Substrate and Inhibitor Profile of BACE (β-Secretase) and Comparison with Other Mammalian Aspartic Proteases
2002; Elsevier BV; Volume: 277; Issue: 7 Linguagem: Inglês
10.1074/jbc.m109266200
ISSN1083-351X
AutoresFiona Grüninger-Leitch, Daniel C. Schlatter, Erich Küng, Peter Nelböck, Heinz Döbeli,
Tópico(s)Phytase and its Applications
ResumoThe full-length and ectodomain forms of β-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the β-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2′ and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM↓DAEFR,K m = 7 μm,K cat = 0.002 s−1; Swedish mutant, SEVNL↓DAEFR, K m = 9 μm,K cat = 0.02 s−1). A new substrate (VVEVDA↓AVTP, K m = 1 μm,K cat = 0.004) was identified by serendipity. The full-length and ectodomain forms of β-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the β-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2′ and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM↓DAEFR,K m = 7 μm,K cat = 0.002 s−1; Swedish mutant, SEVNL↓DAEFR, K m = 9 μm,K cat = 0.02 s−1). A new substrate (VVEVDA↓AVTP, K m = 1 μm,K cat = 0.004) was identified by serendipity. Alzheimer's disease is characterized by the extracellular deposition of insoluble amyloid plaques. The main component of amyloid plaques is the 39–43-amino acid β-amyloid peptide (Aβ), 1The abbreviations used are:Aββ-amyloid peptideAPPamyloid precursor proteinDMAN,N-dimethylacetamideFmocfluorenylmethoxycarbonylHATUN-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridino-1-yl-methylmethanaminium hexafluorophosphate N-oxideHPLChigh performance liquid chromatographyFRETfluorescence resonance energy transferMES4-morpholinoethanesulfonic acidBACEβ-site APP cleavage enzyme which derives from a larger protein precursor (amyloid precursor protein, APP). Aβ is excised from APP by the sequential action of two proteases known, respectively, as β-secretase, which cuts amino-terminal to Aβ, and γ-secretase, which cleaves at the carboxyl terminus. Several reports appeared recently describing the cloning and characterization of β-secretase (1Vassar 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. 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Nature. 1999; 402: 533-537Crossref PubMed Scopus (1340) Google Scholar, 5Lin X. Koelsch G., Wu, S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (743) Google Scholar). This protein, designated Asp-2, BACE, or memapsin 2, according to the laboratory in which it was discovered, is a novel transmembrane aspartic protease that cleaves APP at the β-secretase site. BACE possesses all the characteristics expected for β-secretase in terms of substrate preference, pH optimum for activity, tissue distribution, and subcellular localization. In addition, two recent reports indicate that Aβ levels in the brains of BACE knockout mice are reduced by more than 90% compared with control mice (6Roberds 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. Nichols N.F. Power M. Robertson D.W. Schenk D. Schoor M. Shopp G.M. Shuck M.E. Sinha S. Svensson K.A. Tatsuno G. Tintrup H. Wijsman J. Wright S. McConlogue L. Hum. Mol. Genet. 2001; 10: 1317-1324Crossref PubMed Google Scholar, 7Luo 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: 231-232Crossref PubMed Scopus (956) Google Scholar). In addition to cleaving APP at the β-secretase site, BACE cuts APP further downstream within the amyloid region (between Tyr-10 and Glu-11 of Aβ), generating a truncated form of Aβ that is probably still amyloidogenic (3Sinha 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. Zhao J. McConlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1488) Google Scholar, 8Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (349) Google Scholar). Parallel to the discovery of BACE, a second, homologous transmembrane aspartic protease termed Asp-1, BACE2, memapsin 1, or down region aspartic protease was reported (4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1340) Google Scholar, 5Lin X. Koelsch G., Wu, S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (743) Google Scholar, 9Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.R. