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(The FASEB Journal. 2000;14:1837-1847.)
© 2000 FASEB

Human bleomycin hydrolase regulates the secretion of amyloid precursor protein

ILIYA M. LEFTEROV1, RADOSVETA P. KOLDAMOVA1 and JOHN S. LAZO2

Department of Pharmacology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261, USA

2Correspondence: Department of Pharmacology, University of Pittsburgh School of Medicine, Biomedical Science Tower E1340, Pittsburgh, PA 15261, USA. E-mail: lazo+{at}pitt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human bleomycin hydrolase (hBH) is a neutral cysteine protease genetically associated with increased risk for Alzheimer disease. We show here that ectopic expression of hBH in 293APPwt and CHOAPPsw cells altered the processing of amyloid precursor protein (APP) and increased significantly the release of its proteolytic fragment, ß amyloid (Aß). We also found that hBH interacted and colocalized with APP as determined by subcellular fractionation, in vitro binding assay, and confocal immunolocalization. Metabolic labeling and pulse-chase experiments showed that ectopic hBH expression increased secretion of soluble APP{alpha}/ß products without changing the half-life of cellular APP. We also observed that this increased Aß secretion was independent of hBH isoforms. Our findings suggest a regulatory role for hBH in APP processing pathways.—Lefterov, I. M., Koldamova, R. P., Lazo, J. S. Human bleomycin hydrolase regulates the secretion of amyloid precursor protein.


Key Words: Alzheimer disease • protease • ß-amyloid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SS AMYLOID (ASS) is the principal component of the amyloid neuritic plaques found in Alzheimer’s disease (AD) brain. Aß is 4 kDa peptide derived by constitutive proteolytic processing of a large type I integral transmembrane glycoprotein, the amyloid precursor protein (APP), which is encoded by a gene on chromosome 21 (1 2 3) . The major APP isoforms (APP695, APP751, and APP770) are produced by alternatively spliced transcripts (1 , 4 , 5) , and the segregation of several genetic mutations with rare familial forms of early onset AD suggests that APP metabolism plays a causative role in the pathogenesis of the disease (6 7 8) . Not all of these mutations cause an increase in Aß secretion when studied in primary or transfected cells (9 10 11) . Transgenic mice that overexpress familial AD mutated forms of human APP proteins show early cognitive and memory deficits or amyloid pathology in the brain typical for AD (12 13 14 15) . At least three proteases, designated {alpha}, ß, and {gamma}, participate in the processing of APP (16 , 17) . The normal secretory processing of APP results in a cleavage of the precursor by {alpha}-secretase within the Aß coding region. This cleavage leads to the secretion of the large soluble ectodomain of APP (sAPP{alpha}) and the retention of a small 10 kDa carboxyl-terminal fragment (CTF) within the membrane. A protease designated {gamma}-secretase cleaves the membrane-bound 10 kDa CTF within the transmembrane domain of APP and releases a small 3 kDa peptide (p3), which can be identified in the conditioned media of cultured cells together with sAPP{alpha} (18 19) . Alternatively, cleavage by ß- and {gamma}-secretases generates the majority of Aß variants and releases sAPPß in the media (20) . The major proteolytic processing of APP is {alpha}-secretase-mediated cleavage that occurs through the secretory pathway in the late trans-Golgi, trans-Golgi network compartments or at the plasma membrane (20 21 22) . Aß is produced mainly through endosomal/lysosomal pathway, but part of Aß is generated in the secretory pathway in endoplasmic reticulum (ER)/Golgi intermediate compartment and trans-Golgi network (21 , 23 , 24) .

Bleomycin hydrolase (BH) is a cysteine proteinase from the papain superfamily. The protein is highly conserved among eukaryotes with >40% identity between yeast BH (yBH) and human BH (hBH) and more than 98% between mammalian species. The physiological function of BH, however, remains unknown. Using a two-hybrid screen, we found that hBH interacts with a number of secreted proteins, among which are serum amyloid A protein and {alpha}1-antichymotrypsin (unpublished data). BH does not contain a signal peptide or transmembrane domain; its lumenal localization has not been established and it is believed that BH is not secreted in the medium or extracellular fluids. Recently it has been suggested that one of the two isoforms of hBH (hBHval443) is associated with an increased risk of sporadic AD in non-ApoE4 patients (25) . In other populations, however, this association was not obvious (26 , 27) . None of these genetic studies provided molecular or cellular information on a possible role for hBH. There are, however, recent immunohistochemical data (28 , 29) showing the distribution of hBH in regions of the human brain relevant to AD. In one of the studies, the staining pattern in normal controls and AD patients appeared to be different, suggesting higher levels in the diseased brains (29) .

Here we have analyzed the influence of hBH on APP secretion and Aß release. Therefore, we expressed hBHIle443 and hBHVal443 isoforms in cells stably transfected with APP wild-type or APP Swedish mutant APP695(K595M596-> N595L596), and analyzed the amount of secreted APP and amyloidogenic Aß fragment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid constructions and site-directed mutagenesis
To generate some of the recombinant expression vectors, we used full-length hBH cDNA (a gift from Dieter Brömme, Mt. Sinai School of Medicine, New York, N.Y.) that contained T7 epitope sequences (Novagen, Madison, Wis.) in-frame preceding the ATG start codon. For expression in mammalian cells, hBH cDNA was subcloned into a pcDNA3.1Zeo vector (Invitrogen, Carlsbad, Calif.), thus generating pcDNA3.1hBHZeo, as described previously (30) .

