Neprilysin Degrades Both Amyloid beta  Peptides 1-40 and 1-42 Most Rapidly and Efficiently among Thiorphan- and Phosphoramidon-sensitive Endopeptidases*

Keiro ShirotaniDagger §, Satoshi TsubukiDagger , Nobuhisa IwataDagger , Yoshie TakakiDagger , Wakako HarigayaDagger , Kei Maruyama, Sumiko Kiryu-Seo||, Hiroshi Kiyama||, Hiroshi Iwata**, Taisuke Tomita**, Takeshi Iwatsubo**, and Takaomi C. SaidoDagger

From the Dagger  Laboratory for Proteolytic Neuroscience, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, the ** Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, the || Department of Anatomy, Asahikawa Medical College, Asahikawa 078-8510, and the  Department of Pharmacology, Saitama Medical School, Moroyama 350-0495, Japan

Received for publication, September 18, 2000, and in revised form, February 7, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To identify the amyloid beta  peptide (Abeta ) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha , NEPLP beta , and NEPLP gamma  cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha , and NEPLP beta , as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta 1-40 and Abeta 1-42 most rapidly and efficiently. PEX degraded cold Abeta 1-40 and NEPLP alpha  degraded both cold Abeta 1-40 and Abeta 1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta -degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is characterized by the accumulation of amyloid beta  peptide (Abeta ) in the brain. Abeta is composed of 39-43 amino acids and is constitutively produced by proteolysis of the beta -amyloid precursor protein (APP). Alterations in either synthesis or clearance of Abeta may potentially contribute to increased levels of Abeta and amyloid deposits. Although much attention has been focused on the production of Abeta , little is known about how Abeta is degraded and cleared, especially in the brain. Identification of the peptidases involved in Abeta catabolism in vivo is important for the development of therapeutics designed to prevent or treat AD.

Proteases, including cathepsin D (1), serine protease-alpha 2-macroglobulin complex (2) and insulin-degrading enzyme (3), were purified and identified as Abeta -degrading enzymes in vitro. Several other recombinant or purified peptidases have also been shown to degrade Abeta in vitro (4-14). We focused on the in vivo catabolism of Abeta 42, because this specific form, rather than Abeta 40, is considered to be the primary pathogenic agent in AD. Recently, we demonstrated that an endopeptidase(s) similar or identical to neprilysin (NEP), whose activity is sensitive to thiorphan and phosphoramidon, is involved in the catabolism of Abeta 1-42 in vivo (15, 16).

NEP is a type II membrane protein on the cell surface and is classified as a member of the M13 family. NEP hydrolyzes and inactivates several circulating peptides, such as enkephalin, atrial natriuretic peptide, endothelin, and substance P, and has wide tissue distribution and substrate specificity (17). The M13 family comprises six zinc-dependent metalloproteases, NEP, endothelin-converting enzyme (ECE-1) (18), ECE-2 (19), KELL antigen (20), phosphate regulating gene with homologies to endopeptidases on the X chromosome (PEX) (21), and the recently identified damage-induced neuronal endopeptidase (DINE)/X-converting enzyme (XCE) (22, 23). Among these, only NEP and DINE (22) have been shown to be sensitive to both thiorphan and phosphoramidon. Because ECE-1 and ECE-2 are not inhibited by thiorphan (18, 19), they can be excluded as candidates for Abeta 1-42-degrading enzymes in vivo. KELL is only partially inhibited by phosphoramidon (24) and is mainly present in erythroid tissues. Although PEX degrades parathyroid hormone-derived peptides (25), its sensitivity to thiorphan or phosphoramidon is unknown. These facts indicate that NEP, DINE, and PEX are candidates for Abeta 1-42-degrading enzymes in vivo. We also need to consider the possible presence of an unidentified protease(s), whose activity is sensitive to thiorphan and phosphoramidon and responsible for Abeta 1-42 degradation.

