The Pro Domain of beta -Secretase Does Not Confer Strict Zymogen-like Properties but Does Assist Proper Folding of the Protease Domain*

Xiao-Ping Shi, Elizabeth Chen, Kuo-Chang Yin, Sang Na, Victor M. GarskyDagger , Ming-Tain Lai, Yue-Ming Li, Michael Platchek, R. Bruce Register, Mohinder K. Sardana, Mei-Jy Tang, James Thiebeau, Theresa Wood, Jules A. Shafer, and Stephen J. Gardell§

From the Departments of Biological Chemistry and Dagger  Medicinal Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486

Received for publication, October 9, 2000, and in revised form, December 1, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta -Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta  peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The kcat/Km of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The amyloid precursor protein (APP)1 is cleaved sequentially by two proteolytic activities, beta - and gamma -secretase, to generate the N and C termini, respectively, of the amyloid beta  peptides. Aberrant production or compromised clearance of the amyloid beta  peptides, which give rise to neuritic plaques in brain parenchyma, may be a root cause of Alzheimer's disease (1). beta -Secretase (BACE; Asp2; memapsin 2) was recently cloned (2-6) and shown to be a novel type I transmembrane aspartyl protease. The cDNA encoding BACE predicts a precursor protein with a modular structure containing Pre, Pro, protease, transmembrane, and cytosolic tail domains (see Fig. 1).

Pro domains are commonly found in protease precursors where they have been shown to virtually abolish catalytic activity (7) and assist in protein folding (8). The Pro domain is typically cleaved from the protease precursor to generate the mature active protease. Edman degradation of purified human brain BACE revealed a single N-terminal protein sequence that began at the protease domain (3) (this species is designated mature BACE). BACE isolated from human embryonic kidney 293T cells that were transfected with the full-length BACE cDNA exhibited an N-terminal sequence that began mostly at the protease domain, whereas only ~10% began at the Pro sequence (3). In addition, N-terminal sequencing of full-length Asp2 (i.e. BACE), which was expressed in Chinese hamster ovary cells, also revealed proteolytic processing at the junction between the putative Pro and protease domains (4). These results establish that processing of membrane-anchored BACE after Arg-45 to release the Pro segment and generate mature BACE is prevalent in mammalian cells.

In the present study, we expressed in insect cells a soluble form of the BACE precursor that is truncated at the junction between the putative protease and transmembrane regions (PreProBACE460). A BACE-related species, ProBACE460, that displays an N-terminal sequence that corresponds exactly to mammalian cell-derived ProBACE was purified from the conditioned medium (CM). The studies described herein show that the Pro domain of ProBACE460, rather than abolishing catalytic activity as in the case of true zymogens, appears to facilitate the proper folding of the BACE protease domain.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of the BACE cDNA-- The BACE cDNA was cloned by reverse transcription-polymerase chain reaction amplification of human brain mRNA (CLONTECH; Palo Alto, CA) using oligonucleotides that were designed from the published BACE cDNA sequence (2-6). In particular, the 5'-oligonucleotide (5'-CGGGATGCGGCCGCATGGCCCAAGCCCTGCCCTGGCTCCTGCTG-3') contained a NotI site upstream of the ATG start codon and the 3'-oligonucleotide (5'-GGATCGGTCGACTTAAATTTGAGGTGACCAAGA-3') spanned the SalI site located in the 3'-untranslated region (262 base pairs downstream from the TGA stop codon). The 1771 base pair polymerase chain reaction product was subcloned into pLHCX (CLONTECH) to create the BACE retrovirus expression construct, pRBR133. The integrity of the BACE cDNA was confirmed by DNA sequencing (Midland Reagent Company, Midland TX).

Expression of BACE by Recombinant Baculovirus in Insect Cells-- The cDNA encoding truncated PreProBACE (amino acids 1-460) was polymerase chain reaction amplified from pRBR133 using a 5'-oligonucleotide containing a BamHI site upstream of the ATG start codon, 5'-GCTAGAGGATCCATGGCCCAAGCCCTGCCCTGGCTCCTGCTGTGG-3' and a 3'-oligonucleotide containing a stop codon after the Tyr460 residue followed by an XhoI site, 5'-GTCGCACTCGAGTCAATAGGCTATGGTCATGAGGGTTGACTC-3'. The 1389-base pair fragment was subcloned into the BamHI and XhoI sites of a baculovirus expression vector, pBacPAC8 (CLONTECH), to generate pRBR136. The integrity of the BACE cDNA clone was again confirmed by DNA sequencing (Midland Reagent Company). pRBR136 was transfected into Spodoptera frugiperda (Sf21) insect cells along with BacPAK6 viral DNA to generate recombinant virus (BACE460 virus stock) according to the manufacturer's instructions. Recombinant virus was harvested 5 days post-transfection, plaque-purified and titered as described in the CLONTECH manual. Sf21 cells (30 liters) were grown in Grace's insect medium, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1× penicillin/streptomycin to ~9.0 × 105 cells/ml and were infected with BACE460 virus stock at a multiplicity of infection of 4 (Cell and Molecular Technologies, Inc., Lavallette NJ). After 72 h, the CM was harvested by filtration and immediately stored at 4 °C.