For in vitro transcription and translation of 35S-labeled proteins, we exploited pCite4 vectors (Novagen). pCite4APP695wt vector was generated by polymerase chain reaction (PCR) subcloning using as a template pRK5APP695 (originally developed by Efrat Levy-Lahad and provided by Robert Bowser, University of Pittsburgh) and the following primers (the restriction enzyme recognition sites within the primers are underlined): forward-5'-GT-GGAATCCATATGCTGCCCGGTTT-3' and reverse-5'-GCGCGTCGACCTAGTTCTGCATCTGCTC-3'. The in vitro APP695 was synthesized according to the manufacturer’s directions for the TnT Rabbit Reticulocyte Lysate system (Promega, Madison, Wis.) as described previously (31) . We used pNSE-APP (kindly provided by Carmela Abraham, Boston University School of Medicine, Boston, Mass.) for subcloning of APP751 cDNA into HindIII-XbaI digested pcDNA3.1Hygro mammalian expression vector (Invitrogen). The recombinant vector was created by standard ligation of a PCR amplification product created by using the following primers: forward-5'-GTGGAAGCTTGCGATGCTGCCCGGTTTG-3' and reverse-5'-GCGCTCTAGACTAGTTCTGCATCTGCTC-3'. cDNA for APP695sw (K595M596-> N595L596; 695 numbering), kindly provided by Christian Haass (Ludwig-Maximilians University, Munich, Germany), was subcloned by PCR amplification into pcDNA3.1Hygro using the same primer set, thus generating pcDNA3.1APPsw. pcDNA3.1APP751 and pcDN3.1AAPsw were used later for stable or transient transfections of mammalian cells.

Oligonucleotide-directed site specific mutagenesis was performed using the QuickChange mutagenesis kit (Stratagene, San Diego, Calif.). Oligonucleotides complementary to the both strands of hBH were synthesized to change Ile443 to Val443: forward-5'-GAACCCATTGTCCTGCCAGCAT-3' and reverse-5'-ATGCTGGCAGGACAATGGGTTC-3'. The template used for the PCR was pcDNA3.1hBHZeo and the procedure was performed as described previously (31) . The resultant pcDNA3.1hBHval443Zeo expression vector was used for mammalian cell transfections. The in-frame position of all cDNA inserts was confirmed by dye terminator labeling and sequencing using ABI Prism 373 DNA Sequencer (University of Pittsburgh Research Facility).

Antibodies
Anti-APP mAb LN27, with an epitope within the first 200 amino acids on the NH2 terminus of APP, was from Zymed (South San Francisco, Calif.). Anti-Aß1–16 mAb, anti-Aß17–26 mAb and anti-Aß40 polyclonal antibody were from Biosource International (Camarilo, Calif.). Anti-T7 mAb was from Novagen. Polyclonal anti-ß-COP antibody was from Affinity Bioreagents (Golden, Colo.). Anti-hBH polyclonal antibody was a generous gift from Stephen A. Johnston, University of Texas (Dallas, Tex.). AlexaTM546 goat anti-rabbit and AlexaTM488 goat anti-mouse secondary antibodies were from Molecular Probes (Eugene, Oreg.). Goat anti-rabbit, alkaline phosphatase-labeled secondary antibody was from Jackson Immunoresearch (West Grove, Pa.).

Stable cell lines and transfection procedures
Untransfected Chinese hamster ovary cells (CHO-K1) and stable hBH expressing CHOhBH cell line were maintained in Ham F-12 medium, supplemented with 2 mM of L-glutamine, 100 U/ml penicillin, 10 µg/ml streptomycin sulfate, and 10% v/v heat-inactivated fetal bovine serum in a humidified atmosphere of 95% air:5% CO2 at 37°C as described (30) . For transient transfection and expression of APP751, CHO-K1 or CHOhBH cells were treated with 8 µg vector DNA per 25 cm2 growth area using SuperFect (Qiagen, Carlsbad, Calif.) according to the manufacturer’s protocol. Cells were washed 24 h after the transfection and were subjected to metabolic labeling for the appropriate time (see below). CHO-K1 were stably transfected with pcDNA3.1APP751 or pcDN3.1AAPsw using the same amount of DNA and initially maintained for selection in 250 µg/ml Hygromycin (Life Technologies, Inc., Gaithersburg, Md.). Polyclonal cell lines with the highest level of APP751 or APPsw expression were chosen for second transfection with pcDNA3.1BHZeo or pcDNA3.1BHval443Zeo. The levels of expression of both proteins were verified by Western blotting and the cells were constantly maintained in 500 µg/ml of Zeocin (Invitrogen) and 250 µg/ml Hygromycin (Life Technologies, Inc.). We used 293APP cells overexpressing APP751 (kindly provided by D. Selkoe, Harvard Medical School, Boston, Mass.) for transfection with pcDNA3.1hBHZeo and generated 293APP/BH cells maintained in 500 µg/ml of Zeocin and 500 µg/ml Geneticin (Life Technologies, Inc.). We also used 293 cells (ATCC Number CRL-1573) for stable transfection with pcDN3.1AAPsw and subsequently for double transfection with pcDNA3.1Zeo, pcDNA3.1BHZeo, or pcDNA3.1BHval443Zeo. All 293 cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) with 25 mM HEPES buffer, high glucose content, antibiotics, and the appropriate selection agent.