In this study, considering the redundancy of peptidases, we investigated the presence of unidentified peptidase(s), which is homologous to NEP, DINE, or PEX. We cloned novel cDNAs, which were termed neprilysin-like peptidases (NEPLPs) and examined their sensitivities to thiorphan and phosphoramidon. We compared the ability to proteolyse Abeta among NEP, PEX, DINE, and NEPLPs and found that NEP was the most potent Abeta -degrading enzyme.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNA Cloning-- To obtain cDNAs homologous to those of NEP and PEX, two degenerate oligonucleotides corresponding to amino acid residues 646-654 (GENIADNGG) and 685-696 (QLFFLNFAQVWC) in the mouse NEP were used; 5'-GGAGAAAA(C/T)ATTGCTGATAATGG(A/G)GG-3' and 5'-CACC(A/T)(C/T)AC(G/C/A)TG(A/G)GCA(A/T)A(G/ A)(T/C)TCA(G/A)GAA(GA)AA(G/T)AG(T/C)TG-3'. PCR was performed in a 50-µl 1× EX-Taq buffer (Takara, Kyoto, Japan) containing 2 mM MgCl2, 250 µM each of dNTPs, 2 µl of ICR mouse brain Marathon Ready cDNA (CLONTECH, Palo Alto, CA), 2.5 units of EX-Taq polymerase (Takara), and 2 µM of each primer. Amplifications were carried out according to initial denaturation of 1 min at 94 °C, 30 cycles of 30 s at 94 °C, 1 min at 50 °C, and 1 min at 72 °C and a final extension of 2 min at 72 °C. PCR products were analyzed by 6% acrylamide gel electrophoresis. Products ~150 bp in length were excised from the gel, cloned into a pCR-TOPO vector (Invitrogen, San Diego, CA), and sequenced. A novel cDNA was named NEPLP. 5' and 3' regions of the NEPLP gene were amplified by nested PCR with an Advantage cDNA PCR kit (CLONTECH) using AP-1/AP-2 as sense primers at the cDNA adaptor and 5'-TGGGCATAGGTCAGGTTCAGTCCCG-3'/5'-CAGTCGCTGATCTTTGCCGCCATCA-3' as antisense primers and 5'-GGTGTGCGACAGGCATACAAGG-3'/5'-CCTACGGTGGCTGGCTGATGGCGGC-3' as sense primers and AP-1/AP-2 as antisense primers, respectively. Both products of the 5' and 3' regions were cloned into a pCR- TOPO vector and sequenced. Full-length NEPLP cDNAs were amplified using the primer pairs 5'-GCGGGTACCGGAAAGTCTGAGAGCCCAGTGGGG-3' and 5'-TTGGCTACCAGATGCGACATCGCTT-3' and subcloned into the SmaI site of pBluescript II KS (+).

A similar procedure was performed to obtain cDNA homologous to NEP and DINE/XCE. Briefly, two oligonucleotides, 5'-AA(T/C)CA(A/G)AT(A/C/G/T)GT(A/C/G/T)TT(C/T)CC(A/C/G/T)GC-3' and 5'-CC(A/C/G)(A/T)T(A/G)TC(A/C/G/T)GC(A/G/T)AT(A/G)TT(C/T)TC-3' corresponding to amino acid residues 551-557 (NQIVFPA) and 647-653 (ENIADNG) of mouse NEP, respectively, were prepared, and PCR was performed under the same conditions described above. Products ~300 bp in length were sequenced.

Human NEP and PEX cDNAs were amplified from human hippocampus Marathon Ready cDNA (CLONTECH). Rat DINE cDNA was obtained as described previously (22). FLAG sequences (MDYKDDDDK) were introduced at the C-terminal end of each cDNA by PCR using appropriate primers containing FLAG nucleotides. All the cDNAs were subcloned into the mammalian expression vector pcDNA3.1 (+) (Invitrogen).

Cell Culture and Transfection-- 293 human embryonic kidney cell lines were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies, Inc.). The 293 cells were transfected with either an empty vector, NEP, PEX, NEPLP alpha , NEPLP beta , NEPLP gamma , or DINE cDNAs using a calcium phosphate method. Stable 293 cell lines were obtained by selection with 600 µg/ml G418. Stable mouse neuroblastoma N2a cells expressing both human APP695 with the Swedish mutation and presenilin 2 (PS2) with the N141I mutation were obtained by selection with 150 µg/ml hygromycin and 500 µg/ml G418, respectively.

Antibodies-- The rabbit polyclonal anti-NEPLPC (named as 065P and M97P) and anti-PEXC (M96G) antibodies were raised against a synthetic peptide, CCPRGSPMHPMKRCRIW corresponding to the C-terminal amino acid residues of the NEPLP, glutathione S-transferase fusion protein encompassing residues 534-646 of the NEPLP alpha , and glutathione S-transferase fusion protein encompassing residues 700-749 of human PEX, respectively. 56C6 (Novocastra Laboratory, Tyne, UK) and M2 (Stratagene, La Jolla, CA) are mouse monoclonal antibodies against the extracellular domain of human NEP and FLAG, respectively. DINE antibody was characterized previously (22).

Western Blot Analysis-- Cells were homogenized in solution A (0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A) and centrifuged at 500 × g for 5 min. Membranes were prepared by precipitation of the postnuclear supernatant at 100,000 × g for 30 min. The supernatant was used as a cytosolic fraction. The resulting pellet was lysed in solution A containing 1% Triton X-100 for 1 h at 4 °C and centrifuged at 100,000 × g for 15 min. The supernatant was used as a membrane fraction. Aliquots of the membrane fraction (3 µg) or 10 µl of culture medium (CM) were separated on 7.5% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane. The blotted membrane was blocked with 5% skim milk in a buffer containing 10 mM Tris-HCl, pH 8.0, and 0.15 M NaCl and sequentially incubated with the primary antibody in the buffer with 0.05% Tween 20 and with horseradish peroxidase-goat anti-rabbit or anti-mouse IgG (Amersham Pharmacia Biotech, Buckinghamshire, UK) and visualized using an ECL-plus kit (Amersham Pharmacia Biotech) according to the manufacturer's directions. Cell lysates were also prepared using the solution A containing 1% Triton X-100.

