Degradation of the Alzheimer's Amyloid beta  Peptide by Endothelin-converting Enzyme*

Elizabeth A. Eckman, Dana Kim Reed, and Christopher B. EckmanDagger

From the Mayo Clinic Jacksonville, Jacksonville, Florida 32224

Received for publication, August 19, 2000, and in revised form, May 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Deposition of beta -amyloid (Abeta ) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of Abeta is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of Abeta . Much less, however, is known about the mechanisms responsible for Abeta removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Abeta -degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular Abeta concentration that appears to be due to inhibition of intracellular Abeta degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular Abeta concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Abeta 40 and Abeta 42 in vitro at multiple sites.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is the most common cause of dementia in the elderly and is characterized pathologically by the accumulation of beta -amyloid peptides (Abeta ) in the brain in the form of senile plaques. Abeta is normally produced from the beta -amyloid precursor protein (beta APP) through the combined proteolytic actions of beta - and gamma -secretase and is then secreted into the extracellular milieu (1, 2). The degree of Abeta accumulation is dependent not only on its production but also on the mechanisms responsible for its removal. While considerable effort has been directed at elucidating the enzymes and pathways contributing to the production of Abeta , much less is known regarding Abeta catabolism.

Abeta catabolism is likely to involve proteases at multiple sites, both intracellular and extracellular. Proteases acting at the site of Abeta generation and/or within the secretory pathway may degrade the peptide intracellularly, thus limiting the amount of the peptide available for secretion. The concentration of secreted Abeta may be further regulated by direct degradation by extracellular proteases and by receptor-mediated endocytosis or phagocytosis followed by lysosomal degradation. Catabolism of Abeta peptides at each of these steps would limit the accumulation of extracellular Abeta , and disruption of this catabolism may be a risk factor for AD. Additionally, the identification of enzymes that degrade Abeta intracellularly and extracellularly may lead to development of novel therapeutics aimed at reducing Abeta concentration by enhancing its removal.

Recent reports suggest a role for both insulin-degrading enzyme and neprilysin (NEP) in the degradation of extracellular Abeta (3-10). Matrix metalloproteinase-9, EC 3.4.24.15, and alpha 2-macroglobulin complexes have also been reported to play a role in Abeta degradation (11-13). In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Abeta -degrading enzyme. The endothelin converting enzymes are a class of type II integral membrane zinc metalloproteases (active site luminal) named for their ability to hydrolyze a family of biologically inactive intermediates, big endothelins (big ETs), exclusively at a Trp21-Val/Ile22 bond to form the potent vasoconstrictors endothelins (14). In addition to this specific cleavage event, ECE-1 has been reported to hydrolyze several biologically active peptides in vitro, including bradykinin, neurotensin, substance P, and oxidized insulin B chain by cleaving on the amino side of hydrophobic residues (15, 16).

Two different endothelin-converting enzymes have been cloned. The first identified, ECE-1, is abundantly expressed in the vascular endothelial cells of all organs and is also widely expressed in nonvascular cells of tissues including lung, pancreas, testis, ovary, and adrenal gland (17-19). A comprehensive analysis examining both ECE activity and expression in human brain has not been reported. Studies have, however, detected human ECE-1 immunoreactivity in fibers within the glial limitans and neuronal processes and cell bodies of the cerebral cortex (18). In rats, ECE-1 immunoreactivity has been detected in pyramidal cells of the hippocampus and in cultured primary astrocytes (20).

Four isoforms of human ECE-1 differing only in the cytoplasmic tail are produced by a single gene located on chromosome 1 (1p36) through the use of alternate promoters (17, 21-25). The four isoforms cleave big ETs with equal efficiency but differ primarily in their subcellular localization and tissue distribution (24, 25). Human ECE-1a is localized predominantly to the plasma membrane (24, 25). Human ECE-1c and ECE-1d have also been reported to be localized predominantly to the plasma membrane with additional intracellular expression detected (24, 25). In contrast, human ECE-1b appears to be localized exclusively intracellularly. Co-immunolocalization studies performed by Schweizer et al. (24) on human ECE-1b-transfected CHO cells indicate the presence of this isoform in the trans-Golgi network (TGN). Azarani et al. similarly demonstrated that human ECE-1b was located in an intracellular compartment when expressed in Madin-Darby canine kidney cells (26), and Cailler et al. (27) demonstrated that a dileucine motif in the cytosolic tail of ECE-1b was probably responsible for its intracellular localization. In an endogenous ECE-1b- and ECE-1c-expressing cell line, ECV304, ECE-1 immunoreactivity was detected in intracellular Golgi-like structures as well as at the cell surface (24).