Production of Anti-BACE Antibodies-- A peptide corresponding to the Pro segment of BACE (TQHGIRLPLRSGLGGAPLGLRLPR-C) was conjugated to keyhole limpet hemocyanin and used as antigen for production of rabbit polyclonal antibodies (Covance Inc., Denver PA). Another peptide (RDLRKGVYVPC) that is situated within the protease domain of BACE (amino acids 122-131) was similarly used as an antigen for production of rabbit polyclonal antibodies (Covance Inc.). The anti-Pro antibody was purified by passing the serum over an affinity column containing the immobilized hapten.

BACE Activity Assay-- CM from Sf21 cells infected by BACE460 virus stock (or control virus stock), purified ProBACE460, or purified BACE460 were incubated with acetyl-TTRPGSGLTNIK(6-(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVNLDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide (L-405,492), a modified 31-residue polypeptide that is designed from the amino acid sequence of APP in the immediate vicinity of the beta -secretase cleavage site (the residues that flank the scissile bond are underlined). L-405,492 contains the mutations NL in the P2 and P1 positions, respectively, that are observed in the Swedish-type familial Alzheimer's disease kindreds (9). An alternative substrate, L-405,525, that was used to assay purified BACE460 and ProBACE460 is identical to L-405,492 except that it has KM at the P2 and P1 positions, respectively, which are the residues that are present in the wild type APP sequence. All assays were performed in the presence of 50 mM sodium acetate, 0.15 M NaCl, pH 4.5, 0.2% 3-[(3cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid 0.1 mg/ml bovine serum albumin. Where indicated, BACE assays were also performed in the presence of the BACE inhibitor (3), P10-P4'Stat(Val) (Midwest Biotechnology, Fishers IN and Enzyme Systems Products, Livermore, CA). Reactions were incubated at 37 °C for 2 h unless otherwise indicated and terminated by heating the samples at 75 °C for 5 min. Samples were analyzed by reverse-phase HPLC using a Zorbax Extend C18 column (Waters, Milford, MA). Coumarin-tagged product and substrate were monitored by fluorescence detection with excitation and emission wavelengths of 340 and 440 nm, respectively.

Purification of Recombinant Baculovirus Expressed BACE-- The CM from BACE460 virus stock-infected Sf21 cells was spiked with NaCl (350 mM), aprotinin (0.5 µM), and leupeptin (1 µM) and applied to a concanavalin A-Sepharose (Amersham Pharmacia Biotech, Piscataway NJ) column (1.5 liters of bed volume). The lectin affinity column was washed with Buffer A (20 mM Tris-HCl, pH 7.4, and 0.5 M NaCl) plus 0.5 µM aprotinin, 1 µM leupeptin and eluted with Buffer A plus 0.3 M alpha -D-glucopyranoside, 0.3 M methyl-alpha -D-mannopyranoside, 0.5 µM aprotinin, and 1 µM leupeptin. EDTA (1 mM) was added to the eluate from the lectin affinity column. Diafiltration with a hollow fiber 10,000 molecular weight cut-off column (A/G Technologies, Needham MA) was used to concentrate the lectin affinity column eluate ~15-fold and buffer exchange into 50 mM sodium acetate, pH 5.0, 0.5 M NaCl, 0.5 µM aprotinin, 1 µM leupeptin, and 1 mM EDTA. The diafiltration retentate was applied to an affinity column (HiTrap N-hydroxysuccinimide-activated; Amersham Pharmacia Biotech) containing immobilized P10-P4' Stat(Val) (3). The affinity column was washed sequentially with (a) Buffer B (20 mM sodium acetate, pH 5.0, 125 mM NaCl) plus 0.5 µM aprotinin, 1 µM leupeptin, 1 mM EDTA, 100 µM pepstatin, (b) Buffer B, (c) Buffer C (20 mM sodium acetate, pH 5.0, and 750 mM NaCl), and (d) Buffer B. The P10-P4' Stat(Val) affinity column was eluted with Buffer D (20 mM Tris-HCl, pH 7.2, and 125 mM NaCl). Fractions were collected and evaluated for BACE activity with L-405,492. Peak activity fractions were pooled and stored as aliquots at -70 °C.

Generation of BACE460 by Treatment of ProBACE460 with Furin-- ProBACE460 or BACE460 (500 nM) were incubated with furin (20 units/ml; Affinity Bioreagents Inc, Golden, CO) at 30 °C in 0.1 M Hepes, 2.5 mM CaCl2, and 0.5% Triton X-100, pH 7.5. At increasing times, aliquots were removed and furin activity was quenched with EDTA (5 mM). The production of BACE460 by furin treatment of ProBACE460 was assessed by SDS-PAGE/immunoblotting using antibodies against the Pro or protease regions of BACE. Signals from the immunoblots were quantified by scanning analysis using a densitometer SI (Molecular Dynamics, Sunnyvale, CA). In addition, the level of BACE activity after furin treatment was evaluated with L-405,492. For the isolation of preparative amounts of BACE460, ProBACE460 (2 µM) was incubated with furin (100 units/ml) in 0.1 M Hepes, pH 7.6, 2.5 mM CaCl2, 0.5% Triton X-100 (final volume, 6.5 ml) for 14 h at 30 °C. The sample was dialyzed versus 20 mM sodium acetate, pH 5.0, 125 mM NaCl. In vitro generated BACE460 was purified by affinity chromatography with immobilized P10-P4' Stat(Val) as described above (except that pepstatin was eliminated from the column wash step).