Peptidase Assay-- Membrane fractions (0.5 µg) from the 293 cells transfected with the various cDNAs were incubated with 50 µM Z-Ala-Ala-Leu-p-nitroanilide (ZAAL-pNA) (Peptide Institute, Osaka, Japan) in 100 µl of 100 mM MES (pH 6.5) or 50 mM HEPES (pH 7.2) for 1 h at 37 °C. The reaction mixture was added with 0.4 milliunit of leucine aminopeptidase (Sigma Chemical Co., St. Louis, MO), further incubated for 20 min at 37 °C and measured at 405-nm absorption. For the inhibition study the membrane fractions were preincubated with 10 µM thiorphan or 10 µM phosphoramidon for 5 min before the addition of ZAAL-pNA.

Digestion Assay of 3H/14C-radiolabeled Abeta 1-42-- Membrane fractions (0.5 µg) were incubated with 3H/14C-radiolabeled Abeta 1-42 (20,000 dpm, 1.3 µM) in 50 µl of 50 mM HEPES (pH 7.2) containing 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1× complete without EDTA (Roche Molecular Biochemicals) at 37 °C for 1 h. The reaction was stopped by adding 250 µl of a solution containing 0.05% triethylamine, 10 mM betaine, and 0.1 mM EDTA, followed by boiling for 5 min, and was analysed using HPLC as described (15). For the inhibition studies, membrane fractions were preincubated with 10 µM thiorphan for 5 min before adding the 3H/14C-radiolabeled Abeta 1-42.

Digestion Assay of Cold Abeta 1-40 and Abeta 1-42-- Abeta (Bachem, Torrance, CA) was dissolved in dimethyl sulfoxide at 5 mg/ml as a stock solution. 2 µg of Abeta 1-40 (4.6 µM) or 5 µg of Abeta 1-42 (11 µM) was incubated with 10 µg of the membrane fractions in 100 µl of 50 mM HEPES (pH 7.2) containing 1 µg/ml leupeptin and 1 µg/ml pepstatin A at 37 °C for the times indicated. The reaction was stopped by adding 200 µl of buffer A (0.1% trifluoroacetic acid), and the solution was injected into a reverse-phase HPLC (CAPCELL PAK C18 UG 120, particle size 5 µm, column dimensions 4.6 × 250 mm) (Shiseido, Tokyo, Japan) equilibrated with the buffer A. The HPLC protocol used to analyze 3H/14C-radiolabeled Abeta 1-42 was not suitable for analyzing cold Abeta peptides, because it produced high background noise when analyzed at the absorbance of 210 nm. Abeta and its proteolytic products were eluted with a linear solvent gradient of 0-70% buffer B (0.1% trifluoroacetic acid in acetonitrile) over 10-40 min at a flow rate of 0.5 ml/min at 50 °C. Peptides were detected by absorbance at 210 nm. 10 µM thiorphan was preincubated for the inhibition.

Mass Spectrometric Analysis and Protein Sequencing-- HPLC peaks in digestion assays of cold Abeta peptides were collected, dried under vacuum, and analyzed by a mass spectrometer and a protein sequencer as described previously (15).

Kinetic Analysis-- Various amounts of cold Abeta peptides were incubated with membrane fractions of pcDNA-transfected cells or NEP-transfected cells, and the remaining peaks of the Abeta peptides on the HPLC chart were used to determine the initial Abeta concentrations (S0) or Abeta concentrations at time t (St), respectively. Note that membrane fractions of pcDNA-transfected cells showed no detectable degradation to Abeta . Km and Vmax were determined according to the following first degree equation (26),
(S<SUB>0</SUB>−S<SUB><UP>t</UP></SUB>)/t=<UP>−</UP>K<SUB>m</SUB>/t×2.303 <UP>log </UP>(S<SUB>0</SUB>/S<SUB><UP>t</UP></SUB>)+V<SUB><UP>max</UP></SUB>

Digestion Assay of Secreted Abeta from N2a Cells-- CM from the N2a cells in the absence of fetal bovine serum was collected, diluted 5-fold with medium, and used as a substrate. The CM was incubated with 4 µg of membrane fractions in 100 µl of 50 mM HEPES (pH 7.2) at 37 °C for 16 h. The reaction was stopped by boiling and the samples were subjected to BAN50/BA27 and BAN50/BC05 sandwich ELISAs to quantify Abeta 1-40 and Abeta 1-42, respectively (27).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNA Cloning of NEPLPs-- To clone peptidases homologous to NEP and PEX, we designed degenerate oligonucleotide primers. With mouse brain cDNA as a template, the PCR resulted in the amplification of DNA with the predicted size (150 bp). After subcloning the PCR products and sequencing individual clones, we found that 11 of the 42 clones had a novel identical sequence that was similar to but distinct from members of the M13 family. The other 16 clones were identical to ECE-1, 11 clones were NEP, 2 clones were ECE-2, and 2 clones were PEX. During sequencing the full-length of the novel gene, we noted the presence of three isoforms, which probably resulted from alternative splicing. We termed them NEPLP alpha , NEPLP beta , and NEPLP gamma , and their structural characteristics are outlined in Fig. 1. Of 42 isolated full-length clones, 21 were NEPLP alpha , 20 were NEPLP beta , and 1 was NEPLP gamma . Compared with NEPLP alpha , NEPLP beta  and NEPLP gamma  had an insertion of 23 amino acids immediately following the putative transmembrane region and 37 amino acids near the center of the protein, respectively. All the isoforms were predicted to be type II transmembrane proteins with a putative zinc-binding domain (HEXXH) in the extracellular portion. NEPLPs showed the highest homology (~54%) to NEP among the members of the M13 family. The degenerate primers, which were designed to amplify the peptidases homologous to NEP and DINE/XCE, did not amplify a novel gene. Recently, three research groups independently reported a novel member of the M13 family (28-30). NEPLP alpha  and NEPLP beta  were identical to SEPDelta /splice 1 and SEP/NL1/NEPII, respectively. NEPLP gamma  was a novel isoform.