In vitro binding and in vitro APP cleavage assays
The expression and purification of the glutathione S-transferase (GST)-hBH and His-hBH fusion constructs as well as the in vitro binding assays were performed as described previously (30 , 31) . Briefly, 35S-labeled APP (3 µl of a standard TnT reaction) was incubated with the GST-hBH fusion construct prebound to glutathione-Sepharose beads (25 µl) in 50 mM NaCl and bovine serum albumin (1 mg/ml) at 4°C for 1 h. As a control, 35S-labeled proteins were incubated with GST bound to glutathione-Sepharose. The beads were washed four times with 0.1% Nonidet P-40 in phosphate-buffered saline (PBS), boiled, and loaded on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were soaked in fluorographic reagent Amplify (Amersham, Arlington Heights, Ill.), dried, and exposed to Kodak X-ray film. For in vitro cleavage assays 10 µl of 35S-labeled APP was incubated with 5 µg recombinant His-tagged hBH in 100 µl reaction buffer (Dulbecco’s phosphate-buffered saline) at 37°C for different periods of times. At each time point, 10 µl aliquots were mixed with an equal amount of Tricine sample buffer and resolved on 10–20% Tricine gradient gel. The gels were dried, scanned on Molecular Dynamics PhosphorImager and images were processed using ImageQuant.

Bleomycin (BLM) hydrolase assays
The metabolism of BLM was determined by our previously described method that separates BLM A2 from its inactive metabolite deamidobleomycin A2 (BLM dA2). Briefly, His-hBH (2 µg/ml) was incubated with 70 µM BLM A2 (Nippon Kayku Co. Ltd., Tokyo, Japan) in 50 µl reaction buffer (20 mM Tris pH 7.5) at 37°C for 10 h. The reaction was stopped by adding 40 µl methanol and 10 µl of 7.5 mM CuSO4 and injected onto a Symmetry C8 reverse-phase high-performance liquid chromatography (HPLC) column. BLM A2 and BLM dA2 were eluted at 1 ml/min with 17% acetonitrile, 0.8% acetic acid, 2 mM heptane sulfonic acid, and 25 mM triethylamine (pH 5.5) and detected by absorbance at 292 nm.

Metabolic labeling and immunoprecipitation
For metabolic labeling, ~ 2.5 x 105 cells were plated in T75 culture flasks and then grown to a level of 85–90% confluence. After washing and starvation for 1 h in methionine/cysteine free DMEM (ICN, Aurora, Ohio), the cells were incubated with 200 µCi/ml [35S]methionine/cysteine EXPRE35S35S Protein labeling mix (NEN, Boston, Mass.) in methionine/cysteine-free DMEM with 5% dialyzed fetal bovine serum. For pulse-chase experiments cells were labeled for 1 h and chased with complete DMEM for different periods of time. For steady-state experiments, cells were labeled continuously for 16 h. Total labeled cellular holoAPP (APPfl) was determined by lysing cells with RIPA buffer immediately after labeling, followed by immunoprecipitation with amino-terminal anti-APP mAb LN27 and protein G agarose (Sigma, St. Louis, Mo.). Secreted APP{alpha}/ß was immunoprecipitated from the medium with amino-terminal anti-APP mAb LN27 and secreted APP{alpha} was immunoprecipitated from the medium with anti-Aß1–16 mAb, followed by protein G agarose. Aß was immunoprecipitated from the medium with anti-Aß17–26 monoclonal antibody and protein G agarose. Values obtained for APP{alpha}/ß, APP{alpha}, and Aß were normalized to this total labeled APPfl in each experiment.

Enzyme-linked immunoassay (ELISA)
For the sandwich ELISA, cell culture medium was changed to fresh complete DMEM or F12. After incubation for 48 h at 37°C, the conditioned medium was collected and subjected to a sandwich ELISA for Aß using anti-Aß1–16 mAb as the capture antibody. For detection, samples were incubated with anti-Aß40 polyclonal antibody recognizing specifically the carboxyl terminus of Aß1–40, followed by incubation with goat anti-rabbit, alkaline phosphatase-labeled secondary antibody, and the Attophos substrate (Roche Molecular Biochemicals, Indianapolis, Ind.). The Aß1–40 peptide used as a standard was from Biosource International. The values obtained were normalized to the total cellular APPfl determined by immunoblotting with anti-APP (LN27), followed by densitometry and quantification using ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).