Immunoprecipitation of ProBACE460-- CM (100 µl) from the BACE460 virus stock-infected Sf21 cells was spiked with control antibody (normal rabbit IgG; 0.3 µM) or anti-Pro antibody (0.3 µM; affinity purified) and immobilized protein G (10 µl of a 75% slurry) (Amersham Pharmacia Biotech). After overnight storage at 4 °C with mixing, the samples were centrifuged, and the supernatant fractions were evaluated by SDS-PAGE/immunoblotting (probing with either anti-Pro antibody or anti-protease antibody) and assayed for BACE activity using L-405,492. Immunoprecipitation of the purified ProBACE460 and BACE460 preparations (0.5 µM) was performed similarly except that 2.7 µM control and anti-Pro antibody were used. The pellet fractions were extensively washed with phosphate-buffered saline and assayed for BACE activity using L-405,492.

Denaturation/Renaturation of ProBACE460 and BACE460-- ProBACE460 and BACE460 (7 mg/ml) were denatured for 1 h at 23 °C with 6 M guanidine HCl in Buffer D in the absence or presence of 50 µM BACE Pro peptide, NH2-TQHGIRLPLRSGLGGAPLGLRLPR-COOH (Synpep Inc., Dublin CA). The samples were renatured by rapid addition of Buffer D (60 volumes) to yield a final concentration of 0.1 M guanidine HCl and set at room temperature for 2 h. Control ProBACE460 and BACE460 samples to determine maximal BACE activity were adjusted to 7 mg/ml in an equivalent volume of Buffer D and treated similarly. Samples were assayed for BACE activity using L-405,492. Aliquots of the mock-denatured/renatured or denatured/renatured ProBACE460 and BACE460 samples were analyzed by SDS-PAGE/immunoblotting using the anti-protease antibody. In addition, aliquots of ProBACE460 with and without treatment by guanidine denaturation/renaturation were loaded onto a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) using an AKTAexplorer chromatography system (Amersham Pharmacia Biotech). The gel filtration column was eluted with 20 mM Tris-HCl, pH 7.2, 125 mM NaCl, and 0.1 M guanidine HCl.

Analytical Analyses-- The amino acid compositions of protein/peptide samples were determined with a Beckman 6300 analyzer after hydrolysis of the samples using 6 N HCl containing 0.2% phenol in sealed evacuated vials at 110 °C for 20 h. N-terminal sequence analysis of purified protein samples was performed by Edman degradation using an Applied Biosystems PROCISE Model 494 protein sequencer. Verification of product identity by liquid chromatography/mass spectometry analysis was performed with an Agilent Technology 1090 liquid chromatograph coupled to a PE Sciex API-3000 triple quadrupole mass spectrometer.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of PreProBACE460-- A truncated BACE precursor (encompassing amino acids 1-460) that contains the Pre, Pro, and protease domains (PreProBACE460) was produced in Sf21 cells using a baculovirus expression construct (Fig. 1). This expression system was designed to direct the secretion of a soluble nonmembrane tethered form of BACE from the cells. SDS-PAGE/immunoblot analysis of CM from cells that were infected with BACE460 virus stock revealed the presence of a protein (molecular mass, ~55,000) that reacted with anti-BACE antibodies raised against the Pro domain (Fig. 2A, lane 2) or protease domain (data not shown). In contrast, no immunoreactivity was evident with CM from Sf21 cells infected with control virus stock when probed similarly using anti-BACE antibodies versus the Pro domain (Fig. 2A, lane 1) or protease domain (data not shown).


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Fig. 1.   Schematic representation of the protein structure of the BACE precursor protein as deduced from its cDNA sequence. Full-length BACE is composed of several domains that include the Pre (residues 1-21), Pro (residues 22-45), protease (residues 46-460), transmembrane (TM; residues 461-477) and cytosolic tail (residues 478-501) regions. The amino acid sequence of the putative Pro region is shown. Also shown is the schematic structure of the BACE derivative, PreProBACE460, that was produced in insect cells using baculovirus-mediated expression.