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Fig. 1.   Schematic representation of the primary sequences of the NEPLP alpha , NEPLP beta , and NEPLP gamma  proteins. The black rectangles represent the inserted 23 and 37 amino acids in the NEPLP beta  and gamma  proteins, respectively. The inserted nucleotide and amino acid sequences in NEPLP gamma  are shown by underlined letters. TM and HEITH represent the predicted transmembrane domain and the HEXXH zinc-binding motif, respectively. Arrowheads indicate the positions of the degenerate primers to amplify the NEPLP gene.

Expression of NEPLPs, NEP, PEX, and DINE in the 293 Cell Line-- We stably transfected NEPLP cDNAs in 293 cells and determined the expression using the 065P antibody, which was raised against the C-terminal portion of NEPLP. In the 293 cells transfected with pcDNA vector alone (referred to as pcDNA cells), no endogenous NEPLP protein was detected (Fig. 2A, lane 5). In membrane fractions of 293 cells stably transfected with NEPLP alpha  (NEPLP alpha  cells), 100- and 120-kDa broad bands were detected (Fig. 2A, lane 6). Because NEPLP alpha  was expressed as a single 100-kDa band in COS cells (not shown) and Chinese hamster ovary cells (28), the 120-kDa band probably represented a highly glycosylated or modified protein specific for the 293 cells. NEPLP beta  and NEPLP gamma  proteins were expressed in membrane fractions as 110- and 115-kDa bands, respectively (Fig. 2A, lanes 7 and 8). This corresponded to the increasing sizes of amino acids, although proteins with higher molecular mass did not accumulate as seen in NEPLP alpha  cells. The amounts of NEPLP beta  and NEPLP gamma  proteins were lower than that of NEPLP alpha . NEPLP alpha , NEPLP beta , and NEPLP gamma  were not detected in the cytosolic fractions (not shown). In the culture medium, we could detect only NEPLP beta  (Fig. 2C, lanes 13) with a molecular mass of 125 kDa, which was larger than that of membrane-associated NEPLP beta . This result suggested that the 23-amino acid sequences inserted in NEPLP beta  possessed a secretion signal and that NEPLP beta  is much more glycosylated or modified in the secretory pathway than in the membrane. Indeed Ikeda et al. (28) reported that the molecular mass of membrane-associated and secreted NEPLP beta /SEP were reduced to the same mass (89 kDa) by peptide-N-glycanase F treatment.


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Fig. 2.   Western analysis of NEP, PEX, NEPLP alpha , NEPLP beta , NEPLP gamma , and DINE expressed in the 293 cell line. A, equal amounts (3 µg) of membrane fractions of 293 cells stably transfected with cDNAs indicated below each lane were subjected to Western analysis using the antibodies 56C6 (lanes 1 and 2), M96G (lanes 3 and 4), and 065P (lanes 5-8). B, membrane fractions of 293 cells transiently transfected with pcDNA and DINE (lanes 9 and 10, respectively) were subjected to Western analysis using the DINE antibody. C, culture medium (CM) of 293 cells stably transfected with pcDNA, NEPLP alpha , NEPLP beta , and NEPLP gamma  were subjected to Western analysis using 065P (lanes 11-14). The 66-kDa bands indicated with an asterisk may be nonspecific staining of albumin derived from the serum. D and E, estimation of relative amounts of each protein expressed in 293 cells. Cell lysates of 293 cells transiently transfected with cDNAs tagged with FLAG were subjected to Western analysis using the anti-FLAG antibody M2 (D). The amounts of lysates transfected with NEP, PEX, NEPLP alpha , and DINE without FLAG were 0.5, 0.5, 0.5, and 1.5 µg, respectively (E). Each protein was blotted with the same antibodies as in A and B with the exception of NEPLP alpha . NEPLP alpha  was blotted with the M97P antibody. Note that almost equal amounts of peptidases were loaded.