Subcellular fractionation
Iodixanol gradients were formed by layering (30, 25, 20, 15, 10%) of Optiprep (Life Technologies, Inc.) in a centrifuge tube and then placing the tube on its side for 10 h. The 293 APP751/hBH cells, grown to 90% confluence on T150 tissue culture flasks, were detached from the surface by incubation with 10 mM EDTA in PBS for 5 min. Cells were pelleted at 1000 g for 10 min, resuspended in homogenization buffer (0.25 M sucrose, 10 mM Tris, pH 7.4, 0.2 mM MgCl2, 5 mM KCl and protease inhibitors 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 µg/ml pepstatin), and disrupted with 25–30 passes in a Dounce homogenizer with a tight pestle until > 90% of the cells were broken as determined by trypan blue dye exclusion. Nondisrupted cells and cellular nuclei were pelleted by centrifugation at 3000 g for 10 min. The pellet was washed with homogenization buffer, and postnuclear supernatants from both centrifugations were combined and centrifuged at 80,000 g for 1 h. The membrane pellet was resuspended in 0.8 ml homogenization buffer. The 80,000 g pellet was loaded on the top of a preformed 10–30% continuous iodixanol gradient and centrifuged at 200,000 g for 3 h at 4°C using SW41 rotor in Beckman XL-70 ultracentrifuge. The gradients were unloaded from the bottom of the gradient. After centrifugation of the fractions at 100,000 g for 1 h to collect the membranes, the 100,000 g pellets were resuspended in SDS sample buffer and analyzed by SDS-PAGE and immunoblotting.

Immunocytochemistry
Immunofluorescence was performed as described previously (32) . Briefly, CHOAPP751/hBH cells grown to 50–80% confluence were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After incubation in blocking solution containing 2% bovine serum albumin/0.5% normal goat serum in PBS, cells were incubated with the primary antibodies: anti-APP mAb LN27 (Zymed) and a rabbit polyclonal anti-hBH antibody. We used AlexaTM546 goat anti-rabbit (red) and AlexaTM488 goat anti-mouse (green) secondary antibodies. Slides were washed and mounted in Mowiol (Calbiochem, San Diego, Calif.). Images were collected on 3-dimensional data sets using a Photometrics cooled CCD camera and Zeiss Axiovert microscope and processed with ONCOR Image software at 0.2 µm vertical separation, which provided confocal quality images. For hBH/ß-COP immunostaining, we used CHO cells stably transfected and expressing T7-tagged hBH. The cells were fixed in 95% ethanol:5% acidic acid (v/v) for 30 min, rehydrated in PBS for 1 h, and incubated with polyclonal anti ß-COP (Affinity Bioreagents) and monoclonal anti-T7 (Novagen) primary antibodies. We used Cy3-labeled goat anti-rabbit (red) (Amersham) and FITC-conjugated goat anti-mouse (green) (Sigma) secondary antibodies. Slides were mounted as above and images were collected and processed on a Molecular Dynamics Confocal Laser Microscope.

Analytical procedures
Protein concentrations in cell lysates were determined by the BCA method (Pierce, Rockford, Ill.). SDS-PAGE was performed on 8%, 12% Tris glycine gels or 10–20% Tris-Tricine gels (Novex, San Diego, Calif.). For Western blots, proteins were transferred to nitrocellulose membrane, probed with antibodies, and detected by Renaissance chemiluminescence reagent (NEN Life Science Products, Inc.). Radioactive gels were scanned on Molecular Dynamics PhosphorImager and the images were processed using ImageQuant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
hBH interacts with APP in vitro
To look for interaction between hBH and APP, we performed in vitro binding assays using full-length hBH expressed as a GST fusion protein in Escherichia coli. GST fusion proteins were first immobilized on glutathione-Sepharose beads, then incubated with in vitro transcribed and translated 35S-labeled APP protein. As a negative control, 35S-labeled proteins were incubated with GST protein alone bound to glutathione-Sepharose beads. Figure 1 shows that 35S-labeled APP bound specifically to GST-hBH but did not bind to GST. Lack of binding between hBH and irrelevant control proteins, namely, 35S-labeled luciferase and 35S-labeled serum amyloid A protein, confirmed the specificity of hBH–hAPP interaction (data not shown). We have also detected in vivo interactions between hBH and APP in a yeast two-hybrid system (unpublished results). Attempts to coimmunoprecipitate APP with our current hBH antibody have not been successful, which we presume reflects on the limited characteristics of this antibody.



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  		Figure 1. In vitro binding assays of 35S-labeled APP695wt and GST-hBH fusion proteins immobilized on glutathione-Sepharose. 35S-labeled APP695wt was precipitated with GST tagged hBH (lane 3), but not with GST alone (lane 2). Precipitated proteins were separated on 12% SDS-PAGE gels, dried, and exposed to X-ray film. The resulting film was scanned.

hBH does not process 35S-labeled APP in vitro
To investigate whether hBH cleaves APP in vitro, we incubated 35S-labeled APP with recombinant His-tagged hBH (His-hBH). The activity of His-hBH was confirmed by its ability to convert BLM A2 to metabolite BLM dA2 (Fig. 2A ). As is visible from Fig. 2B , a concentration of hBH that completely degraded BLM A2, did not cleave APP. Examining the scanned gel, we were unable to find bands migrating with a size of the carboxyl-terminal fragments corresponding to {alpha}- or ß-secretase cleavage of APP or Aß.