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (144) Google Scholar, 10Acquati F. Accarino M. Nucci C. Fumagalli P. Jovine L. Ottolenghi S. Taramelli R. FEBS Lett. 2000; 468: 59-64Crossref PubMed Scopus (121) Google Scholar). Preliminary analysis of BACE2 indicated that it can also function as a β-secretase in vitro (8Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (349) Google Scholar, 9Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.R. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (144) Google Scholar). β-amyloid peptide amyloid precursor protein N,N-dimethylacetamide fluorenylmethoxycarbonyl N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridino-1-yl-methylmethanaminium hexafluorophosphate N-oxide high performance liquid chromatography fluorescence resonance energy transfer 4-morpholinoethanesulfonic acid β-site APP cleavage enzyme In seeking to develop a disease-modifying therapy for Alzheimer's Disease, BACE presents itself as an ideal drug target. It belongs to a well understood class of protease where inhibitors have previously been developed for therapeutic use (renin, human immunodeficiency virus protease). Two different peptidomimetic inhibitors of BACE with nanomolar activity have already been described (3Sinha 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. Zhao J. McConlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1488) Google Scholar, 11Ghosh A.K. Shin D. Downs D. Koelsch G. Lin X. Ermolieff J. Tang J. J. Am. Chem. Soc. 2000; 122: 3522-3523Crossref PubMed Scopus (250) Google Scholar). In addition, an x-ray crystal structure has been published (12Hong L. Koelsch G. Lin X., Wu, S. Terzyan S. Ghosh A.K. Zhang X.C. Tang J. Science. 2000; 290: 150-153Crossref PubMed Scopus (703) Google Scholar), which should facilitate rational design of new inhibitors. However, because the treatment of Alzheimer's disease will be a long term therapy, a β-secretase inhibitor has to be very selective. Aspartic proteases are widely distributed in the body. BACE2, for instance, is found at low levels in most peripheral organs (13Bennett B.D. Babu-Khan S. Loeloff R. Louis J.C. Curran E. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 20647-20651Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Here we describe the enzymatic properties of BACE in comparison with its homologue, BACE2, and other relevant mammalian aspartic proteases, namely pepsin, cathepsin D, cathepsin E, napsin A and renin. cDNAs encoding the aspartyl proteases BACE and BACE2 were modified by PCR in the 5′ non-coding region to optimize ribosomal recognition and at the 3′ end by adding sequences encoding 6×His residues to enable rapid purification of the recombinant proteins. Expression in Sf9 insect cells via recombinant baculovirus resulted in higher yields than expression in Escherichia coli, Schizosaccharomyces pombe, or HEK293 cells. Thus the cDNAs were cloned into the pFASTBAC1 vector (Invitrogen) as BamHI × XbaI fragments for expression in insect cells and the PCR products were confirmed by sequencing. After recombination into the baculovirus genome, the purified viral DNA was transformed into the insect cells. Sf9 cells were cultured at 27 °C in TC100 medium (BioWhittaker) with 5% (v/v) fetal calf serum. Virus stocks were generated with a titer of 1.5 × 109 plaque-forming units/ml. For large scale production of BACE and BACE2, 24-liter fermenters of Sf9 cells were infected with a multiplicity of infection of 1. The first step in purification of BACE ectodomain is immunoaffinity chromatography using a monoclonal antibody (BSC-1) generated in-house by Dr. M. Brockhaus. The antibody was generated by standard methods from a mouse immunized with BACE purified from E. coli. The specificity of BSC-1 (IgG1) was established by enzyme-linked immunosorbent assay and Western blot assays. Thus 5 liters of cell-free Sf9 fermentation broth was concentrated 10-fold by ultrafiltration and loaded directly onto a 1.6 × 4-cm BSC-1-Sepharose immunoaffinity column that had been equilibrated in 