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Fig. 2.   Expression of PreProBACE460 by recombinant baculovirus-infected insect cells. A, SDS-PAGE/immunoblot analysis of CM. CM (5 µl) from Sf21 cells infected with control virus stock (lane 1) or BACE460 virus stock (lane 2) was probed by immunoblotting (IB) with the anti-Pro antibody. CM from Sf21 cells infected with BACE460 virus stock was immunoprecipitated (IP) with control antibody (lanes 3 and 5) or anti-Pro antibody (lanes 4 and 6) and treated CM was probed by immunoblotting using anti-Pro antibody (lanes 3 and 4) or anti-protease antibody (lanes 5 and 6). B, assay of BACE activity in CM from baculovirus-infected Sf21 cells. BACE substrate (L-405,492; 0.3 µM) was incubated with increasing amounts of CM from cells infected with control virus stock (open circle ) or BACE460 virus stock (). The BACE460 virus stock was also assayed in the presence of 10 µM P10-P4' Stat(Val) BACE inhibitor (+). Product formation as detected by HPLC is expressed as a percentage of the initial substrate concentration. Each sample was assayed in triplicate; the mean ± S.D. is shown. C, CM from BACE460 virus stock-infected Sf21 cells was immunoprecipitated (IP) with control antibody (lane 1), anti-Pro antibody (lane 2), or anti-Pro antibody in the presence of Pro-peptide (lane 3) and the "cleared" CM samples were assayed for BACE activity using L-405,492. Data show the means of duplicate values, which are normalized to the activity observed after treatment with the control antibody (assigned a value of 100%).

Treatment of CM from BACE460 virus stock-infected Sf21 cells with the antibody versus the Pro region of ProBACE in the presence of immobilized protein G fully depleted the BACE-related species that cross-reacts with the anti-Pro antibody (Fig. 2, lanes 3 and 4). Nevertheless, the ProBACE460-immunodepleted CM still possessed a less abundant species that reacts with the anti-protease antibody and migrates in the vicinity of ProBACE460 (which is indistinguishable from the mobility of BACE460, see below) (Fig. 2A, lanes 5 and 6). These observations indicate that most but not all of the BACE-related protein in the CM contains the Pro region.

BACE activity in the CM was measured using L-405,492, the modified 31-mer polypeptide derivative designed from the APP sequence (Swedish-type familial Alzheimer's disease variant) in the vicinity of the beta -secretase scissile bond. The CM from Sf21 cells infected with BACE460 virus stock catalyzed the cleavage of L-405,492 (Fig. 2B). A linear dependence of product formation on the volume of CM was observed. In contrast, BACE activity was not evident with CM from Sf21 cells that were infected with control virus stock (Fig. 2B). Mass spectrometric analysis confirmed that cleavage of L-405,492 was occurring at the beta -secretase scissile bond (data not shown). The addition of the protease inhibitors, EDTA, aprotinin, and leupeptin did not block BACE activity in the CM from BACE460 virus stock-infected cells. However, the BACE activity in the CM was completely suppressed by the BACE inhibitor, P10-P4' Stat(Val) (Fig. 2B).

CM from cells infected with the BACE460 virus stock that was extensively immunodepleted of proteins possessing the Pro region of BACE (as described above) exhibits an approximate 60% reduction of apparent BACE activity when compared with control antibody-treated CM (Fig. 2C). This result provided the initial indication that a Pro region-containing form of BACE displayed activity. The decrease of BACE activity in CM caused by the immunocapture step with anti-Pro antibody was totally blocked when the hapten, Pro peptide, was also present (Fig. 2C). The presence of both residual BACE activity and anti-protease (but not anti-Pro) immunoreactivity after exhaustive immunodepletion of ProBACE460 in the CM indicates that a lesser non-Pro region-containing BACE species in the CM also displays BACE activity.

Isolation of ProBACE460 and BACE460-- The CM from BACE460 virus stock-infected Sf21 cells was spiked with aprotinin/leupeptin and fractionated by lectin affinity chromatography using immobilized concanavalin A. The BACE activity was partially captured and eluted with alpha -D-glucopyranoside and methyl-alpha -D-mannopyranoside (approximate 60% recovery). The fractions containing the BACE activity were pooled, concentrated, and subjected to affinity chromatography using immobilized P10-P4' Stat(Val) (3). The BACE activity was bound and subsequently eluted at pH 7.2. The final recovery of BACE activity was ~15%.

The BACE preparation that was isolated from the CM of BACE460 virus stock-infected Sf21 cells exhibited a single band when subjected to SDS-PAGE and visualized by Coomassie Blue staining (Fig. 3A, lane 1). SDS-PAGE/immunoblot analysis using the antibodies against the Pro or protease regions of BACE both reacted with this species (Fig. 3B, lane 1, upper and lower panels, respectively). The purified BACE-related species is a glycoprotein as judged by its capture by immobilized concanavalin A and its increased mobility during SDS-PAGE after treatment with endoglycosidases (data not shown). Edman degradation of purified BACE, which was isolated from CM revealed an N terminus that began exclusively at the Pro region of BACE (Fig. 3C).2 Control N-terminal sequencing runs with ProBACE460 spiked with varying amounts of mature BACE460 (prepared as described below) established that a 5% or greater contamination of ProBACE460 with BACE460 would be detected. The apparent homogeneity of the ProBACE460 preparation was corroborated by the absence of BACE immunoreactive material, using either the anti-Pro (Fig. 3B, lane 3, upper panel) or anti-protease (Fig. 3B, lane 3, lower panel) antibodies, after the ProBACE460 preparation was immunodepleted using antibody versus the Pro region.