When we expressed other members of the M13 family (NEP, PEX, and DINE) in the 293 cells, these proteins were detected as 110-, 110-, and 95-kDa bands in membrane fractions (Fig. 2A, lanes 2, 4, and 10), whereas they were not detected in the pcDNA cells (Fig. 2A, lanes 1, 3, and 9). NEP, PEX, and DINE were not detected in cytosolic fractions or CM (not shown). To estimate the relative amounts of the peptidases, we expressed each cDNA to which FLAG sequence was added at its C-terminal end. Because nearly identical intensities of bands were detected with the anti-FLAG antibody (Fig. 2D), equal amounts of peptidases with FLAG were loaded onto the gels. Fig. 2E shows the results of loading the same amounts of FLAG-tagged peptidases as those in Fig. 2D. The gel shown in Fig. 2E was also loaded with 0.5 µg of lysates from NEP, PEX, and NEPLP alpha  cells and 1.5 µg from DINE cells and was then stained with the corresponding antibodies. Almost identical band intensities resulted regardless of whether or not the peptidases contained FLAG. We estimated from the results that the relative amounts of peptidases were 1:1:1:1/3 for NEP:PEX:NEPLP alpha :DINE. The addition of FLAG to PEX and NEPLP alpha  reduced their molecular weight compared with their weights in the absence of FLAG (Fig. 2E), suggesting immature glycosylation or modification resulting from the FLAG sequence at the C terminus. We also observed reduced proteolytic activity of NEP-FLAG than that of NEP (not shown). Therefore, we used each peptidase without FLAG in the following proteolytic analyses.

Proteolytic Activity to Synthetic Peptide (ZAAL-pNA)-- We investigated the peptidase activity of NEPLPs, and their sensitivities to thiorphan and phosphoramidon. Membrane fractions of NEPLPs and other peptidases expressed in the 293 cells were incubated with ZAAL-pNA at pH 6.5 (Fig. 3, A and B). Membrane fractions of pcDNA cells showed a very low level of thiorphan- and phosphoramidon-sensitive activity due to the presence of an endogenous enzyme. A high degree of proteolytic activity was detected in the membrane fractions of the NEP cells (Fig. 3, A and B). PEX showed a low but significant proteolytic activity compared with that of the control (pcDNA) (Fig. 3A). Both NEPLP alpha  and NEPLP beta  proteolyzed the peptide, and their activities were almost the same and one-third compared with that of NEP, respectively. In contrast, NEPLP gamma  had no proteolytic activity compared with the control, although the level of the NEPLP gamma  protein was similar to that of NEPLP beta  (Fig. 2A). This suggested that NEPLP gamma  protein has different substrate specificity or that it is a zymogen that requires proteolytic activation. It is also possible that the 37-amino acid sequence inserted into NEPLP gamma  inhibits its activity by interfering with proper folding. DINE had no proteolytic activity in this system (Fig. 3B), although its endopeptidase activity and sensitivity to thiorphan and phosphoramidon were demonstrated previously in a baculovirus expression system (22). Proteolytic activities of NEP, PEX, NEPLP alpha , and NEPLP beta  were inhibited by thiorphan and phosphoramidon (Fig. 3A), suggesting that they could all be candidates for Abeta -degrading enzymes in vivo. The secreted form of NEPLP beta  also had the proteolytic activity (not shown). We obtained identical results at pH 7.2, too.


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Fig. 3.   Proteolytic activity of the peptidases expressed in the 293 cell line. A, membrane fractions of 293 cells stably transfected with cDNAs indicated below each column were analyzed for peptidase activity using ZAAL-pNA as a substrate in the absence (white columns) or presence of thiorphan (black columns) and phosphoramidon (hatched columns). Data are representative of at least three independent experiments and shown with means ± S.D. B, membrane fractions of 293 cells transiently transfected with pcDNA, NEP, and DINE were analyzed for peptidase activity.

Degradation of Radiolabeled Abeta 1-42-- We characterized the Abeta -degrading activity of the peptidases using 3H/14C-radiolabeled Abeta 1-42, which we had synthesized previously (15). To exclude the effects of endogenous peptidases, equal amounts of membrane fractions (0.5 µg) were analyzed for their ability to proteolyze Abeta . Membrane fractions of the pcDNA cells exhibited almost no proteolytic activity to the radiolabeled Abeta 1-42 (peak at 42 min in Fig. 4A, closed triangle) in 1 h at 37 °C. NEP decreased the radioactive peak at 42 min and increased the peaks at 7 and 37 min, suggesting that NEP efficiently proteolyzed the Abeta 1-42 into free amino acids/small peptides and a catabolic intermediate (Fig. 4B, open triangles). This degradation by NEP was very similar to the in vivo proteolysis as described previously (15). The degradation was completely inhibited by thiorphan, suggesting that NEP directly proteolyzed the peptide (Fig. 4H). In 4 h, NEP almost completely digested the Abeta 1-42 (not shown). In contrast PEX, NEPLP alpha , NEPLP beta , NEPLP gamma , and DINE did not degrade the Abeta 1-42, because their HPLC profiles were almost the same as that of the control (pcDNA) (Fig. 4, C-G). HPLC profiles in the 3H mode were essentially identical to the 14C mode (not shown), as reported previously (15), suggesting that NEP proteolyzed the Abeta 1-42 from both the N and C termini.


