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The Protease Inhibitor, MG132, Blocks Maturation of the Amyloid Precursor Protein Swedish Mutant Preventing Cleavage by β-Secretase

2001; Elsevier BV; Volume: 276; Issue: 6 Linguagem: Inglês

10.1074/jbc.m008793200

1083-351X

Michelle Leigh Steinhilb, Raymond Scott Turner, James R. Gaut,

Amyloidosis: Diagnosis, Treatment, Outcomes

Amyloid (Aβ) peptides found aggregated into plaques in Alzheimer's disease are derived from the sequential cleavage of the amyloid precursor protein (APP) first by β- and then by γ-secretases. Peptide aldehydes, which inhibit cysteine proteases and proteasomes, reportedly block Aβ peptide secretion by interfering with γ-secretase cleavage. Using a novel, specific, and sensitive enzyme-linked immunosorbent assay for the β-secretase-cleaved fragment of the Swedish mutant of APP (APPSw), we determined that the peptide aldehyde, MG132, prevented β-secretase cleavage. This block in β-secretase cleavage was not observed withclasto-lactacystin β-lactone and thus, cannot be attributed to proteasomal inhibition. MG132 inhibition of β-secretase cleavage was compared with the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF inhibition of β-secretase cleavage was immediate and did not affect α-secretase cleavage. With MG132, inhibition was delayed and it decreased secretion of α-cleaved APPSw as well. Furthermore, MG132 treatment impaired maturation of full-length APPSw. Both inhibited intracellular formation of the β-cleaved product. These results suggest that peptide aldehydes such as MG132 have multiple effects on the maturation and processing of APP. We conclude that the MG132-induced decrease in β-secretase cleavage of APPSw is due to a block in maturation. This is sufficient to explain the previously reported peptide aldehyde-induced decrease in Aβ peptide secretion. Amyloid (Aβ) peptides found aggregated into plaques in Alzheimer's disease are derived from the sequential cleavage of the amyloid precursor protein (APP) first by β- and then by γ-secretases. Peptide aldehydes, which inhibit cysteine proteases and proteasomes, reportedly block Aβ peptide secretion by interfering with γ-secretase cleavage. Using a novel, specific, and sensitive enzyme-linked immunosorbent assay for the β-secretase-cleaved fragment of the Swedish mutant of APP (APPSw), we determined that the peptide aldehyde, MG132, prevented β-secretase cleavage. This block in β-secretase cleavage was not observed withclasto-lactacystin β-lactone and thus, cannot be attributed to proteasomal inhibition. MG132 inhibition of β-secretase cleavage was compared with the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF inhibition of β-secretase cleavage was immediate and did not affect α-secretase cleavage. With MG132, inhibition was delayed and it decreased secretion of α-cleaved APPSw as well. Furthermore, MG132 treatment impaired maturation of full-length APPSw. Both inhibited intracellular formation of the β-cleaved product. These results suggest that peptide aldehydes such as MG132 have multiple effects on the maturation and processing of APP. We conclude that the MG132-induced decrease in β-secretase cleavage of APPSw is due to a block in maturation. This is sufficient to explain the previously reported peptide aldehyde-induced decrease in Aβ peptide secretion. a 40- or 42-amino acid peptide derived from APP peptide derived from α- and γ-secretase cleavage of APP 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride COOH-terminal fragment amyloid precursor protein APP bearing the Swedish mutation (K651N/M652L) β-site APP-cleaving enzyme enzyme-linked immunosorbent assay soluble β-secretase-cleaved APPSw fragment intracellular soluble β-secretase-cleaved APPSw fragment soluble α-secretase-cleaved APPSw fragment soluble α-secretase-cleaved APP fragment soluble β-secretase-cleaved APP fragment Chinese hamster ovary human embryonic kidney polyacrylamide gel electrophoresis Dulbecco's modified Eagle's medium phosphate-buffered saline phosphate-buffered saline plus Tween 20 endoplasmic reticulum brefeldin A Evidence continues to accumulate supporting the hypothesis that amyloid plaques in the brain have a causative role in the generation of Alzheimer's disease (for review, see Ref. 1Selkoe D.J. Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). Increased brain levels of amyloid peptide and cognitive decline are strongly correlated (2Naslund J. Haroutunian V. Mohs R. Davis K.L. Davies P. Greengard P. Buxbaum J.D. JAMA. 2000; 283: 1571-1577Crossref PubMed Scopus (1106) Google Scholar). Amyloid plaques largely consist of peptides of 40 (Aβ40)1 and 42 (Aβ42) amino acids in length that are derived by the enzymatic processing of a type I transmembrane protein called amyloid precursor protein (APP). Two enzymatic cleavages of APP are necessary to produce amyloid peptides. First, β-secretase cleaves APP to create the amino-terminal end of the peptide. A double mutation (K651N/M652L; 751 isoform numbering) just amino-terminal to this β-secretase cleavage site has been identified in a Swedish pedigree of familial Alzheimer's disease (3Mullan M. Crawford F. Axelman K. Houlden H. Lilius L. Winblad B. Lannfelt L. Nature Genetics. 1992; 1: 345-347Crossref PubMed Scopus (1177) Google Scholar). This double mutation of APP, known as the “Swedish” mutation (APPSw), elevates intracellular and secreted levels of Aβ peptide from 6- to 8-fold (4Forman M.S. Cook D.G. Leight S. Doms R.W. Lee V.M. J. Biol. Chem. 1997; 272: 32247-32253Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This appears to be a consequence of increased cleavage of APPSw by β-secretase compared with wild type APP. Following β-secretase cleavage, γ-secretase subsequently cleaves the COOH-terminal membrane-bound fragment (CTF) of APP within the transmembrane sequence to release the Aβ peptide. Thus, inhibitors that specifically block the cleavage of APP by these secretases have enormous therapeutic potential. Several laboratories have now cloned an enzyme that cleaves APP and APPSw at the β-secretase site (5Vassar 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: 735-741Crossref PubMed Scopus (3271) Google Scholar, 6Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier JR Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi LA Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1329) Google Scholar, 7Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar). Referred to as BACE (β-site APP cleavingenzyme), this enzyme is a member of a unique family of transmembrane aspartic proteases. A second related protein designated ASP1 or BACE2 has also been identified (6Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier JR Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi LA Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1329) Google Scholar, 8Acquati F. Accarino M. Nucci C. Fumagalli P. Jovine L. Ottolenghi S. Taramelli R. FEBS Lett. 2000; 468: 59-64Crossref PubMed Scopus (121) Google Scholar, 9Bennett B.D. Babu-Khan S. Loeloff R. Louis J.C. Curran E. Citron M. Vassar R. J. Biol. Chem. 2000; 275 (20511): 20647Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). However, the expression pattern of BACE2 in the brain appears to exclude it from playing a major role in Alzheimer's disease (9Bennett B.D. Babu-Khan S. Loeloff R. Louis J.C. Curran E. Citron M. Vassar R. J. Biol. Chem. 2000; 275 (20511): 20647Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). The mature, fully glycosylated form of BACE has a half-life in the cell of greater than 9 h (10Haniu M. Denis P. Young Y. Mendiaz E.A. Fuller J. Hui J.O. Bennett B.D. Kahn S. Ross S. Burgess T. Katta V. Rogers G. Citron M. J. Biol. Chem. 2000; 275: 21099-21106Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). BACE appears to localize to the Golgi apparatus (5Vassar 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: 735-741Crossref PubMed Scopus (3271) Google Scholar). Despite about 40% amino acid similarity between BACE and pepsin proteases (9Bennett B.D. Babu-Khan S. Loeloff R. Louis J.C. Curran E. Citron M. Vassar R. J. Biol. Chem. 2000; 275 (20511): 20647Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), the