Steiner H. Haass C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5913-5918Google Scholar, 50Lammich S. Okochi M. Takeda M. Kaether C. Capell A. Zimmer A.-K. Edbauer D. Walter J. Steiner H. Haass C. J. Biol. Chem. 2002; 277: 44754-44759Google Scholar, 51Okochi M. Steiner H. Fukumori A. Tanii H. Tomita T. Tanaka T. Iwatsubo T. Kudo T. Haass C. Takeda M. EMBO J. 2002; 21: 5408-5416Google Scholar). Samples were analyzed on MALDI-target plates by matrix-assisted laser desorption ionization (Brucker Reflex III). In order to analyze the Aβ species secreted by BACE-expressing cells we collected conditioned media from HEK 293 cells stably transfected with Swedish mutant βAPP695 (βAPPsw) and BACE. Cells expressing either endogenous levels of BACE or moderate or high levels of transfected BACE were investigated (Fig.1 A). To prove the catalytic activity of BACE in these cell lines we performed in vitroactivity assays using solubilized membranes of the respective cell lines (46Capell A. Meyn L. Fluhrer R. Teplow D.B. Walter J. Haass C. J. Biol. Chem. 2002; 277: 5637-5643Google Scholar). As expected we found substantially increased β-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-Aβ antibody 3926. Surprisingly, increasing BACE expression negatively correlated with Aβ production (Fig. 1 B). This is consistent with previous findings by Vassar et al. (2Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Google Scholar) who observed reduced Aβ production despite increased BACE activity in cells transfected with βAPPsw (2Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Google Scholar). This paradoxical finding raised the possibility that Aβ 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 Aβ species by a combined immunoprecipitation/MALDI-TOF MS method. As expected cells expressing endogenous BACE secreted predominantly Aβ-(1–40) (Fig. 1 C). In addition we also obtained small amounts of Aβ-(1–42) and Aβ-(1–37/38/39) (Fig. 1 C). Upon expression of moderate levels of BACE we observed an additional Aβ species (Aβ-(1–34); Fig. 1 C). In order to analyze if the production of this truncated species is related to BACE expression levels, we next investigated Aβ species secreted from cells expressing higher levels of BACE (Fig. 1 A). This revealed robust amounts of Aβ-(1–34), which was accompanied by reduced levels of Aβ-(1−40), Aβ-(1−42), and Aβ-(1–37/38/39) (Fig. 1 C). Similar results were obtained using cell lines co-expressing wtAPP and BACE (data not shown). The detection of robust levels of Aβ-(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 Aβ peptide has been removed by the additional cleavage. Although the above described results suggest that BACE is directly involved in the enhanced production of Aβ-(1–34), previous observations indicated that C-terminally truncated Aβ species including Aβ-(1–34) are generated by the γ-secretase complex in a PS-dependent manner (52Vandermeeren M. Geraerts M. Pype S. Dillen L. Van Hove C. Mercken M. Neurosci. Lett. 2001; 315: 145-148Google Scholar, 53Beher D. Wrigley J.D. Owens A.P. Shearman M.S. J. Neurochem. 2002; 82: 563-575Google Scholar). In order to analyze if a PS-dependent γ-secretase activity is required for Aβ-(1–34) generation, we co-expressed BACE with either PS1 wt or the non-functional PS1 D385N mutant (Fig.2 A). As shown previously (42Steiner H. Romig H. Pesold B. Philipp U. Baader M. Citron M. Loetscher H. Jacobsen H. Haass C. Biochemistry. 