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Fig. 4.   Proteolysis of synthetic 3H/14C-radiolabeled Abeta 1-42 by the peptidases. Radioactive patterns in HPLC are shown in the 14C mode. A-G, membrane fractions (0.5 µg) of 293 cells transfected with the cDNAs indicated in each panel were analyzed for degrading activity to 3H/14C-radiolabeled Abeta 1-42. H, thiorphan was preincubated with membrane fractions of NEP-transfected cells. Black and white triangles represent intact 3H/14C-radiolabeled Abeta 1-42 and degrading products, respectively. Data are representative of at least two independent experiments.

Degradation of Cold Abeta 1-40 and Abeta 1-42-- We examined the degradation of cold Abeta 1-40 and Abeta 1-42 using HPLC under different conditions from those used for analyzing the degradation of 3H/14C-radiolabeled Abeta 1-42 (see "Experimental Procedures"). Membrane fractions of the pcDNA cells were used as a negative control (Fig. 5A, panel 1). NEP almost completely degraded the Abeta 1-40 peptide corresponding to the peak at 35.7 min (Fig. 5A, panel 2) in 2 h at 37 °C. The degradation was accompanied by the appearance of multiple peptides between 23 and 28 min (Fig. 5A, panel 2, open triangles). NEPLP alpha  showed relatively low but significant proteolytic activity to cold Abeta 1-40 as discerned by the decrease of the peak at 35.7 min and the appearance of new peaks between 26 and 29 min (Fig. 5A, panel 4). Thiorphan (Fig. 5A, panels 8 and 9) and phosphoramidon (not shown) completely abolished the proteolytic activity of NEP and NEPLP alpha . PEX, NEPLP beta , NEPLP gamma , DINE (Fig. 5A, panels 3, 5-7) and secreted form of NEPLP beta  (not shown) showed no proteolytic activity to Abeta 1-40. Although we assayed 3-fold amounts of membrane fractions of NEPLP beta , NEPLP gamma , and DINE cells to normalize the amounts of peptidases, they showed no detectable proteolytic activity to cold Abeta 1-40 in 2 h (not shown). With a longer incubation time (Fig. 5C, 6-12 h), NEP degraded much larger amounts of Abeta 1-40, although NEPLP alpha  did not further degrade Abeta 1-40. PEX showed very low proteolytic activity, and NEPLP beta , NEPLP gamma , and DINE showed no detectable proteolytic activity to Abeta 1-40 in 6-12 h (Fig. 5C).


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Fig. 5.   Proteolysis of synthetic cold Abeta 1-40 and Abeta 1-42 by the peptidases. A and B, membrane fractions (10 µg) of 293 cells transfected with the cDNAs indicated in each panel were analyzed using HPLC for degrading activity to cold Abeta 1-40 for 2 h (A) or Abeta 1-42 for 4 h (B). Thiorphan was preincubated with membrane fractions of NEP-transfected cells (A and B, panels 8) or with those of NEPLP alpha -transfected cells (A, panel 9). Black and white triangles indicate the peaks of intact Abeta and degrading products, respectively. An arrow indicates the peak of thiorphan. Data are representative of at least two independent experiments. C and D, time course of degradation of cold Abeta 1-40 (C) or Abeta 1-42 (D) by membrane fractions (10 µg) of each peptidase. The Y axis represents the remaining peak of the Abeta peptides compared with pcDNA cells (% of control) after indicated incubation periods.

When cold Abeta 1-42 was used as a substrate, NEP degraded it in a reproducible manner as demonstrated by the decrease of the original peak at 36.4 min and the appearance of a new peak at 26.4 min (Fig. 5B, panel 2) compared with the control (Fig. 5B, panel 1) in 4 h at 37 °C. In contrast, PEX, NEPLP alpha , NEPLP beta , NEPLP gamma , and DINE (Fig. 5B, panels 3-7), and the secreted form of NEPLP beta  (not shown) showed almost no proteolytic activity. Degradation of Abeta 1-42 by NEP was completely inhibited by thiorphan (Fig. 5B, panel 8) and phosphoramidon (not shown). Again, 3-fold amounts of membrane fractions of NEPLP beta , NEPLP gamma , and DINE cells showed no detectable proteolytic activity to cold Abeta 1-42 in 4 h (not shown). NEP degraded greater amounts of Abeta 1-42 with longer incubation times (Fig. 5D, 8-12 h). Among the other peptidases, only NEPLP alpha  degraded very small amounts of Abeta 1-42 in 8-12 h (Fig. 5D). N-terminal amino acid sequence and the molecular mass of the peak at 26.4 min produced by NEP (Fig. 5B, panel 2) were DAE--- and 1033.92, suggesting that the peak contained Abeta 1-9 (calculated mass MH+ = 1034.02). This result is consistent with the previous study, which indicated that 3H/14C-radiolabeled Abeta 1-42 was cleaved at G9/Y10 by thiorphan- and phosphoramidon-sensitive peptidase(s) in vivo (15). The other degrading products by NEP could not be identified probably due to further degradation.