50 mm Tris-HCl, pH 7.2, 150 mmNaCl. The column was washed with several column volumes each of 50 mm Tris HCl, pH 7.2, 150 mm NaCl then 50 mm sodium citrate, pH 5.0, 500 mm NaCl. The column was eluted with 50 mm sodium citrate, pH 3.0. Fractions containing BACE were pooled, neutralized with sodium bicarbonate, then dialyzed against 50 mm Tris-HCl, pH 7.4. This material was passed over a Mono S HR 5/5 column (Amersham Biosciences, Inc.) that had been previously equilibrated in 50 mm Tris-HCl, pH 7.4. BACE, which is obtained in the column flow-through, was subsequently concentrated by ultrafiltration and chromatographed on a Superdex 200 HR 16/60 column (Amersham Biosciences, Inc.) in 50 mm Tris-HCl, pH 7.2, 100 mm NaCl. The purified BACE was stored at 4 °C. 50 g (wet weight) of Sf9 cells was suspended in 750 ml of phosphate-buffered saline, 2% Triton X-100 and homogenized with a hand-held glass homogenizer. The homogenate was stirred on ice for 30 min and then centrifuged at 100,000 ×g for 20 min. The supernatant was adjusted to pH 8.0 and loaded on a 2.6 × 2.5-cm Ni2+-nitrilotriacetic acid-Sepharose column (Qiagen, Germany) that had been equilibrated in 50 mm sodium phosphate, 10 mm Tris, 100 mm NaCl, 0.1% Triton X-100, pH 8.0. The column was subsequently washed with this buffer and then with 50 mmsodium phosphate, pH 7.4, 100 mm NaCl, 0.1% Triton X-100. The column was then eluted with 50 mm sodium phosphate, pH 7.4, 100 mm NaCl, 200 mm imidazole, 0.1% Triton X-100 (10 column volumes). Pooled fractions containing full-length BACE were passed over a 5-ml HiTrap Q column (Amersham Biosciences, Inc.), and the unbound material was collected. This material was then diluted 10-fold into 50 mm Tris-HCl, pH 7.4, 10 mm NaCl, 0.1% Triton, and re-loaded onto a second 5-ml HiTrap Q column that had been equilibrated in 50 mmTris-HCl, pH 7.4, 10 mm NaCl, 0.1% Triton X-100. The column was washed in this buffer and eluted with a gradient of 50 mm Tris-HCl, pH 7.4, 1 m NaCl, 0.1% Triton X-100 (20 column volumes). Fractions containing BACE were pooled, dialyzed against 50 mm Tris-Cl, pH 8.0, 0.1% Triton X-100 and loaded on a Mono S HR5/5 column (Amersham Biosciences, Inc.). The unbound material, containing BACE, was pooled and stored at 4 °C. 50 g (wet weight) of Sf9 cells were washed with 10 volumes of ice-cold phosphate-buffered saline and then suspended in 750 ml of phosphate-buffered saline, 1.5% dodecyl β-d-maltoside and homogenized with a hand-held glass homogenizer. The homogenate was stirred on ice for 30 min and then centrifuged at 100,000 × g for 20 min. The supernatant was adjusted to pH 8.0, and imidazole was added to a final concentration of 10 mm. The extract was then loaded on a 2.6 × 2.5-cm Ni2+-nitrilotriacetic acid-Sepharose column (Qiagen) that had been equilibrated in 50 mm sodium phosphate, 10 mm Tris 100 mm NaCl, 0.1% Triton X-100, pH 8.0. The column was subsequently washed with this buffer and then with 50 mm sodium phosphate, pH 7.4, 100 mm NaCl, 0.1% Triton X-100. The column was then eluted with 50 mm sodium phosphate, pH 7.4, 100 mmNaCl, 200 mm imidazole, 0.1% Triton X-100 (10 column volumes). BACE2-containing fractions were dialyzed against 25 mm sodium acetate, pH 4.5, 25 mm NaCl and further purified by affinity chromatography on P10-P4′ StatVal-Sepharose, as described by Sinha et al. (3Sinha 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. Zhao J. McConlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1488) Google Scholar) for BACE. Protein concentration was determined using the BCA assay (Pierce). Pepsin from hog stomach was purchased from Fluka (Buchs, Switzerland), and cathepsin D was purchased from Calbiochem. Recombinant mouse cathepsin E was a gift from Prof. John Kay and was purified according to Hillet al. (14Hill J. Montgomery D.S. Kay J. FEBS Lett. 1993; 326: 101-104Crossref PubMed Scopus (29) Google Scholar). Recombinant human renin was purified according to Mathews et al. (15Mathews S. Döbeli H. Pruschy M. Bosser R. D'Arcy A. Oefner C. Zulauf M. Gentz R. Breu V. Matile H. Schlaeger J. Fischli W. Protein Expression Purif. 