1999; 38: 14600-14605Google Scholar), PS1 wt undergoes endoproteolysis whereas no endoproteolysis was obtained in cells expressing PS1 D385N (Fig. 2 A, right panel; Ref. 27Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Google Scholar). The non-functional PS1 D385N fully replaced biologically active endogenous PS (Fig. 2 A,right panel). Whereas robust levels of Aβ-(1–34) and Aβ-(1–40) (and all other minor Aβ species) were produced from cells co-expressing PS1 wt and BACE, Aβ-(1–34) generation as well as generation of all other Aβ species was almost completely inhibited in the presence of the non-functional PS1 D385N (Fig. 2 B,right panel). This clearly demonstrates that a PS-dependent γ-secretase activity is involved directly or indirectly in the production of Aβ-(1–34). However, the results described in Fig. 1, demonstrated that upon BACE expression Aβ-(1–34) generation occurs to the expense of the production of all other Aβ variants and thus suggests a direct involvement of BACE in the cleavage of Aβ at position 34. This apparent paradox may indicate that γ-secretase activity is required first to produce secreted Aβ species, which are then trimmed at their C termini by a so far unknown BACE activity. In order to prove this hypothesis, synthetic Aβ-(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 (43Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Google Scholar). To prove the catalytic activity of secreted BACE we carried out in vitro assays (46Capell A. Meyn L. Fluhrer R. Teplow D.B. Walter J. Haass C. J. Biol. Chem. 2002; 277: 5637-5643Google Scholar). Soluble BACE was fully active in the in vitro assay, whereas no activity was obtained in control media (Fig.3 A). This activity was fully blocked by the BACE-specific inhibitor GL189 (Fig. 3 A). Upon incubation of synthetic Aβ-(1–40) with soluble BACE an additional peptide, which co-migrated with synthetic Aβ-(1–34) was detected on a gel system capable to separate low molecular weight peptides (Fig.3 B; Ref. 54Wiltfang J. Smirnov A. Schnierstein B. Kelemen G. Matthies U. Klafki H.W. Staufenbiel M. Huther G. Ruther E. Kornhuber J. Electrophoresis. 1997; 18: 527-532Google Scholar). In vitro generation of Aβ-(1–34) was completely inhibited upon addition of the specific BACE inhibitor GL189 (Fig. 3 B). Using MALDI-TOF MS we confirmed a time-dependent generation of Aβ-(1–34) during incubation of soluble BACE with Aβ-(1–40) (Fig. 3 C). In contrast incubation of synthetic Aβ-(1–40) with conditioned media, not containing soluble BACE does not reveal truncated Aβ species (Fig.3 C). This finding demonstrates that BACE has the ability to cleave Aβ after amino acid 34 and, together with the results shown in Fig. 1, excludes artificial trimming by exopeptidases. Because in vivo, only very minor amounts of BACE are secreted (data not shown and Ref. 55Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chretien M. Seidah N.G. J. Biol. Chem. 2001; 276: 10879-10887Google Scholar) we next investigated if membrane-bound BACE can convert secreted Aβ-(1–40/42) to Aβ-(1–34) in living cells. To do so we collected conditioned media (Fig. 4 A) from cells expressing βAPPsw 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 γ-secretase activity in the latter cell line almost no de novo synthesis of any Aβ species occurs (Ref. 42Steiner H. Romig H. Pesold B. Philipp U. Baader M. Citron M. Loetscher H. Jacobsen H. Haass C. Biochemistry. 1999; 38: 14600-14605Google Scholar; compare also Fig. 2 B). Therefore any truncation of Aβ should occur independent of γ-secretase activity. In conditioned media incubated with cells expressing PS1 D385N no detectable conversion of Aβ-(1–40) to Aβ-(1–34) was observed (Fig. 4 B). However, upon addition of the conditioned media to cells expressing both PS1 D385N and BACE, Aβ-(1–34) was readily produced (Fig. 4 C). Taken together these results demonstrate that BACE can proteolytically modify Aβ species, which were originally produced by a γ-secretase-dependent pathway. While BACE is the protease with the major β-secretase activity (15Luo Y. Bolon B. Kahn S. Bennett B.D. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Martin L. Louis J.C. Yan Q. Richards W.G. Citron M. Vassar R. Nat. Neurosci. 2001; 4: 231-232Google Scholar, 16Roberds S.L. Anderson J. Basi G. Bienkowski M.J. Branstetter D.G. Chen K.S. Freedman S.B. Frigon N.L. Games D. Hu K. Johnson-Wood K. Kappenman K.E. Kawabe T.T. Kola I. Kuehn R. Lee M. Liu W. Motter R. Nicho
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