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Fig. 3.   Characterization of purified ProBACE460 and BACE460. A, Coomassie Blue staining of purified ProBACE460 (1 µg; lane 1) and BACE460 (1 µg; lane 2). The protein molecular weight markers and their corresponding apparent molecular mass values are shown. B, Immunoprecipitation (IP) of isolated ProBACE460 and BACE460 with anti-Pro antibody. Lane 1, ProBACE460; lane 2, ProBACE460 + control antibody; lane 3, ProBace460 + anti-Pro antibody; lane 4, BACE460; lane 5, BACE460 + control antibody; lane 6, BACE460 + anti-Pro antibody. The unbound protein was analyzed by SDS-PAGE/immunoblot (IB) analysis using the anti-Pro antibody (top panel) or anti-protease antibody (bottom panel). C, N-terminal analysis of the purified ProBACE460 and BACE460 preparations. The depicted residues are those obtained at each cycle of the Edmund degradation and correspond to the residues expected from the amino acid sequence (2-6).

The cellular protease that processes ProBACE to yield mature BACE was recently shown to be furin (10). The minimal consensus sequence for furin-mediated cleavage, Arg-Xaa-Xaa-Arg (11), is situated at the C terminus of the Pro region of BACE. We thus treated purified ProBACE460 with furin and used SDS-PAGE/immunoblotting to monitor for time-dependent processing. The reactivity of ProBACE460 with the anti-Pro antibody is markedly diminished and eventually eliminated as the incubation time with furin is lengthened (Fig. 4A, upper panel). On the other hand, the reactivity with the anti-protease antibody was largely unaffected (Fig. 4A, lower panel). Importantly, N-terminal sequence analysis established that furin cleaves ProBACE460 at the junction between the putative Pro and protease domains to yield BACE460 (Fig. 3C).


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Fig. 4.   In vitro activation of ProBACE460 to yield BACE460 by treatment with furin. A, SDS-PAGE/immunoblot (IB) analysis showing time-dependent, furin-mediated conversion of ProBACE460 to BACE460. Protein blots were probed with the anti-Pro (top panel) and anti-protease (bottom panel) antibodies. The incubation times with furin are indicated. Lane B contains BACE460. B, impact of furin treatment of ProBACE460 on BACE activity (open circle ) as measured with the BACE substrate (L-405,492) and HPLC analysis. Also shown is the residual amount of ProBACE460 using the data in A and gel scanning densitometric analysis (). C, assay of BACE activity of the ProBACE460 or BACE460 preparations using L-405,492 after incubation with and without furin for the indicated times.

A larger amount of BACE460 was obtained for further characterization by treating ProBACE460 (625 µg) with furin and rechromatographing the sample on the P10-P4' Stat(Val) affinity column. The protein recovered from the affinity column (386 µg) was shown to be BACE460 from its failure to react with anti-Pro antibody by immunoblot analysis (Fig. 3B, lane 4, upper panel) and N-terminal sequencing analysis (data not shown). Moreover, in contrast to the ProBACE460 preparation, treatment of BACE460 with immobilized anti-Pro antibody failed to deplete the BACE-related species as detected by immunoblotting using the anti-protease antibody (Fig. 3B, compare lanes 3 and 6, lower panel).

ProBACE460 and BACE460 Both Display Catalytic Activity-- The purified ProBACE460 preparation exhibits appreciable activity toward L-405,492. Indeed, assaying the proteolytic activity of ProBACE460 enabled its isolation. Treatment of the purified ProBACE460 preparation with immobilized anti-Pro antibody resulted in the loss of >90% of the BACE activity (data not shown). Moreover, assay of the pellet containing ProBACE460 tethered to the immobilized anti-Pro antibody revealed appreciable BACE activity (data not shown). The results of this immunocapture experiment establish that ProBACE460 itself, and not a non-Pro region-containing contaminant, is responsible for the observed activity in the ProBACE460 preparation.

Processing of ProBACE460 to BACE460 by furin is accompanied by a modest increase in BACE activity toward L-405,492, which inversely mirrored the loss in anti-Pro reactivity that was detected by immunoblotting (Fig. 4B). There was an approximate 2-fold increase in BACE activity after a 4-h incubation with furin. In an independent experiment, treatment of ProBACE460 with furin for 4 and 20 h increased BACE activity by 2.0- and 2.8-fold, respectively (Fig. 4C). As expected, treatment of BACE460 with furin did not increase BACE activity (Fig. 4C). Incubation of L-405,492 with furin alone did not cause the generation of product (data not shown).

Cleavage of L-405,492 by purified ProBACE460 and BACE460 displayed a linear dependence on substrate concentration up to 100 µM of substrate (Fig. 5). The absence of detectable saturation is in agreement with the results from a previous BACE study that used a polypeptide substrate of similar length (3). The estimated kcat/Km values of ProBACE460 and BACE460 for L-405,492 are 3110 and 7300 M-1 s-1, respectively. Hence, ProBACE460 appears to display ~43% of the activity of BACE460 for this particular substrate. Mass spectrometric analysis confirmed that L-405,492 is cleaved at the beta -secretase scissile bond by both purified ProBACE460 and BACE460 (data not shown).