1996; 7: 81-91Crossref PubMed Scopus (19) Google Scholar) and recombinant human napsin A was purified according to Schauer-Vukasinovic et al.(16Schauer-Vukasinovic V. Bur D. Kitas E. Schlatter D. Rossé G. Lahm H.W. Giller T. Eur. J. Biochem. 2000; 267: 2573-2580Crossref PubMed Scopus (16) Google Scholar). The BACE inhibitor P10-P4′ StatVal (=Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe, where Sta is a statin transition state mimetic), originally described by Sinhaet al. (3Sinha 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. Zhao J. McConlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1488) Google Scholar), was purchased from Bachem (Bubendorf, Switzerland). The BACE inhibitor OM99-2 (=Glu-Val-Asn-Mim-Ala-Glu-Phe, where Mim is a transition state mimetic containing a Leu-Ala motif and where the peptide bond is replaced by a CHOH-CH2 group), originally described by Hong et al. (12Hong L. Koelsch G. Lin X., Wu, S. Terzyan S. Ghosh A.K. Zhang X.C. Tang J. Science. 2000; 290: 150-153Crossref PubMed Scopus (703) Google Scholar) was synthesized in-house by Dr. Eric Kitas and co-workers. Saquinavir (Ro 31-8959) and Remikiren (Ro 42-5892) were taken from the Roche bulk products, and pepstatin was purchased from Fluka. Peptides were synthesized on TentaGel S-Rinkamide resin (0.25 mmol/g, Rapp Polymer, Tübingen) following a standard procedure consisting of the repetition of the following steps: 1) deprotection with 20% piperidine in N,N-dimethylacetamide (DMA); 2) washing with DMA, isopropyl alcohol, and DMA; 3) coupling with fluorenylmethoxycarbonyl (Fmoc)-protected amino acid usingN-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridino-1-yl-methylmethanaminium hexafluorophosphate N-oxide (HATU) and diisopropylethylamine in N-methylpyrrolidone. Double coupling of the Fmoc-protected amino acid was performed by usingO-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TPTU) and diisopropylethylamine inN-methylpyrrolidone. Boc-Lys(dabsyl)-OH (where Boc istert-butoxycarbonyl) was prepared by the reaction of Boc-Lys-OH with dabsyl chloride. Fmoc-Glu-Lucifer Yellow was obtained by condensation of the N-hydroxysuccinimide ester of Fmoc-Glu-(OtBu)-OH (where tBu istert-butyl) with Lucifer Yellow and subsequent treatment with trifluoroacetic acid; 4) same washing as step 2. After the last cycle, the resins were treated with trifluoroacetic acid/H2O 95:5. Crude peptides were purified by reversed-phase HPLC using a Nucleosil-C18 column (7 μm, 10 × 250 mm, Machery Nagel, gradient of H2O and acetonitrile). Identity and purity of the peptides were analyzed by HPLC and electrospray ionization-mass spectroscopy (using a PE SCIEX, model API-100 liquid chromatograph). All enzyme assays were performed at 20 °C on a FLUOstar (BMG Lab Technologies, D-77656 Offenburg) using 96-well microtiter plates (DYNEX Microfluor 2, Chantilly, VA). The assay volume was 100 μl. Typically, inhibitors dissolved in dimethyl sulfoxide were added into a well followed by buffer and enzyme. The dimethyl sulfoxide concentration was kept below 4%. The enzymatic reaction was started by adding the substrate. pH and buffer conditions under which the experiments were carried out are indicated in the figure and table legends. The progression of the fluorescence increase was measured at λemission = 520 nm with fluorescence excitation at λexcitation = 430 nm. Reaction kinetics were followed periodically for 30 min at various substrate concentrations. The detected signals were converted into moles of substrate hydrolyzed per second. Kinetic data were determined graphically from Lineweaver-Burk plots. It should be noted that data obtained by varying substrate concentrations had to be corrected for the effect of excess quenching capacity, which is typical of all the FRET substrates used here. Assays were performed at enzyme concentrations that warranted a linear progression of product formation. The samples were dried in a Speed-Vac and then dissolved in 110 μl of 50% formic acid. 100 μl were subjected to reversed-phase HPLC using