cysteine residues in BACE involved in intramolecular disulfide bonds are not conserved with other pepsin family members (10Haniu M. Denis P. Young Y. Mendiaz E.A. Fuller J. Hui J.O. Bennett B.D. Kahn S. Ross S. Burgess T. Katta V. Rogers G. Citron M. J. Biol. Chem. 2000; 275: 21099-21106Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Such fundamental structural differences may explain why β-secretase is insensitive to pepstatin, a specific inhibitor of pepsin proteases (7Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar). The search for such a specific inhibitor of β-secretase cleavage of APP as a possible treatment for Alzheimer's disease has intensified with the discovery of BACE. Before the cloning of the aspartic protease, BACE, cellular studies using a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), suggested that it blocked Aβ peptide generation by directly inhibiting β-secretase activity (11Citron M. Diehl T.S. Capell A. Haass C. Teplow D.B. Selkoe D.J. Neuron. 1996; 17: 171-179Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). There is little information published about other potential specific inhibitors of β-secretase. Several studies have shown that peptide aldehyde protease inhibitors affect the secretion of Aβ peptides (12Higaki J. Quon D. Zhong Z. Cordell B. Neuron. 1995; 14: 651-659Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 13Klafki H.W. Paganetti P.A. Sommer B. Staufenbiel M. Neurosci. Lett. 1995; 201: 29-32Crossref PubMed Scopus (31) Google Scholar, 14Yamazaki T. Haass C. Saido T.C. Omura S. Ihara Y. Biochemistry. 1997; 36: 8377-8383Crossref PubMed Scopus (56) Google Scholar). Of these peptide aldehydes, MG132 was one of the most potent inhibitors of Aβ peptide secretion (15Klafki H. Abramowski D. Swoboda R. Paganetti P.A. Staufenbiel M. J. Biol. Chem. 1996; 271: 28655-28659Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Studies focusing specifically on the secretion of Aβ40 and Aβ42 revealed peptide aldehydes to have a complex effect on APP processing. Curiously, at low concentrations, these peptide aldehydes produced an increase in Aβ40 and Aβ42 peptide secretion, whereas, at higher concentrations, a decrease in Aβ peptide secretion was observed (14Yamazaki T. Haass C. Saido T.C. Omura S. Ihara Y. Biochemistry. 1997; 36: 8377-8383Crossref PubMed Scopus (56) Google Scholar, 15Klafki H. Abramowski D. Swoboda R. Paganetti P.A. Staufenbiel M. J. Biol. Chem. 1996; 271: 28655-28659Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 16Yamazaki T. Ihara Y. Neurobiol. Aging. 1998; 19: S77-S79Crossref PubMed Scopus (13) Google Scholar, 17Zhang L. Song L. Parker E.M. J. Biol. Chem. 1999; 274: 8966-8972Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The increase in Aβ peptide secretion at low concentrations of peptide aldehydes has been postulated to result from inhibition of degradation of the CTFs generated by BACE cleavage, making more of them available for γ-secretase cleavage (17Zhang L. Song L. Parker E.M. J. Biol. Chem. 1999; 274: 8966-8972Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The inhibition of Aβ peptide secretion with higher concentrations of peptide aldehydes is attributed to an impairment of γ-secretase cleavage of the CTFs (12Higaki J. Quon D. Zhong Z. Cordell B. Neuron. 1995; 14: 651-659Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 13Klafki H.W. Paganetti P.A. Sommer B. Staufenbiel M. Neurosci. Lett. 1995; 201: 29-32Crossref PubMed Scopus (31) Google Scholar, 17Zhang L. Song L. Parker E.M. J. Biol. Chem. 1999; 274: 8966-8972Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 18Skovronsky D.M. Pijak D.S. Doms R.W. Lee V.M. Biochemistry. 