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  		Figure 2. hBH did not degrade 35S-labeled APP695wt in vitro. A) Enzymatic activity of hBH in BLM degradation assays. The amount of BLM A2 metabolized to BLM dA2 was determined by HPLC as described in Materials and Methods. a) Control assay with no enzyme; b) affinity-purified His-hBH was incubated with BLM A2 for 10 h. B) In vitro cleavage assay. His-hBH was incubated with TnT expressed 35S APP695wt for different periods of time (+ lanes). As a control 35S APP695wt was incubated in the buffer, without enzyme (- lanes). Aliquots, corresponding to the respective h time points, were loaded on 10–20% Tricine gels. The gels were dried and scanned on Molecular Dynamics PhosphorImager and images processed using ImageQuant.

Effect of hBH overexpression on APP turnover
To study the effect of hBH on the half-life of full-length intracellular APP (APPfl) and the amount of secreted soluble APP (sAPP{alpha}/ß), we performed pulse-chase experiments with 293APP751wt and 293APP751wt/hBH cells, which were selected for stable expression of APPwt or APPwt and hBH, respectively. We estimate that the 293APP751wt/hBH cells expressed ~fivefold more BH compared with the 293APP751wt cells based on the hBH HPLC enzyme assay. Cells were metabolically labeled with 35S methionine/cysteine for 1 h and chased for different period of times. Cellular APPfl and secreted APP{alpha}/ß from the conditioned media were immunoprecipitated with amino-terminal antibody LN27, which recognizes soluble products cleaved by {alpha}- and/or ß-secretase. Among the three experiments performed, there was no significant reproducible difference in APPfl expression between the cell lines; nevertheless, the secreted proteins were normalized to the level of the total APPfl precursor at the 0 h time point for each experiment. Results of the SDS-PAGE temporal profile and the quantitation of the scanned protein bands are shown in Fig. 3A, B . Immunoprecipitation of APPfl from cell lysates after the pulse-chase revealed a continuous decrease in radioactivity with a t1/2 of ~90 min. Overexpression of hBH did not significantly affect the decrease of cellular APPfl or the ration between mature and immature APP forms. Immunoprecipitation of sAPP{alpha}/ß from the medium, however, showed that the secretion of soluble APP{alpha}/ß products was significantly increased in 293APP751wt/hBH cells as compared to the 293APP751wt cells at all time points. At 6 h 293APP751wt/hBH cells secreted almost twofold more relative to 293APP751wt cells.



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  		Figure 3. Temporal profile of APP expression and secretion by 293APP751wt (-hBH and +hBH). 293APP751wt (-hBH and +hBH) cells were metabolically labeled with 35S methionine/35S cysteine; 200 µCi/ml) for 1 h and chased for different time periods. Cell lysates and media were immunoprecipitated with amino-terminal antibody LN27 separated by 8% SDS-PAGE, and exposed to X-ray film. A) The autoradiogram represents results from one of three experiments conducted. B) Quantitative analysis of APP precursor (APPfl) in cell lysate and total secreted APP in the media, as a result of {alpha}- and ß-secretase cleavage (sAPP{alpha}/ß). Relative intensity of the bands was calculated as percentage of the intensity of APPfl protein band in cell lysate at the beginning of the chase (time 0). Mean ± SE, n = 3, **P < 0.005, *P < 0.05.

hBH alters the proteolytic processing of APPwt
To examine the influence of hBH on steady-state levels of secreted sAPP{alpha}/ß, we used continuous metabolic labeling of 293APP751wt and 293APP751wt/hBH cells for 16 h and immunoprecipitation of sAPP{alpha}/ß products from conditioned medium with the same amino-terminal antibody used for pulse-chase experiments. The results shown on Fig. 4A demonstrate that overexpression of hBH causes a twofold increase in secretion of sAPP{alpha}/ß during the 16 h labeling period (P<0.0001, n=9). Because this antibody does not discriminate between soluble products cleaved by {alpha}- or ß-secretase, we used an anti-Aß1–16 antibody, which specifically recognizes the carboxyl terminus of sAPP{alpha} and therefore immunoprecipitates only soluble products generated by {alpha}-secretase(s). We labeled 293APP751wt cells and 293APP751wt/hBH as above; at the end of the labeling period, the lysates were immunoprecipitated with LN27 antibody and media were immunoprecipitated with anti-Aß1–16 antibody. As is visible from Fig. 4B , the overexpression of hBH increased by 1.5-fold the amount of sAPP{alpha} (P<0.005, n=9).