Bovine ECE-1b, which corresponds to human ECE-1c, is also localized predominantly on the plasma membrane (28). However, in contrast to human ECE-1a, bovine ECE-1a has convincingly been shown to be constitutively targeted to the lysosome (28). This difference between the localization of human and bovine ECE-1a may be due to the fact that the isoform-specific N-terminal region of ECE-1a is poorly conserved between the species. In fact, Emoto et al. (28) identified lysosome-targeting signals in the N-terminal tail of bovine ECE-1a that are not conserved in human ECE-1a.

ECE-2 is a homologous enzyme with catalytic activity similar to that of ECE-1. Bovine ECE-2 has been cloned and is encoded by a separate gene from ECE-1 (29). The sequence and chromosomal location of the human ECE-2 gene have not been reported. However, Nagase et al. (30) recently reported the cloning of an unidentified human brain cDNA, KIAA0604, that shares 93% identity with the bovine ECE-2 gene and is located on human chromosome 3. Given the similarity to bovine ECE-2, this cDNA probably represents human ECE-2.

ECE-2 is localized intracellularly and has an acidic pH optimum (29). Immunocytochemical analysis of endogenous ECE-2 in HUVECs revealed a punctate pattern of staining consistent with expression of ECE-2 in acidic intracellular vesicles of the constitutive secretory pathway (31). Northern blot analysis of bovine tissues revealed that ECE-2 is most abundantly expressed in neural tissues including cerebral cortex, cerebellum, and adrenal medulla, with low level expression detected in many other tissues (29). In mouse brain, ECE-2 is expressed in heterogeneous populations of neurons in the thalamus, hypothalamus, amygdala, dentate gyrus, and CA3 (32). Like ECE-1, ECE-2 cleaves big ET-1 most efficiently among the three big ETs (24, 29). Another member of the ECE family, ECE-3, has recently been purified from bovine iris microsomes and is highly specific for the conversion of big ET-3 (33). The enzyme responsible for this activity has not yet been cloned.

The role that ECE may play in Alzheimer's disease has not been previously explored. Here we present both pharmacological and biochemical evidence that ECE-1 can hydrolyze Abeta both in vitro and in vivo. These data indicate a potential role for this enzyme family in Abeta catabolism.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Analysis of Abeta Concentration by Sandwich ELISA-- Human Abeta was measured by sandwich ELISA as previously described (34), using the BAN50/BA27 and BAN50/BC05 antibody systems (Takeda) to detect Abeta 40 and Abeta 42, respectively. Hamster Abeta derived from CHO cells was measured using the BNT77/BA27 and BNT77/BC05 antibody systems. The BNT77 antibody (Takeda) was raised against amino acids 11-28 and thus may recognize amino-terminally modified or truncated peptides (35). Abeta concentration was determined by comparing values obtained for samples with those obtained for synthetic Abeta 40 and Abeta 42 standards (Bachem).

Cloning of Human ECE-1a and ECE-1b-- ECE-1a and ECE-1b were cloned by reverse transcription-polymerase chain reaction in two fragments (joined by a unique PvuII site) from human umbilical vein endothelial cell (HUVEC, ATCC) RNA using the following primers (restriction sites underlined): 1) ECE-1a forward, 5'-CAGGAATTCGCCACCATGCCTCTCCAGGGCCTGGGCCTGC-3'; 2) ECE-1b forward, 5'-CAGGAATTCGCCACCATGCGGGGCGTGTGGCCGCCC-3'; 3) ECE-1 PvuII reverse, 5'-CAGCAGCTTCCCCAGCTGGACC-3'; 4) ECE-1 PvuII forward, 5'-GGTCCAGCTGGGGAAGCTGCTG-3'; and 5) ECE-1 reverse, 5'-GCTCTAGATTACCAGACTTCGCACTTGTGAGGCGGG-3'.

RNA was prepared from HUVEC cells using the Qiagen RNeasy miniprep kit and was reverse transcribed using Superscript II reverse transcriptase and an oligo(dT) primer (Roche Molecular Biochemicals). The 5' fragment of ECE-1a was amplified using primers 1 and 3. The 5' fragment of ECE-1b was amplified using primers 2 and 3. The 3' fragment of ECE-1, which is common to both isoforms, was amplified using primers 4 and 5. Pfu polymerase (Stratagene) was used for all amplifications. The 5' and 3' fragments were ligated together at the PvuII site and subcloned into pcDNA3 (Invitrogen) using primer-encoded EcoRI and XbaI sites. The sequences of the constructs were confirmed by dideoxy sequencing by the Mayo Molecular Biology Core Facility.