a AL12S05 column (5 μm, 5 × 100 mm, YMC, gradient of 1% formic acid and acetonitrile). Peak fractions were collected and dried again in a Speed-Vac. The peptide fragments were analyzed by electrospray ionization-mass spectroscopy (using a PE SCIEX, model API-100LC). The FRET substrate combinatorial libraries were synthesized on PEGA1900(copolymer of polyethylene glycol with a molecular weight of 1900 and acrylamide) resin via split synthesis after standard Fmoc chemistry using HATU as the condensing reagent. Fmoc-Gly-OH, Fmoc-Glu-Lucifer Yellow, and all Fmoc-protected amino acids were coupled to the amino PEGA1900 resin using HATU in the presence of diisopropylethylamine in N-methylpyrrolidone. Double coupling was performed usingO-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TPTU) in the presence of diisopropylethylamine inN-methylpyrrolidone. The synthesis was performed on a semi-automated shaking-vessel machine. For variable positions in the peptide sequences the resin was transferred in a Manual Multiple/Library Synthesizer (MULTIBLOCK®) and split into 19 portions. A different and single Fmoc-protected amino acid (except Fmoc-Cys-OH) was coupled to each of the 19 portions. Then the portions were mixed together and washed thoroughly. Depending on the library the splitting was either repeated or synthesis proceeded in a single portion. The last building block wastert-butoxycarbonyl-Lys(dabsyl)-OH. The protecting groups were removed with trifluoroacetic acid:H2O:TIS 95:3:2 for 2 h. A part (∼2000 beads) of the peptide library (either Lys(dabsyl)-SEVXXDAEFR-Glu(Gly-PEGA)-Lucifer Yellow or Lys(dabsyl)-SEVXLXAEFR-Glu(Gly-PEGA)-Lucifer Yellow) was extensively washed with 100 mm sodium acetate, pH 5.0. Then 80 μl of the purified enzyme (ectodomain) was added to the resin in a final volume of 0.8 ml in the reaction buffer. After shaking for 18 h at room temperature, the resin was filtered off, and the beads were washed with the incubation buffer and 100 mm MES buffer, pH 4.8, containing 5% glycerol and 0.05% Triton X-100. The beads were inspected under the fluorescence microscope (Leica MZ12 equipped with Leica 2 video system). Brightly fluorescent beads were isolated and submitted to Edman sequencing. The sequencing was performed on a PE Biosystems Procise 494 HT Sequencer. Portions of 200–400 beads of the six libraries, Lys(dabsyl)-SEXNLDAEFR-Glu(Gly-PEGA)-Lucifer Yellow, Lys(dabsyl)-SEVXLDAEFR-Glu(Gly-PEGA)-Lucifer Yellow, Lys(dabsyl)-SEVNXDAEFR-Glu (Gly-PEGA)-Lucifer Yellow, Lys(dabsyl)-SEVNLXAEFR-Glu(Gly-PEGA)-Lucifer Yellow, Lys(dabsyl)-SEVNLDXEFR-Glu(Gly-PEGA)-Lucifer Yellow, and Lys(dabsyl)-SEVNLDAXFR-Glu(Gly-PEGA)-Lucifer Yellow, were extensively washed and incubated in a volume of 0.1 ml as described above. Then the beads were washed with the incubation buffer and 100 mm MES buffer, pH 4.8, containing 5% glycerol and 0.05% Triton X-100. Each portion of beads was inspected separately, and the exact number of total beads as well as brightly fluorescent beads was counted. Fluorescent beads were submitted to sequencing. Full-length BACE, BACE ectodomain, and full-length BACE2 constructs were made containing a carboxyl-terminal hexahistidine tag to facilitate purification. The purification process was monitored by Western blotting using an anti-6×His antibody. The presence of enzymatically active β-secretase was checked by a mass spectrometry assay (17Grüninger-Leitch F. Berndt P. Langen H. Nelboeck P. Döbeli H. Nat. Biotechnol. 