Kinetic Analysis of Abeta Proteolysis by NEP-- We determined Km and Vmax values for the proteolysis by NEP using varying amounts of cold Abeta peptides (Table I). The Km values were 11.2 and 6.95 µM and Vmax values were 158 and 21.1 nM/min for Abeta 1-40 and Abeta 1-42, respectively. It is difficult to determine the Km and Vmax values for the other peptidases, presumably because the Km values are too large and/or the Vmax values are too small to measure.

                              
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Table I
Kinetic analysis of cold Abeta 1-40 and Abeta 1-42 proteolyzed by NEP
Various concentrations of Abeta 1-40 or Abeta 1-42 were incubated with membrane fractions for 2 or 4 h, respectively. Km and Vmax were calculated as described under "Experimental Procedures."

Degradation of Cell-secreted Abeta 1-40 and Abeta 1-42-- We investigated the degradation of cell-secreted Abeta by the peptidases. We chose the N2a cells as Abeta -producing cells because they were stably co-transfected with APP with the Swedish mutation and PS2 with the N141I mutation and secreted large amounts of both Abeta 1-40 and Abeta 1-42. We tried to examine the effect of NEP expression by co-culturing the NEP-expressing 293 cells with the N2a cells or by culturing the NEP-expressing 293 cells in CM derived from the N2a cells. However, the 293 cells used in the present study by themselves possessed a potent activity that completely removed the Abeta in CM, presumably through internalization and/or extracellular degradation employed by endogenous proteins. To avoid cell-mediated removal of Abeta , the membrane fractions of the 293 cells were incubated with CM from the N2a cells at 37 °C for 16 h. NEP, which was expressed either stably or transiently, reproducibly degraded both Abeta 1-40 and Abeta 1-42 compared with the control (pcDNA) (Fig. 6, A and B). PEX, NEPLP alpha , NEPLP beta , NEPLP gamma , and DINE showed almost no proteolytic activity to the cell-secreted Abeta 1-40 or Abeta 1-42 (Fig. 6, A and B).


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Fig. 6.   Proteolysis of cell-secreted Abeta 1-40 and Abeta 1-42 by the peptidases. Membrane fractions of 293 cells stably (A) or transiently (B) transfected with the cDNAs indicated below each lane were analyzed for degrading activity to Abeta secreted from N2a cells stably transfected with both the APP and PS2 mutants. After incubation with the membrane fractions (4 µg) and Abeta , quantitative ELISA analysis was performed to determine the Abeta 1-40 (white columns) and Abeta 1-42 (black columns). Data (Exp. 1 and 2) are representatives of at least three independent experiments and are shown with means ± S.D.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We previously reported that an endopeptidase(s) similar or identical to NEP is the most probable candidate for an Abeta 1-42-degrading enzyme in vivo (15). In the present study we cloned NEPLP cDNAs and compared Abeta -degrading activity among NEP, PEX, NEPLPs, and DINE whose activities are sensitive to thiorphan and phosphoramidon. Using different methods (HPLC and ELISA) and different substrates (synthetic and cell-secreted Abeta ), we showed that among the peptidases tested NEP most rapidly and efficiently degraded not only Abeta 1-42 but also Abeta 1-40 at concentrations ranging from picomolar to micromolar in vitro. Because picomolar concentrations of Abeta peptides are present in human cerebrospinal fluids and plasmas, NEP is most likely to be an Abeta -degrading enzyme in vivo.

We first demonstrated that enzymatic activities of PEX and NEPLP alpha /SEPDelta /splice 1 were sensitive to thiorphan and phosphoramidon and confirmed that that of NEPLP beta /SEP/NL1/NEPII was sensitive to these inhibitors (28, 29). PEX proteolyzed synthetic Abeta 1-40 more slowly and to an extremely weaker extent than NEP, but it did not proteolyze synthetic Abeta 1-42 or cell-secreted Abeta 1-40 and Abeta 1-42. Although NEPLP alpha  had almost the same proteolytic activity to ZAAL-pNA as NEP, it degraded cold Abeta 1-40 and Abeta 1-42 more slowly and to a weaker extent than NEP and showed no detectable degrading activity to cell-secreted Abeta 1-40 and Abeta 1-42. These results suggest that PEX and NEPLP alpha  have lower affinities and/or smaller Vmax for Abeta than NEP, and therefore, they could not be the major degrading enzymes of endogenous Abeta 1-40 or Abeta 1-42 in vivo as compared with NEP. However, PEX and NEPLP alpha  might degrade Abeta in AD patients or APP transgenic mice brains, where Abeta is accumulated in excess. It seems unlikely that NEPLP beta , NEPLP gamma , or DINE degrade Abeta in vivo, because they showed almost no proteolytic activity to Abeta under any of the conditions we examined.

Our results suggest that NEP directly degrades both Abeta 1-40 and Abeta 1-42 as determined by its inhibitor profiles, consistent with a previous report using purified NEP and synthetic Abeta 1-40 (7). Alternatively, NEP may mediate the initial cleavage of Abeta and another endogenous peptidase may further degrade Abeta . Although it is possible that NEP is indirectly involved in Abeta degradation through, for instance, the proteolytic activation of another peptidase, this is unlikely because NEP is capable of proteolyzing peptides smaller than 4-5 kDa (31). In any case, it is noted that NEP is required for Abeta degradation.