2000; 39: 810-817Crossref PubMed Scopus (71) Google Scholar). By developing an ELISA specific for the soluble β-secretase-cleaved amino-terminal fragment of APPSw (APPSwβ; see Fig. 1 A), we now report that the peptide aldehyde, MG132, prevents β-secretase cleavage of APPSw in a concentration-dependent manner. Furthermore, MG132 is not inhibiting secretion of APPSwβ into the medium since it blocked intracellular production of APPSwβ as well. MG132 is compared with AEBSF and the specific proteasomal inhibitor,clasto-lactacystin β-lactone, on β-secretase cleavage. MG132 impairs maturation, blocking β-secretase cleavage of APPSw in the late Golgi apparatus. This offers an alternative explanation as to how higher concentrations of a peptide aldehyde can decrease Aβ peptide secretion. The mouse monoclonal antibodies referred to in Fig. 1 A as 22C11 andBIOSOURCE were obtained from Roche Molecular Biochemicals and BIOSOURCE International (monoclonal antibody P2-1), respectively. The mouse monoclonal antibody 6E10, which recognizes an epitope in the first 17 amino acids of the Aβ peptide, was obtained from Senetek, Inc. The anti-APP COOH-terminal rabbit polyclonal antibody was obtained from Chemicon International, Inc., and the mouse monoclonal antibody to the amino terminus of APP, LN27, was purchased from Zymed Laboratories Inc. The mouse monoclonal antibody, 8E5, was a generous gift from Dr. Dale Schenk (Elan Pharmaceuticals). The rabbit polyclonal antibody, 945, was raised against a synthetic peptide corresponding to the last 19 amino acid residues of the carboxyl terminus of APP (CMQQNGYENPTYKFFEQMQN) that was cross-linked to keyhole limpet hemocyanin via an amino-terminal cysteine. The polyclonal antibodies, 931 and 932, were raised against a synthetic peptide corresponding to 19 amino acid residues (CRPGSGLTNIKTEEISEVNL) just amino-terminal to the β-secretase cleavage site of APPSw that was similarly cross-linked to keyhole limpet hemocyanin via an amino-terminal cysteine. The peptide aldehyde, MG132, was dissolved in dimethyl sulfoxide (Me2SO) at a concentration of 10 mm (Peptides International). The proteasomal inhibitor,clasto-lactacystin β-lactone (Calbiochem), was also dissolved in Me2SO at 2 mm. Me2SO was used in all experiments as a vehicle control. The serine protease inhibitor AEBSF (Sigma) was dissolved in sterile water at 0.2m. The Chinese hamster ovary cell line, CHOK1, and the HEK293 cell line used for transient transfections were obtained from the American Type Culture Collection. The cell lines stably expressing the 695 isoforms of either APPSw (CHOAPPSw) or APP wild type were a generous gift from Taraneh Haske (Pfizer Pharmaceuticals, Ann Arbor, MI). All CHO cell lines were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, glutamine, nonessential amino acids, and penicillin/streptomycin/fungizone as described (19Yang Y. Turner R.S. Gaut J.R. J. Biol. Chem. 1998; 273: 25552-25555Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Experiments involving transient expression of APPSw or APP were conducted using cDNAs encoding for the 751 isoforms that were cloned into the pCDNA3 mammalian expression vector (Invitrogen, Inc.). Transfections of HEK293 cells grown on 60-mm plates were conducted using LipofectAMINE as described by the Life Technologies, Inc. Forty hours following transfection, cells were metabolically labeled or lysed for immunoblot analysis as described below. Cells stably or transiently expressing APP or APPSw were preincubated in methionine/cysteine-free medium for 15 min prior to labeling. Cells were metabolically labeled by incubating in 2 ml of medium containing [35S]methionine and [35S]cysteine (Tran35S-label; ICN Pharmaceuticals) at 50 μCi/ml for 1 h. In pulse-chase studies, cells were preincubated in methionine/cysteine-free medium for 15 min and then pulsed for 12 min with Tran35S-label (100 μCi/ml). Cells were then washed and incubated in complete medium containing excess methionine and cysteine for the chase times shown. After labeling and chase were complete, the conditioned medium was collected and cells washed in PBS. Cells were lysed in 1 ml of lysis buffer (0.5% Nonidet P-40, 0.5% deoxycholate in 50 mm Tris, 150 mm NaCl, and 5 mm EDTA, pH 8.0) and insoluble cell debris removed by centrifugation as described (19Yang Y. Turner R.S. Gaut J.R. J. Biol. Chem. 1998; 273: 25552-25555Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The resulting cleared supernatant of the cell lysate was then subjected