Cell Culture and Transfections-- Unless otherwise noted, cell culture reagents were purchased from Life Technologies, Inc., and cell lines were purchased from ATCC. HUVECs were cultured in Kaighn's F12K medium (ATCC) supplemented with 10% fetal bovine serum, 0.1 mg/ml heparin, 0.03 mg/ml endothelial cell growth supplement (Sigma), 100 units/ml penicillin, and 100 µg/ml streptomycin. CHO cells were cultured in Ham's F-12 medium (BioWhittaker) supplemented with 10% newborn calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. H4 cells (human neuroglioma origin) were cultured in Opti-MEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. For passaging of cells prior to experiments in which ECE activity was to be measured, a highly purified trypsin (Sigma T-7418) solution was used (17). CHO cells were transfected with FuGENE 6 (Roche Molecular Biochemicals) according to the directions of the manufacturer. Stable lines were generated by selecting pcDNA3-transfected cells (ECE-1a and ECE-1b) with 1 mg/ml Geneticin and pSecTag-transfected cells (solECE-1) with 0.8 mg/ml Zeocin (Invitrogen).

Measurement of ECE Activity in Cell Membrane Fractions-- Cell membrane fractions were prepared as described by Xu et al. (17). Protein concentration was determined by BCA assay (Pierce) in membranes resuspended in buffer B (20 mM Tris-HCl, pH 7.4, containing 250 mM sucrose) or in membranes solubilized in buffer B containing 2.5% C12E10 (polyoxyethylene-10-lauryl ether, Sigma). For measurement of ECE activity, membrane fractions (10-50 µg of protein) were incubated for 30 min at 37 °C with 0.1 µM big ET-1 (1-38) (American Peptide Co.) in 0.1 M sodium phosphate buffer (pH 6.8) containing 0.5 M NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 µM leupeptin, and 20 µM pepstatin. Duplicate reactions were carried out in the presence of phosphoramidon (100 µM). Reactions were stopped by the addition of EDTA (5 mM), and mature ET-1-(1-21) peptide was quantitated by sandwich ELISA (Amersham Pharmacia Biotech).

Treatment of Cells with Metalloprotease Inhibitors-- Cells were passaged into six-well plates 1 day prior to treatment and were utilized at confluence. Triplicate wells were washed twice with Hanks' balanced salt solution and then incubated for 17-24 h with 1 ml of growth medium containing phosphoramidon (Roche Molecular Biochemicals), thiorphan (Sigma), or captopril (Sigma) at the indicated concentrations. Control cells were incubated in growth medium containing an equal concentration of vehicle (phosphate-buffered saline). After treatment, the culture medium was harvested and spun at 14,000 × g, and the supernatant was analyzed for Abeta 40 and Abeta 42 by sandwich ELISA as described and for secreted APP by Western blot. To assess cellular toxicity of the compounds, MTS assays (CellTiter 96®, Promega), which measure the conversion of MTS to formazan by metabolically active cells, were performed on the cells after the indicated time points. Culture medium was subjected to electrophoresis on 10-20% Tricine gels (Novex) and was subsequently transferred to Immobilon P (Millipore Corp.). Western blots on CHO cells were performed using 22C11 antibody (Roche Molecular Biochemicals) to detect secreted APP. Bound antibody was detected by incubation with the appropriate horseradish peroxidase-linked secondary antibody (Amersham Pharmacia Biotech) followed by ECL Western blotting reagents (Amersham Pharmacia Biotech) and exposure to x-ray film.

Expression and Purification of Soluble ECE-1-- A construct encoding a soluble form of ECE-1 similar to that described by Ahn et al. (36) and Korth et al. (37) was generated by amplifying the extracellular domain of ECE-1a using the following primers: SolECE-1 forward, 5'-GAGAGAATTCTCAGTACCAGACAAGATCCCC-3'; SolECE-1 reverse, 5'-CGTTTTCCTTTTGCGGCCGCCCAGACTTCGCACTTGTGAGG-3'.