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  		Figure 4. hBH increased the secretion of sAPP{alpha}/ß and sAPP{alpha}. 293APP751wt (-hBH and +hBH) cells were metabolically labeled with 35S methionine/35S cysteine (200 µCi/ml) for 16 h. Cell lysates were immunoprecipitated with amino-terminal antibody LN27 (A, B) and media were immunoprecipitated with LN27 (A) or anti-Aß1–16 antibodies (B). The immunoprecipitated proteins were separated by SDS-PAGE and exposed to X-ray film. The protein bands were scanned and quantitated. A) Quantitative analysis of sAPP{alpha}/ß. The values of sAPP{alpha}/ß were normalized to the levels of APPfl. B) Quantitative analysis of sAPP{alpha}. The values of sAPP{alpha}/ß and sAPP{alpha} were normalized to the levels of APPfl. The results are the average ± SE of 3 experiments, each in triplicate. n = 9, **P < 0.005, ***P < 0.0001.

hBH increases Aß production
To examine the effect of hBH on Aß secretion, we metabolically labeled 293APP751wt and 293APP751wt/hBH cells as well as CHO and CHOhBH cells transiently transfected with pcDNA3.1APP751. The cells were continuously labeled for 16 h and APPfl was immunoprecipitated from the cell lysates with LN27 antibody while Aß was immunoprecipitated from conditioned media with anti-Aß17–26 antibody. The amount of secreted Aß was normalized to the expression of precursor APPfl. As is visible from Fig. 5A, B , hBH increased the secretion of Aß in 293APP751wt/hBH cells 1.7-fold (P<0.001, n=6). The same increase of Aß secretion was produced by hBH overexpression in CHO cells transiently transfected with APPwt (Fig. 5C , 5P<0.04, n=3). Because we found no evidence for 35S-labeled APP cleavage in vitro by hBH (Fig. 2B ), we believe the increased Aß production is an indirect rather than a direct effect of hBH.



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  		Figure 5. hBH increased the secretion of Aß from APP751wt type. 293APP751wt (-hBH and +hBH) cells and CHO (-hBH or +hBH) cells, transiently transfected with APPwt751, were metabolically labeled with 35S methionine/35S cysteine (200 µCi/ml) for 16 h. Cell lysates were immunoprecipitated with LN27 antibody and media with Aß17–26 antibody. The immunoprecipitated proteins were separated by 10–20% Tricine gels and visualized by PhosphorImager. The values of Aß were normalized to the levels of APPfl. A) The autoradiogram represents results typical of those observed in 21 experiments, each in triplicate. B) Quantitative analysis of Aß (A); the values are the average ± SE, n = 6, ***P < 0.0001. C) Quantitative analysis of Aß, immunoprecipitated in CHO (-hBH or +hBH) cells, transiently transfected with APPwt751. The values are the average ± SE, n=3, *P<0.04.

To confirm that hBH overexpression increased Aß secretion, we used a Swedish mutant of APP (APPsw) that contains a dual amino acid change (Lys595Asp/Met596Leu) known to promote Aß secretion. We used the APPsw, which is a 695 amino acid isoform of APP, because we wanted to probe the isoform dependency of the phenomenon. CHOAPPsw695 cells selected for stable expression of APPsw with or without hBH overexpression were continuously labeled with 35S methionine/cysteine for 16 h and Aß was immunoprecipitated from conditioned media with anti-Aß17–26 antibody. As with 293APP715wt, we found enhanced Aß secretion with hBH coexpression (Fig. 6A ). When the level of Aß was normalized to the expression of cellular APPfl, we calculated that Aß secretion increased 2.9-fold (P<0.002, n=6). The same effect was found in 293APPsw695 cells with or without hBH overexpression (data not shown). Qualitatively similar results were observed by sandwich ELISA. CHOAPPsw695/hBH and CHOAPPsw695 were cultivated for 48 h and conditioned media were subjected to sandwich ELISA. As is visible from Fig. 6B , hBH increased Aß secretion 2.5 fold (P<0.0001, n=9). The same twofold increase in Aß secretion was found in 293APPsw695/hBH cells as compared with 293APPsw695 cells (data not shown). We then examined the effects of hBH on sAPP {alpha} formation and observed increased secretion (Fig. 6C ). Thus, with two APP isoforms, both Aß and sAPP {alpha} secretions were increased by ectopic expression of hBH.



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  		Figure 6. hBH increased in secretion of Aß and sAPP{alpha} from APPsw695 expressing cells. CHO APPsw695 (-hBH and +hBH) cells were metabolically labeled with 35S methionine/35S cysteine (200 µCi/ml) for 16 h. A) Aß was immunoprecipitated from media with Aß17–26 antibody. The immunoprecipitated proteins were separated by 10–20% Tricine gels and visualized by PhosphorImager. The autoradiogram represents results typical of those observed in 2 experiments. B) Conditioned medium (48 h) from CHO APPsw695 (-hBH and +hBH) cells was subjected to a sandwich ELISA for Aß using Aß1–16 as the capture antibody and Aß 40 as the detection antibody. The data represent means ± SE (n=9), ***P < 0.0001. The values of sAPP{alpha} and Aß were normalized to the levels of APPfl. C) sAPP{alpha} was immunoprecipitated from media with Aß1–16 antibody and the immunoprecipitated proteins were separated by 8% Tris-glycine gels and exposed to X-ray film. The protein bands were scanned and quantitated. The results are the average ± SE of six determinations; *P < 0.02.