2000; 18: 66-70Crossref PubMed Scopus (37) Google Scholar), and the enzymatic purity (=absence of contaminating proteases) was determined by a FRET assay using four homologous substrates. The main criteria for purity were the absence of activity with the KL-V and VL-M substrates and presence of activity with the KM-D and NL-D substrates. A further criterion was the preferential cleavage of NL-D over KM-D (see Table I).Table ISoluble substrates and relative activity with BACESubstrateSequenceSubstrate typeCleavage by BACE% of best substrateNL-DSEVNLDAEFRSwedish mutant APP β-cleavage site100EL-DSEVELDAEFRModified APP β-cleavage site87DA-AVVEVDAAVTPArtifical cleavage site at APP carboxyl terminus24NL-ASEVNLAAEFRModified APP β-cleavage site16EF-ASEVEFAAEFRModified APP β-cleavage site15KM-DSEVKMDAEFRWild-type APP β-cleavage site9GY-EHDSGYEVHHQAlternative APP β-cleavage site8EF-DSEVEFDAEFRModified APP β-cleavage site2P4KSKVNLDAEFRModified APP β-cleavage site2VL-MTSVLMAAPCathepsin D substrate (octametric peptide)2KL-VVHHQKLVFFAAPP α-secretase cleavage site1HL-VIHPFHLVIHNRenin substrate0The substrates are listed in the order of cleavage efficiency by BACE. An example of the structure with the decameric peptide sequence, the linker amino acids, the fluorophor, and the quencher is given in Fig.2. Open table in a new tab The substrates are listed in the order of cleavage efficiency by BACE. An example of the structure with the decameric peptide sequence, the linker amino acids, the fluorophor, and the quencher is given in Fig.2. Using the purification procedure described under "Experimental Procedures," essentially homogeneous full-length BACE or BACE ectodomain were obtained (Fig. 1). Amino-terminal sequence analyses showed the both full-length BACE (ETDEEPEE) and BACE2 (ALEPALASPA) are efficiently processed to the mature enzymes in the baculovirus/Sf9 expression system. The respective amino-terminal sequences are identical to those observed by other groups (3Sinha 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. Zhao J. McConlogue L. Varghese J. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1488) Google Scholar, 9Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.R. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (144) Google Scholar, 18Haniu 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. Vassar R. Citron M. J. Biol. Chem. 2000; 275: 21099-21106Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 19Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). BACE ectodomain (TQHGIRL) is secreted from Sf9 cells as the proenzyme. This is in contrast to the situation in mammalian cell lines where three other groups report efficient processing of the ectodomain using either HEK293 or Chinese hamster ovary cells (20Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2000; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 21Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 22Benjannet 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: 10879-10887Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Sf9 cells clearly contain a furin-like protease that is potentially capable of processing the ectodomain since the full-length enzyme is correctly processed in this system. It may be that the soluble ectodomain does not come into extended contact with the furin-like convertase if this protein is, in analogy to furin, membrane-bound. It may also be the case, however, that the very high expression level of BACE ectodomain completely saturates the convertase. The proenzyme as purified here shows almost identical catalytic activity to the mature enzyme, a phenomenon already documented by others, implying that BACE is not a true zymogen (20Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2000; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The purified BACE ectodomain crystallized and the structure was solved to 2.8-Å resolution. 2A. Ruf, unpublished data. BACE2 was not purified to homogeneity but was shown to be almost free of other contaminating protease activities by analysis with a series of aspartic protease substrates (Fig. 3). The FRET assay was based on dodecameric peptides containing 10 amino acids of the putative substrate and a linker amino acid at each end. The linker amino acids, lysine at the amino terminus and glutamine at the carboxyl terminus, are used to couple the fluorescent group Lucifer Yellow and the quenching group dabsyl to the substrate (Fig.2). Because the quenching group is coupled via the ε-amino group of lysine, the peptide can still be analyzed by Edman degradation. The substrates were either used as soluble entit
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