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Fig. 5.   Activity displayed by purified ProBACE460 and BACE460. A, ProBACE460 () or BACE460 (open circle ) (each 10 nM) were incubated with varying concentrations of L-405,492 for 30 min at 37 °C. B, ProBACE460 () or BACE460 (open circle ) (each 500 nM) were incubated with varying concentrations of L-405,525 for 120 min at 37 °C. The amount of product was measured by HPLC. Reactions were performed in triplicate, and the data are reported as the means ± S.D.

ProBACE460 and BACE460 also cleave L-405,525, a substrate that is identical to L-405,492, except that it contains KM at the P2 and P1 positions, respectively, which are the residues that are found in the wild type APP sequence. There was no evidence for saturation of either enzyme up to 30 µM of L-405,525 (Fig. 5B). Mass spectrometric analysis confirmed that cleavage of L-405,525 occurs at the beta -secretase scissile bond (data not shown). The kcat/Km value of ProBACE460 for L-405,525, 46 M-1 s-1, is ~74% of the corresponding value displayed by BACE460, 62 M-1 s-1. Hence, the activities of ProBACE460 and BACE460 with L-405,525 are not markedly different just as was observed with the other substrate, L-405,492. However, the kcat/Km values of ProBACE460 and BACE460 for L-405,525 are 1.5 and 0.9%, respectively, of the corresponding values for L-405,492. This large difference is consistent with earlier observations that BACE displays a striking preference for the Swedish-type mutations at the P2 and P1 positions of the substrate (2-4).

ProBACE460 and BACE460 display similar susceptibility to inhibition by P10-P4' Stat(Val) (Fig. 6). The IC50 values of P10-P4' Stat(Val) for ProBACE460 and BACE460 are 250 and 150 nM, respectively. The inability of P10-P4' Sat(Val) to discriminate between ProBACE460 and BACE460 is concordant with the virtually indistinguishable binding/elution behavior of these BACE derivatives during affinity chromatography with this immobilized ligand (data not shown). Both ProBACE460 and BACE460 are relatively insensitive to inhibition by pepstatin. The residual activities of ProBACE460 and BACE460 in the presence of 100 µM pepstatin were 74 and 58%, respectively, of the uninhibited control activities.


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Fig. 6.   Inhibition of ProBACE460 and BACE460 with P10-P4' Stat(Val). ProBACE460 (20 nM; ) or BACE460 (20 nM; open circle ) was incubated with BACE substrate (L-405,492) in the presence of varying concentrations of P10-P4'Stat(Val). Product formation was measured by HPLC. Data are expressed as the percentages of BACE activity that is observed in the absence of P10-P4' Stat(Val) with either ProBACE460 or BACE460. Reactions were performed in triplicate, and the data are reported as the means ± S.D.

We considered that the apparent activity of ProBACE460 might arise from its conversion to BACE460 during incubation with substrate under standard assay conditions (pH 4.5). To explore this possibility, N-terminal sequencing was performed on ProBACE460 after its incubation with substrate (during which ~20% of the substrate was converted to product). The observed amino acid sequence began exclusively at the N terminus of Pro region; hence, conversion of ProBACE460 to BACE460 because of the presence of substrate or exposure to assay conditions does not appear to occur. SDS-PAGE/immunoblot analysis using the anti-Pro antibody also failed to show diminished immunoreactivity of ProBACE460 after its incubation with substrate under standard assay conditions (data not shown).

The Pro Region of BACE Assists in the Folding of the Protease Domain-- To assess the potential role of the Pro domain in protein folding, ProBACE460 and BACE460 were denatured with 6 M guanidine HCl at neutral pH for 1 h and renatured by rapid dilution into neutral pH buffer. After a 2-h renaturation period, aliquots of the samples were added to BACE assay mixture containing the substrate, L-405,492. The final guanidine HCl concentration during the BACE assay, 0.1 M, has no effect on the activity of ProBACE460 or BACE460. No BACE activity is observed when denatured ProBACE460 or BACE460 is transferred directly to the assay mixture (at pH 4.5) without an intervening renaturation step (at pH 7.2). However, transfer of denatured ProBACE460 to the renaturing milieu leads to recovery of BACE activity. Approximately 35% of the ProBACE460 activity is recovered after a denaturation/renaturation cycle (Fig. 7A, bar 1). In contrast, the recovery of activity from the denatured BACE460 preparation is only ~5% (Fig. 7A, bar 3). SDS-PAGE/immunoblot analysis of the denatured/renatured ProBACE460 and BACE460 samples revealed similar levels of immunoreactivity when probed with the anti-protease antibody (Fig. 7B). Hence, the lower recovery of BACE activity after treatment of BACE460 with guanidine HCl and subsequent renaturation is not due to greater susceptibility to protein degradation.


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Fig. 7.   Recovery of BACE activity after denaturation/renaturation of ProBACE460 and BACE460. A, aliquots of ProBACE460 (bars 1 and 2) and BACE460 (bars 3 and 4) were incubated in 6 M guanidine for 60 min in the absence (bars 1 and 3) or presence (bars 2 and 4) of BACE Pro peptide (50 µM), diluted in renaturation buffer for 2 h, added to assay buffer containing BACE substrate (L-405,492) and incubated at 37 °C for 60 min. Product formation was measured by HPLC. Activity is expressed as the percentage of the mock denatured/renatured ProBACE460 or BACE460. B, immunoblot analysis using the anti-protease antibody of ProBACE460 (lanes 1 and 2) and BACE460 (lanes 3 and 4) before (lanes 1 and 3) and after the denaturation/renaturation (D/R) cycle (lanes 2 and 4). The mobilities of the molecular weight markers are shown.