Effect of hBH isoforms on Aß secretion
To test the role of different isoforms of hBH (i. g. ile443 and val443) on APP processing, we measured the level of Aß secreted in the conditioned media by sandwich ELISA. CHOAPPsw and 293APPsw were selected for expression after transfection with pcDNA3.1hBHval443 or pcDNA3.1hBH. The production of Aß was compared after normalizing to cellular APPfl. There was no difference in the secreted Aß in either cell line overexpressing similar levels of hBH isoforms (Fig. 7A, B ). Aß secretion was increased more than twofold in cells overexpressing hBH as compared with non-hBH-transfected cells regardless of the hBH isoform.



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  		Figure 7. Effect of hBH on Aß secretion is independent of hBH isoform. A) Conditioned medium (48 h) from CHO APPsw695 (-hBH, +hBHile and +hBHval); B) 293 APPsw695 (-hBH, +hBHile and +hBHval) cells was subjected to a sandwich ELISA for Aß using Aß1–16 as the capture antibody and Aß40 as the detection antibody. The data represent means ± SE (n=3); A) -hBH vs. +hBHile, *P < 0.02; -hBH vs. +hBHval, **P<0.002; B) -hBH vs. +hBHile ***P < 0.0001, -hBH vs. +hBHval **P < 0.01.

hBH and APP cofractionate in iodixanol gradients
293APP751wt/hBH were analyzed by subcellular fractionation. Subcellular vesicles were separated on 10–30% continuous Iodixanol gradients as described in Materials and Methods. ER-rich fractions were found at the bottom of the gradient, using an antibody against the ER marker protein calnexin (data not shown). As reported previously (33) , immature forms of APP were found in more dense, ER-rich fractions, whereas the mature APP appeared in the least dense fraction on the top of the gradient. As is visible from Fig. 8 , the highest immunoreactivity of hBH was found in the least dense fractions, corresponding to Golgi, where it cofractionated with mature APP.



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  		Figure 8. hBH and APP cofractionated in iodixanol gradients. The 100,000 g pellet from 293 APP751/hBH cells was fractionated on 10–30% continuous iodixanol gradient (A, B). Membranes collected from each fraction of the gradient were pelleted, separated by SDS-PAGE, and analyzed by immunoblotting with LN27 antibodies to APP (A) and anti-T7 antibody to T7-tagged hBH (B). The fractions are, from left to right, increasingly dense. The results shown are representative from three experiments.

hBH and APP colocalize
To probe whether or not hBH and APP colocalize in the cell, we performed immunofluorescent staining and examined the slides by epifluorescent microscopy. As is visible from Fig. 9 , hBH and APP colocalized in the area surrounding the nucleus. We next studied whether hBH colocalizes with ß-COP, a marker for cis-Golgi. Figure 10 , shows that they colocalized in the area corresponding to Golgi network, although hBH staining penetrated the cytosol more extensively but did not reach the plasma membrane. This demonstrated hBH localized to an early compartment of the secretory pathway.



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  		Figure 9. Colocalization of hBH with APP in CHOAPP751hBH cells. CHO cells were stably transfected with pcDNA3.1hBHZeo and pcDNA3.1APP751 plasmids coding for hBH and APP751 respectively. hBH was visualized with polyclonal anti-hBH antiserum and AlexaTM546-conjugated polyclonal goat anti-rabbit secondary antibodies (A, red). APP751 was immunostained with LN27 mAb and AlexaTM488-conjugated polyclonal goat anti-mouse secondary antibodies (B, green). The orange in panel C shows the regions of overlap. Images were collected on 3-dimensional data sets using a Photometrics cooled CCD camera and Zeiss Axiovert microscope and processed with ONCORTM Image software.



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  		Figure 10. Colocalization of hBH with cis Golgi marker ß-COP in CHOhBH cells. CHO cells were stable transfected with pcDNA3.1hBHZeo plasmid coding for T7-tagged hBH and immunostained A) with anti-T7 (green), and B) anti-ßCOP antibodies (red). The orange in panel C shows the regions of overlap. Images were collected and processed on Confocal Laser Microscope (Molecular Dynamics).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Eukaryotic BH are unique enzymes with almost identical high molecular structure from yeast to humans (34 , 35) . Structurally they are similar to a 20S proteasome and belong to a family of self-compartmentalizing intracellular proteases (36) . Their natural substrates are unknown and the only biologically relevant function is deamidation of the anticancer drug bleomycin due to a demonstrable aminopeptidase activity (37 38 39) . Recently, we have shown that hBH also has intrinsic endopeptidase activity (31) , whereas others using artificial substrates have characterized unusual autocarboxypeptidase and peptide ligase activities of yBH (36) . Thus, BH is a multifunctional enzyme. Using cellular and molecular approaches, we demonstrate here that hBH may significantly influence the proteolytic processing of one protein: APP. Our studies demonstrate that the overexpression of hBH significantly increases the production of Aß in two distinct cell lines transfected with APP wild-type or its Swedish variant. This was paralleled by an increase in the secretion of the soluble long APP fragments.