to further analysis. Full-length APP and APPSw were isolated from the cell lysate supernatants by immunoprecipitation using the 945 rabbit antisera to the carboxyl terminus of APP. Except where noted, lysates were incubated with 4 μl of 945 antisera for 90 min at 4 °C and protein-antibody complexes were isolated by incubation with protein A-Sepharose for 30 min at 4 °C. Intracellular APPSwβ was similarly isolated from cell lysates using 4 μl of 931 rabbit antisera. The 931 antisera was also used to isolate secreted APPSwβ from 0.5 ml of conditioned medium that had Nonidet P-40 and deoxycholate added to a final concentration of 0.5%. Immunoprecipitations using the mouse monoclonal antibodies, 8E5 or 6E10, were conducted as described above except that protein-antibody complexes were isolated using protein G-agarose (Roche Molecular Biochemicals). Isolated proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) using an 8% separating gel. Radiolabeled proteins in SDS gels were detected by fluorography using Amplify (Amersham Pharmacia Biotech). Immunoblot analysis of isolated proteins was conducted essentially as described (19Yang Y. Turner R.S. Gaut J.R. J. Biol. Chem. 1998; 273: 25552-25555Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Briefly, immunoprecipitated proteins were resolved by SDS-PAGE and transferred to PROTRAN (Schleicher & Schuell). Membranes were subsequently blocked in gelatin wash buffer (0.1% gelatin, 15 mm Tris, pH 7.5, 130 mm NaCl, 1 mm EDTA, and 0.1% Triton X-100). Membranes were subsequently incubated with mouse monoclonal antibody 22C11 to detect the amino-terminal end of APP molecules from cell lysates or conditioned medium. Membranes were alternatively incubated with 6E10 to detect APPSwα in conditioned medium. Membranes were subsequently washed and incubated with a sheep anti-mouse IgG antibody conjugated to horseradish peroxidase as described by the manufacturer (Amersham Pharmacia Biotech). The membranes were again washed and signals detected by chemiluminescence using the ECL system (Amersham Pharmacia Biotech). The 945 and 931 antisera were used as capture antibodies for the full-length APP and APPSwβ sandwich ELISAs, respectively. Each was first affinity-purified against the appropriate peptide that had been cross-linked to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) following the manufacturer's instructions. The anti-APP COOH-terminal antibody, 945, was affinity-purified with the same 20-amino acid synthetic peptide used above to inoculate rabbits. The 931 antibody was affinity-purified using a synthetic peptide identical to the last seven amino acids of the neoepitope of the β-secretase-cleaved APPSw soluble fragment (CEIESVNL). The 945 and 931 antibodies that bound to the immobilized peptide were eluted using ActiSep Elution Medium (Sterogene). The eluted antibodies were desalted using a PD-10 column (Amersham Pharmacia Biotech) as directed by the manufacturers. The affinity-purified capture antibodies (945 and 931) were diluted to 1.2 μg/ml in PBS and 100 μl added per well to a 96-well Nunc-Immuno Maxisorp plate (Nalge Nunc International). After incubation to allow binding of antibody, SuperBlock (Pierce) was added to each well to block nonspecific binding sites. The wells were repeatedly rinsed and then stored at 4 °C in PBS, 0.05% Tween 20 (PBS/T) until ready for use. For the full-length APP ELISA, the protein concentration of the cell lysate supernatant was determined using the BCA protein assay (Pierce). Ten micrograms of protein from the cell lysates were aliquoted into each well. The total volume in the well was adjusted to 100 μl with PBS, and samples were incubated overnight at 4 °C. On the following day, the samples were incubated an additional 1 h with constant shaking. The wells were then washed four times with PBS/T and then incubated with 100 μl of diluted detector antibody (the mouse monoclonal antibody, 8E5, at 0.25 μg/ml except where noted). All antibodies used in the ELISA were diluted using a solution of 10% SuperBlock and 90% PBS/T. The detector antibody was incubated