The protein species in the denatured/renatured ProBACE460 sample that displays BACE activity elutes during gel filtration at the same position as unperturbed ProBACE460 (data not shown). The refolded active ProBACE460 species represents only partial recovery of total protein (~37%) based on absorbance measurements and comparison with the elution profile of unperturbed ProBACE460. Importantly, the specific activity of the refolded ProBACE460 that elutes during gel filtration as a monomeric species is indistinguishable from that of ProBACE460 (data not shown). The inability to recover all of the BACE activity after denaturation/renaturation of ProBACE460 probably reflects the formation of misfolded ProBACE460 aggregates.

The presence of the BACE Pro peptide (75 µM) in trans during the denaturation/renaturation of BACE460 serves to increase the recovery of BACE activity to ~23% (Fig. 7A, bar 4), but the Pro peptide essentially has no effect on the recovery of activity from denatured ProBACE460 (Fig. 7A, bar 2). Consistent with the predominant effect of the Pro domain on protein folding and not suppression of activity, the BACE Pro peptide (75 µM) has only a modest effect (16% inhibition) on the activity of BACE460 (data not shown). In stark contrast, Pro peptides for other aspartyl class proteases such as chicken pepsin and human cathepsin D are relatively potent inhibitors (Ki values < 10 nM and 30 nM, respectively) that completely block the activity of their cognate proteases (12).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Comparison of the predicted amino acid sequence of full-length BACE with members of the aspartyl protease family revealed the existence of a 24-residue polypeptide (Pro domain) that is situated between the putative Pre and protease domains (2-5). The peptide bond at the junction of the Pro and protease domains in the BACE precursor is efficiently cleaved in mammalian cells to generate mature BACE (2, 4). A typical function of the Pro region in aspartyl proteases is to confer zymogen-like properties on the unprocessed precursor species (7). In this report, we show that the Pro segment of ProBACE has a relatively minor inhibitory effect on BACE activity, but it does appear to play a role in the proper folding of the protease domain.

The activity of ProBACE460 was not markedly different from that of BACE460 when assayed with modified 31-mer polypeptide substrates corresponding to the wild type and Swedish-variant-type APP sequences (74 and 43% of the BACE460 activity, respectively). The apparent homogeneity of the ProBACE460 preparation that was evident by SDS-PAGE and N-terminal sequencing points to ProBACE460 as being responsible for the observed BACE activity. Consistent with this conclusion, greater than 90% of the BACE activity was removed from the ProBACE460 preparation by immunoprecipitation using the anti-Pro antibody. Moreover, the pellet fraction from the anti-Pro immunocapture step displayed BACE catalytic activity. Conversion of ProBACE460 to BACE460 during incubation with substrate under standard assay conditions was excluded by N-terminal sequencing and immunoblot analysis using the anti-Pro antibody.

The binding of full-length ProBACE (3) and ProBACE460 (shown herein) to the P10-P4' Stat(Val) affinity column is also consistent with the conclusion that ProBACE is catalytically active because only active forms of aspartyl proteases are expected to bind tightly to immobilized transition state analogs. Although some true zymogens of aspartyl proteases bind to immobilized transition state analogs (13-15), in these instances, the precursors invariably display markedly lower affinity for the immobilized ligand, which is not the case with ProBACE460.

Our observation that furin converts purified ProBACE460 to authentic BACE460 is concordant with a recent claim that furin or a furin-like proprotein convertase in mammalian cells is responsible for the processing of ProBACE (10). In addition to using cell-based assays, these investigators showed that furin correctly processed a recombinant soluble BACE precursor in vitro to release the Pro domain (10). Furin-like activity exists in insect cells; in particular, it is present in cells derived from Drosophila melanogaster (16). However, proteins that are expressed recombinantly in insect cells are typically not cleaved at putative furin processing sites unless furin is coexpressed in the cells (17, 18). These results are consistent with the inefficient processing of ProBACE460 to BACE460 by the Sf21 cells.

The finding that ProBACE460 displays activity bears on the prediction as to where beta -secretase mediated cleavage is expected to take place in the cell. However, attempts to pinpoint the subcellular site of beta -secretase activity must also take into account the pH optimum of BACE (2, 3) or ProBACE,3 both approximately pH 4.5. Hence, although it is tempting to attribute the purported beta -secretase-mediated cleavage in the endoplasmic reticulum (19) to colocalized ProBACE (20), the neutral pH value in the endoplasmic reticulum (21) does not appear to be conducive to ProBACE (or BACE) catalysis. Others claim that beta -secretase cleavage is absent in the endoplasmic reticulum but is instead colocalized with markers later in the secretory pathway (22), which is seemingly more compatible with the aforementioned pH dependence of BACE activity. Another recent investigation showed that BACE is actively targeted and localized to endosomes (23), which is an acidic site (pH 5.0) (20) that should be quite favorable for BACE-mediated catalysis (21). It is important to note that furin-mediated processing of ProBACE probably occurs in the secretory pathway upstream of subcellular sites with pH conditions that promote BACE activity (20); hence, the activity displayed by ProBACE in vitro may not be realized within the cell. Further studies are required to establish the significance of cellular ProBACE activity.