The solECE-1 construct was subcloned using primer-encoded EcoRI and NotI sites into pSecTag2B (Invitrogen), which incorporates a leader sequence onto the N terminus of the protein for secretion by mammalian cells and sequential c-Myc and His6 tags at the C terminus to facilitate detection and purification. The sequence of the construct was confirmed by dideoxy sequencing by the Mayo Molecular Biology Core Facility. A stable solECE-1 secreting cell line was generated by transfecting the construct into CHO cells and selecting with Zeocin (0.8 mg/ml). At confluence, the cells were washed twice with Hanks' balanced salt solution and then cultured for 48 h in serum-free medium (CHO-S-SFM II) containing 1 mM sodium butyrate. Conditioned medium was filtered through a 0.2-µm filter and dialyzed against binding buffer, 0.05 M sodium phosphate, pH 8.0, 0.3 M NaCl, prior to purification via the C-terminal His6 tag using Ni2+-nitrilotriacetic acid-agarose (Qiagen). Bound protein was eluted from the Ni2+-nitrilotriacetic acid-agarose with binding buffer containing 100 mM imidazole.

The solECE-1 was initially estimated to be <= 25% pure, as assessed by SDS-polyacrylamide gel electrophoresis and Coomassie staining (data not shown). The concentration of active solECE-1 was further estimated by determining the second order rate of big ET-1 hydrolysis and dividing this rate by the published kcat/Km. Specifically, big ET-1 (0.1 µM) was reacted at 37 °C with solECE-1 in 50 mM MES, pH 6.5, containing 100 mM NaCl, 0.05% bovine serum albumin, 1 mM PMSF, 100 µM leupeptin, and 20 µM pepstatin. The reactions were stopped by the addition of EDTA (5 mM), and mature ET-1 peptide was measured by sandwich ELISA (Amersham Pharmacia Biotech). The second order rate was determined as the apparent rate constant k from Equation 1, 
y=1−e<SUP><UP>−kt</UP></SUP>
where y is the fraction of substrate hydrolyzed and t is the time. Since k = kcat/Km × [E], the calculated k was then divided by the reported kcat/Km for big ET-1 hydrolysis by solECE-1 (2.52 × 104 M-1 s-1 at pH 6.5) (16) to determine the active enzyme concentration. The active enzyme concentration was estimated by this method throughout this report and was ~30-fold lower than that originally estimated from the protein concentration and Coomassie-stained gel, suggesting either that our estimation of purity by Coomassie staining was incorrect or that a majority of the solECE-1 was in an inactive form. To control for the presence of co-purifying native CHO proteins, conditioned serum-free medium from nontransfected CHO cells was purified as above, and the eluted proteins were used in control experiments.

ELISA Analysis of solECE-1-mediated Abeta 40 and Abeta 42 Degradation-- SolECE-1 was preincubated for 15 min at room temperature with the ECE inhibitor PD069185 (synthesized according to published methods (38) by the Organic Chemistry Core Facility at the Mayo Clinic Jacksonville) or phosphoramidon (150 µM) or an equal volume of vehicle (Me2SO and phosphate-buffered saline, respectively), prior to incubation with synthetic Abeta 40 or Abeta 42 (0.01 µM) in 50 mM MES, pH 6.5, containing 0.01% C12E10, 1 mM PMSF, 100 µM leupeptin, and 20 µM pepstatin. The amount of solECE used in this reaction was estimated to be ~6 nM. As a control, Abeta was incubated with Ni2+-nitrilotriacetic acid-purified proteins from nontransfected CHO cells or in reaction buffer alone. Following incubation at 37 °C for the indicated time points, the reactions were stopped by the addition of 5 mM EDTA. Abeta concentration was then analyzed using a highly specific sandwich ELISA, which captures Abeta via binding of antibody BAN50 to the N terminus and detects full-length peptides ending at position 40 (BA27) or 42 (BC05). Thus, Abeta that has been cleaved will not be detected in this assay.

HPLC Analysis of solECE-1-mediated Degradation of 3H-Labeled Abeta 40-- To further analyze the degradation of Abeta by solECE-1, the enzyme was incubated as above with 3H-radiomethylated Abeta 40 (0.56 µM, 2 × 104 dpm), a gift from Dr. T. L. Rosenberry. Abeta peptide and fragments were resolved by reversed-phase HPLC on a Vydac C4 column using a linear gradient of 0-100% B in 60 min (A buffer = 0.1% trifluoroacetic acid in water; B buffer = 0.1% trifluoroacetic acid in acetonitrile). Fractions were collected and counted in a Beckman LS 6500 scintillation counter.