We found that NEP degraded cold Abeta 1-40 more rapidly than cold Abeta 1-42 in vitro (Fig. 5). This may be due to a higher Vmax for Abeta 1-40 than for Abeta 1-42, although NEP had a slightly higher affinity to Abeta 1-42 than to Abeta 1-40 (Table I). The rate of degradation by NEP was different among various Abeta 1-42 peptides (Fig. 4-6). The half-lives of radiolabeled Abeta 1-42, cold Abeta 1-42, and cell-secreted Abeta 1-42 were ~1, 8, and more than 16 h, respectively. Moreover, the degraded products of radiolabeled 1-42 and cold Abeta 1-42 may be different as evaluated by their retention time in HPLC. These different degrees of degradation are probably due to different conformations and concentrations of the peptides and/or different concentrations of the peptidases under the optimum conditions for each assay.

NEP is an ectoenzyme with a large extracellular domain containing a catalytic site. This indicates the direct involvement of NEP in Abeta degradation, because NEP can interact with and degrade Abeta with the correct topology on the cell surface. The fact that a soluble form of NEP is found in human plasma and cerebrospinal fluid (32, 33) also supports the possible interaction between Abeta and NEP in the extracellular space. We could not determine whether NEP degraded extracellular Abeta or intracellular Abeta in this study. To address this issue a further study will be needed to measure intracellular Abeta as well as extracellular Abeta in doubly transfected cells with APP and NEP cDNAs.

In that Abeta 42 is preferentially accumulated in AD, it is important that NEP is capable of degrading Abeta 1-42. The high expression of NEP protein in the brain is restricted to striatum, olfactory tubercle, substantia nigra, choroid plexus, endopeduncular nucleus, and pontine nuclei, and moderate expression in cerebellum and low expression in hippocampus and cerebral cortex (34, 35). The relatively low levels of NEP in hippocampus and cerebral cortex may explain the selective deposition of Abeta 42 in those areas. It is possible that altered activity of NEP in brain upon aging might lead to accumulation of Abeta 42. Indeed NEP mRNA and protein levels in the hippocampus and temporal gyrus were significantly lower in AD patients than those in control cases (36). It would be interesting to examine possible colocalization of NEP protein with senile plaques and neurofibrillary tangles. Genetic analysis of NEP, which is located in chromosome 3q21-27, in sporadic as well as familial AD cases may help understanding of the pathogenesis.

An intriguing finding in this study is that NEP can degrade either synthetic or cell-secreted Abeta 1-40 and Abeta 1-42 over a wide range of concentrations, from picomolar to micromolar. These results suggested not only that NEP degrades endogenous Abeta but also that NEP may be a good therapeutic target for the treatment of AD. Because there is a dynamic balance between monomer and polymer forms of Abeta (37-39), reducing the amounts of monomer Abeta will lead to a reduction of polymer Abeta . It is important to search for methods that would up-regulate NEP activity or inactivate NEP inhibitors. Alternatively, targeting endogenous NEP activity to the site(s) of Abeta production or storage may reduce the Abeta levels in a specific and efficient manner without altering the total NEP activity (40). The use of NEP is advantageous in that NEP is non-destructive due to its limited ability to act only on peptides smaller than 4-5 kDa. Although it remains to be determined whether NEP degrades endogenous Abeta in vivo, knockout and transgenic studies of the NEP gene should elucidate this in future studies. The relative contribution of PEX and NEPLP in the catabolism of Abeta in vivo will also be clarified by such studies of each gene.

    ACKNOWLEDGEMENT

We thank Takeda Chemical Industries, Ltd. for kindly providing the monoclonal antibodies for sandwich ELISA.

    FOOTNOTES

* This work was supported in part by research grants from RIKEN Brain Science Institute, Special Coordination Funds for promoting Science and Technology of Science and Technology Agency, Ministry of Health and Welfare, Ministry of Education, and Takeda Chemical Industries.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF302075, AF302076, and AF302077.

§ Recipient of Special Postdoctoral Researchers Program. To whom correspondence should be addressed: Tel.: 81-48-462-1111; Fax: 81-48-467-9716; E-mail: kshiro@brain.riken.go.jp.

Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M008511200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta  peptide; APP, beta -amyloid precursor protein; NEP, neprilysin; ECE, endothelin-converting enzyme; PEX, phosphate regulating gene with homologies to endopeptidases on the X chromosome; DINE, damage-induced neuronal endopeptidase; XCE, X-converting enzyme; NEPLP, neprilysin-like peptidase; PCR, polymerase chain reaction; PS2, presenilin 2; CM, culture medium; HRP, horseradish peroxidase; pNA, p-nitroanilide; MES, 2-(N-morpholino)ethanesulfonic acid; HPLC, high performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; ELISA, enzyme-linked immunosorbent assay; bp, base pair(s).

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ABSTRACT
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DISCUSSION
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