Whereas the putative Pro region in ProBACE does not confer strict zymogen-like properties on BACE, it does appear to play a role in the proper folding of the protease domain. The remarkable ability of the BACE Pro peptide to assist folding of its cognate mature protease domain in trans is reminiscent of results observed with alpha -lytic protease (24) and subtilisin (25). Our observations indicate that the Pro peptide interacts with BACE460 during refolding and may well serve as a chaperone that thwarts aggregation of unfolded protein. The poor ability of the Pro peptide to block BACE460 activity makes it difficult to attribute the lower activity of ProBACE relative to BACE460 to a direct interaction between the Pro domain and the active site in ProBACE460. The fact that the BACE Pro region promotes folding but is not a potent inhibitor of the protease is unusual but not without precedent. A recent study showed that the inhibitory and chaperone-like functions of the subtilisin Pro peptide are not obligatorily linked (26). It should be noted, however, that additional studies are required to exclude the possibility that membrane-tethered, mammalian-cell derived, full-length ProBACE is a strict zymogen with respect to its ability to process its natural membrane-bound substrate, APP.

During the preparation of this manuscript, a study appeared showing that a purified, soluble (C-terminal truncation 6 residues upstream of the putative transmembrane domain) derivative of ProBACE (pro-memapsin 2) expressed in Escherichia coli displays catalytic activity (27). Surprisingly, pro-memapsin 2 was not cleaved by furin, a finding that differs from that of our study and Vassar and colleagues (10). The inability of furin to process pro-memapsin 2 may reflect differences in the structure of this BACE derivative. Pro-memapsin 2 contains a 8amino acid extension (i.e. part of the putative signal peptide) upstream of the established N terminus for ProBACE in mammalian cells (2). In addition, the absence of protein glycosylation on the E. coli-derived pro-memapsin 2 might influence processing by converting enzymes. These investigators also studied two "mature" BACE analogs of memapsin 2, Leu28p-memapsin 2 and Gly45p-memapsin 2, which possess a 5-residue N-terminal extension and 13-residue N-terminal deletion, respectively, as compared with mammalian-cell derived BACE (27). The potential impact on activity because of these deviations from the structure of the bona fide mammalian cell-derived mature BACE remains to be determined.

Our current investigation provides novel insight into molecular mechanisms that impact the catalytic activity of BACE, a protease that purportedly plays a pivotal role in the etiology of Alzheimer's disease. The observation that ProBACE460 displays substantial activity relative to BACE460 distinguishes this aspartyl protease from typical members of this general protease class. However, from a teleological perspective, the anticipated role of the Pro segment in blocking premature exposure of BACE activity within the cell may be superfluous because of overriding pH-mediated control of enzymatic activity. Nevertheless, the Pro domain in ProBACE is not completely anachronistic because it continues to operate to promote the proper folding of the protease domain. Finally, the activity displayed by ProBACE460 suggests that blocking the conversion of ProBACE to BACE might have little or no impact on the production of the amyloid beta  peptide.

    FOOTNOTES

* 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.

§ To whom correspondence should be addressed: Merck Research Laboratories, WP42-213, Dept. of Biological Chemistry, West Point, PA 19486. Tel.: 215-652-3237; Fax: 215-652-3082; E-mail: steve_gardell@merck.com.

Published, JBC Papers in Press, December 28, 2000, DOI 10.1074/jbc.M009200200

2 Attempts to produce ProBACE460 in Sf21 cells by baculovirus-mediated expression using serum-free CM (Kemp Biotechnologies Inc., Frederick, MD) resulted in an approximate 50:50 mixture of ProBACE460 and mature BACE460 after purification by sequential concanavalin A and P10-P4'Stat(Val) affinity chromatography. ProBACE460 was further purified to apparent homogeneity from this mixture by immunoaffinity chromatography with anti-Pro antibody that is cross-linked to protein G-agarose (using the Immunopure Protein G IgG Plus Orientation kit; Pierce). The ProBACE460 was eluted from the antibody column using IgG elution buffer (Pierce). The ProBACE460 that was purified in this manner was homogenous as judged by N-terminal sequencing. Moreover, it displayed activity that was similar to ProBACE460, which was purified and characterized as described elsewhere in the manuscript (unpublished data).

3 X.-P. Shi, unpublished data.

    ABBREVIATIONS

The abbreviations used are: APP, amyloid precursor protein; CM, conditioned medium; BACE, beta -site APP cleaving enzyme; L-405, 492, acetyl-TTRPGSGLTNIK(6-(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVNLDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis L-405, 525, acetyl TTRPGSGLTNIK(6(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVKMDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide.

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ABSTRACT
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RESULTS
DISCUSSION
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