How does hBH alter APP processing and secretion? The APP processing is complex and includes APP posttranslational modifications and the activity of at least three secretases in different subcellular compartments through the secretory as well as endocytic pathways. Although papain superfamily cysteine proteases resembling hBH have been implicated in the formation of the amyloidogenic peptides through APP cleavage at the ß-secretase cleavage site in a model synthetic peptide (40) , we think it is unlikely that hBH is a ß-secretase. First, hBH does not share structural or enzymatic resemblance with the recently identified ß-secretase (41) . Second, we have failed to document ß-secretase in vitro activity with hBH and APP. Third, there are no convincing data so far in the literature showing intralumenal localization of eukaryotic BH. Thus, we believe it is more likely hBH alters other aspects of APP processing. We have recently shown that hBH binds to ubiquitin-conjugating enzyme 9 and may have a role in the posttranslational modification of a variety of proteins (esp. RAN-GAP1), due to a covalent binding of small ubiquitin-like molecules, such as SUMO-1. Protein conjugation to SUMO-1/Smt3 is involved in many physiological processes including cell cycle progression and protease activities different from proteolytic degradation through the ubiquitin-proteasomal pathway. This particular type of posttranslational modification, however, has not been shown to influence trafficking between the cytosol and ER/Golgi vesicular structures. A number of proteins, however, that are involved in the translocation and vesicular transport machinery require one or more modifications (42) . hBH may well participate in such modifications by providing amino-, carboxy-endopeptidase, or even ligase activities. We observed that the altered APP processing required the catalytic cysteine in hBH (unpublished results). Thus, hBH may affect the recruitment of coatomer subunits and/or assembling of the vesicular structures. Colocalization by confocal microscopy of hBH and ß-COP I provides indirect support for a role of hBH in the APP processing. It is now widely accepted that the generation of Aß takes place in different compartments within the secretory pathway (21 , 24) . The retention of APP is due to protein–protein interactions, and thus the availability of APP at the plasma membrane for internalization may influence significantly the amount of Aß detectable in the conditioned media (43 , 44) . Therefore, the subcellular colocalization of hBH in close proximity to APP supports the idea that hBH may regulate the proteolytic processing of APP.

It is worth noting that Magdolen et al. (45) copurified yBH with Gce1p, a cAMP binding ectoprotein associated with the plasma membrane by a GPI anchor. It has long been known that GPI-anchored proteins together with caveolin-1 and other proteins initially join Golgi membrane structures and initiate the biogenesis of caveolae (46) . Primarily, the function of those microdomains is to import molecules and deliver them to specific locations within the cell, thus forming a unique endocytic and exocytic compartment at the cell surface of most cells types. Recently they have been implicated in the {alpha}-secretase-mediated proteolysis of APP (47) . Moreover, in yeast {alpha}-secretase, activity was attributed to GPI-anchored aspartyl proteases (48) . In CHO cells, however, strong evidence has been provided that one or more GPI anchored proteins play an important role in ß- not {alpha}-secretase activity (49) . It was not clear from those experiments if GPI-anchored proteins were proteases, and the authors left open the possibility for posttranslational modifications, chaperone function, or the existence of a multisubunit ß-secretase as reasonable alternatives. Taken together, these data suggest it may not be necessary for hBH to be localized on the lumenal site of a membrane structure or at the cell surface to influence the proteolytic processing of APP and especially Aß secretion.

Our study shows hBH influenced the proteolytic processing of APP. The observed increase in the amounts of soluble sAPP{alpha}/ß as well as Aß may be due to increased flux of APP through the secretory pathway, thus providing more APP for the endocytic pathway and generation of more Aß. Such a mechanism has recently been demonstrated for FE65, a brain-enriched protein that binds the cytoplasmic domain of APP (44) with no obvious sequence homology to hBH. The localization of APP and hBH in a very close proximity, however, raises the possibility that hBH may produce its effects by targeting some other protein(s) to APP.

It is obvious that future experiments are necessary to clarify the exact mechanism governed by hBH and responsible for the increased Aß production. Such studies will permit the design and implementation of drug discovery approaches aiming at influencing the secretion of Aß and possibly its deposition in amyloid plaques, thus preventing or at least slowing the progression of AD.


   ACKNOWLEDGMENTS
 
We thank Stephen A. Johnston for providing anti-human BH antibody, Christian Haass for APP695sw cDNA, and Carmela Abraham for pNSE-APP. We also thank S. Watkins for providing us the opportunity to work on the microscopes, and Marc DiSabella, Martina Lefterova, and John Skoko for their excellent technical assistance. Ruth Perez is thanked for the critical reading and suggestions in the preparation of the manuscript. Carmela Abraham is thanked for discussing her observations about APP and BH. This work was supported by United States Public Health Service Grant CA43917, Pilot Study Program of the University of Pittsburgh Alzheimer’s Disease Research Center Grant A605133, and the Fiske Drug Discovery Drug Fund.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication October 28, 1999. Revision received February 17, 2000.
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 DISCUSSION
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