with each sample for 4 h with constant shaking. The wells were again washed four times with PBS/T and subsequently incubated for 1 h with rabbit anti-mouse IgG conjugated to horseradish peroxidase (diluted 1:4000; Southern Biotechnology Associates, Inc.). Following three washes with PBS/T and two washes with PBS alone, 100 μl of 3,3′,5,5′-tetramethylbenzidine (Pierce) solution was added to each well. The reaction was stopped by the addition of an equal volume of 2m sulfuric acid. The relative amount of full-length APP in the sample was then quantified colorimetrically at 450 nm. The levels of secreted APPSwβ were assayed in the exact same way except that plates coated with affinity-purified 931 antibody were used to capture the β-secretase-cleaved protein and just 25 μl of conditioned medium was loaded per well. Each experiment was repeated at least three times, and the indicated values are averages of triplicate measurements ± S.D. As shown in Fig. 1, β-secretase cleavage of APPSw generates a large soluble amino-terminal fragment (APPSwβ) that is secreted into the medium and a CTF that is subsequently cleaved by γ-secretase to derive the Aβ peptide. Two novel rabbit polyclonal antisera were generated to perform the experiments described below. The binding sites of these and other antibodies are also shown in Fig. 1. The first polyclonal antisera (945) was raised to the last 20 amino acids of the APP carboxyl terminus. The specificity of the anti-COOH-terminal antibody, 945, is shown in Fig. 2 A. CHO cells stably expressing APPSw (CHOAPPSw) were metabolically labeled for 1 h with Tran35S-label. Following cell lysis, equal amounts of supernatant were incubated with 1, 2, or 4 μl of 945 antiserum or with 4 μl of preimmune serum followed by protein A-Sepharose. The immunoprecipitated APPSw was compared with that obtained using the Chemicon anti-COOH-terminal antibody by SDS-PAGE. The resulting autoradiograph shows that 945 specifically immunoprecipitates the N-glycosylated immature (I) and completely glycosylated mature (M) forms of APPSw. The second antisera specifically recognizes only the carboxyl terminus of the soluble amino-terminal fragment (APPSwβ) created when β-secretase cleaves APPSw just COOH-terminal to Leu652 (751 numbering). Results shown in Fig. 2 B demonstrate that antisera raised in two rabbits (931 and 932) against the 20-amino acid sequence just amino-terminal to the β-secretase cleavage site are capable of immunoprecipitating a soluble APPSw fragment from the conditioned medium of CHOAPPSw cells. Culture medium conditioned for 24 h by CHOAPPSw cells was divided into equal aliquots and incubated with preimmune sera from rabbit 931, 931 antisera, or 932 antisera. The mouse monoclonal antibody, 8E5, which recognizes an epitope in the lumenal region of APP (Fig. 1), served as a positive control. The immunoprecipitates were resolved by SDS-PAGE and the APPSw amino-terminal fragments were identified by Western blot analysis using the mouse monoclonal antibody 22C11. Both 931 and 932 antisera, but not preimmune sera, immunoprecipitated a specific APP soluble fragment from the conditioned media. The 931 antisera appeared to have a higher titer than 932. Thus, all subsequent experiments utilized only 931. As others have demonstrated, the α- and β-cleaved soluble fragments are difficult to electrophoretically resolve from one another (20Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L Selkoe D.J. Nature Med. 1995; 1: 1291-1296Crossref PubMed Scopus (440) Google Scholar, 21Chyung A.S.C. Greenberg B.D. Cook D.G. Doms R.W. Lee V.M.Y. J. Cell Biol. 1997; 138: 671-680Crossref PubMed Scopus (136) Google Scholar). Therefore, it was unclear from this Western blot analysis of conditioned medium whether the soluble APPSw fragment immunoprecipitated by 931 was limited to secreted APPSwβ or also included α-secretase-cleaved APPSw (APPSwα). Furthermore, it was not known if 931 could immunoprecipitate wild type APPβ and APPα. Consequently, HEK293 cells were transiently transfected with either pCDAPP or pCDAPPSw. Medium that had been conditioned for 36 h by these transiently transfected cells was collected and soluble APP fragments were immunoprecipitated with 931 or the mouse monoclonal antibody 8E5. Since the epitope recognized by 8E5 is amino-terminal to the β-secretase cleavage site, it is capable of immunoprecipitating APPα, APPβ, APPSwα, and APPSwβ. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted using the mouse monoclonal antibody, 22C11 to detect all forms of soluble APP and APPSw (see Fig.1). As expected, 8E5 was able to immunoprecipitate both secreted APP and APPSw from the conditioned media (Fig. 2 C,panel 1). However, 931 only immunoprecipitated soluble APPSw amino-terminal fragments from the conditioned medium of cells transiently transfected with pCDAPPSw and did not recognize wild type secreted APP fragments. A set of immunoprecipitations from conditioned medium identical to that conducted in Fig. 2 C(panel 1) was immunoblotted with the mouse monoclonal antibody 6E10, which detects only secreted APP and APPSw soluble fragments that have been cleaved by α-secretase. Although both APPα and APPSwα were detected in the 8E5 immunoprecipitates, neither form was immunoprecipitated using the 931 antibody (Fig.2 C, panel 2). Taken together, these results demonstrate that the 931 antibody specifically recognizes only the neoepitope derived with β-secretase cleavage of APPSw. It does not cross-react with full-length APPSw (compare Fig. 4, A and C, chase at 0 min), soluble APPα, APPβ, or APPSwα. Since 931 specifically recognized the neoepitope of β-secretase-cleaved APPSw in conditioned medium, it was used to create an enzyme-linked immunosorbent assay (ELISA) to quantitatively measure the amount of secreted APPSwβ. A similar ELISA was also developed to measure the amount of full-length APP or APPSw present in cell lysates using 945. To identify the best detector antibody to use in the ELISA, the affinity-purified 945 was coated on 96-well plates to capture full-length APP from cell lysates of either CHO cells or CHO cells stably expressing APPSw. After rinsing, triplicate wells were incubated with no detector, 8E5 (0.25 μg/ml), BIOSOURCE(0.25 μg/ml), or LN27 (0.5 μg/ml) mouse monoclonal antibodies and developed as described under “Experimental Procedures.” The signal level observed using CHO cell lysates with these detector antibodies did not significantly differ from background, demonstrating the specificity of the ELISA for the stably expressed, human APPSw (Fig.2 D). Full-length APPSw was specifically detected in CHOAPPSw lysates with 8E5 and the BIOSOURCE mouse monoclonal antibodies showing the greatest sensitivity. A total protein concentration of 10 μg of cell lysate was found to be optimal to detect full-length APPSw (data not shown). The 8E5 and BIOSOURCE mouse monoclonal antibodies were also evaluated as detector antibodies in the 931 ELISA to measure secreted APPSwβ. Both 8E5 and BIOSOURCEantibodies were sensitive detectors of APPSwβ captured in wells coated with affinity-purified 931 (Fig. 2 E). The addition of the eight-amino acid synthetic peptide corresponding to the carboxyl terminus of APPSwβ to the conditioned medium from CHOAPPSw cells blocked detection of the β-cleaved product. When using 6E10 as the detector antibody, no signal above background was observed (data not shown). This indicated that no APPSwα was captured in the ELISA by the affinity-purified 931. Together, these results demonstrate the high specificity of this novel ELISA for APPSwβ. Experiments were next conducted to determine whether 931 recognized intracellular APPSwβ. Intracellular APPSwβ was detectable in the cell lysate as a slightly diffuse band migrating just above a nonspecific band (Fig.3 A, panel 2). This nonspecific 35S-labeled band was detected in both CHOAPP and CHOAPPSw cell lysates (data not shown). Therefore, an additional experiment was conducted to verify that this protein was not recognized by antibodies specific for the APPSwβ neoepitope. CHOAPPSw cells were pulse-labeled with Tran35S-label for 12 min and chased for 45 min as described under “Experimental Procedure