Characterization of Abeta 40 Proteolytic Fragments by HPLC, Mass Spectrometry, and Edman Sequencing-- solECE-1 (~20 nM) was incubated as above with synthetic Abeta 40 (100 µM) for 17 h at 37 °C. The digest samples were run on an Applied Biosystems 130A Separation System (Applied Biosystems, Foster City, CA) using a C4 reversed-phase column (Brownlee Aquapore BU-300; 2.1 × 220 mm). The peptides were eluted with a gradient of 5-80% B over a period of 69 min (A buffer = 0.1% trifluoroacetic acid; B buffer = 80% acetonitrile, 20% water, 0.09% trifluoroacetic acid). The flow rate was 200 µl/min, and absorbance was monitored at 215 nm. Half-minute fractions were collected, and peaks were analyzed by mass spectrometry and Edman sequencing. The masses of the collected peptides were determined with a PerSeptive Biosystems MALDI-TOF Voyager DE-RP Mass Spectrometer (Framingham, MA) operated in the delayed extraction and reflector mode using alpha -cyano-4-hydroxycinnamic acid. The first two amino acids of each peptide were determined using an Applied Biosystems Procise 492 sequencer.

Kinetics of Abeta Hydrolysis by solECE-1-- In an attempt to determine the Km of Abeta 40 hydrolysis by solECE-1, we incubated the enzyme (~17 nM) with synthetic Abeta 40 (3, 10, and 20 µM) for 0 or 6 h at 37 °C in 50 mM MES buffer, pH 6.5, containing 0.05% bovine serum albumin, 1 mM PMSF, 100 µM leupeptin, and 20 µM pepstatin. Control reactions were carried out in the absence of enzyme or in the presence of solECE-1 inhibited by phosphoramidon (100 µM). The reactions were stopped by the addition of EDTA (5 mM), and Abeta 40 concentration was determined by sandwich ELISA using the BAN50/BA27 system. The rate of Abeta hydrolysis in these assays was linear with respect to substrate concentration, precluding a determination of Km and Vmax.

We next determined the second order rate constant, kcat/Km, for Abeta 40 hydrolysis relative to that for big ET-1 hydrolysis by solECE-1. The second order rate for hydrolysis of each substrate was determined at substrate concentrations well below Km. Specifically, Abeta 40 (2.5 µM) and big ET-1 (0.1 µM) were incubated in triplicate alone or with solECE-1 (0.3-8.3 nM) at 37 °C at either pH 6.5 or pH 5.6 in 50 mM MES buffer containing 100 mM NaCl, 0.05% bovine serum albumin, 1 mM PMSF, 100 µM leupeptin, and 20 µM pepstatin. The reactions were stopped by the addition of EDTA (5 mM). Remaining full-length Abeta 40 was determined by BAN50/BA27 sandwich ELISA, and the amount of ET-1 peptide generated was measured by ET-1 sandwich ELISA (Amersham Pharmacia Biotech). The apparent rate constant k was again determined from Equation 1. Under these conditions, no detectable ET-1 peptide was generated in the absence of solECE-1. However, we consistently observed some loss of Abeta 40 in the absence of enzyme, presumably due to adsorption to the reaction tube or other nonspecific mechanism. Therefore, the rate of loss in the absence of enzyme was determined and subtracted from the rate determined in the presence of solECE-1. For subsequent calculations the average k ± S.E. for Abeta 40 hydrolysis at each pH was determined from three experiments, with triplicate reactions in each experiment. For big ET-1 hydrolysis, the k is an average of two separate experiments, with triplicate reactions in each experiment. From the enzyme concentration [E] determined from the k for big ET-1 at pH 6.5 as outlined under Equation 1, we determined the kcat/Km for Abeta 40 hydrolysis at pH 6.5 and 5.6 and for big ET-1 at pH 5.6 with the equation kcat/Km k/[E].

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphoramidon, but Not Thiorphan or Captopril, Increases Abeta Accumulation by H4 Neuroglioma Cells-- Our group and others have previously shown that treatment with metalloprotease inhibitors, in particular phosphoramidon, results in a rapid 2-3-fold increase in the concentration of Abeta 40 and Abeta 42 in the conditioned medium of neuronal cell lines without affecting the concentration of secreted APP (sAPP) (39, 40). Importantly, this increase in Abeta concentration is as large or larger than that seen in cells expressing most AD-causing mutations (2). The enzyme(s) responsible for the phosphoramidon-induced elevations in Abeta has not previously been reported. Phosphoramidon is known to inhibit several metalloproteases including NEP (IC50 = 0.034 µM), angiotensin-converting enzyme (ACE; IC50 = 78 µM), ECE-1 (IC50 = 1-3.5 µM), and ECE-2 (IC50 = 0.004 µM) (29, 41) but does not inhibit insulin-degrading enzyme (42). A role for NEP in extracellular Abeta catabolism has been highlighted in a recent report from Iwata and colleagues (9). Infusion of the metalloprotease inhibitor thiorphan into the hippocampus of rats resulted in a significant increase in the amount of Abeta and in the deposition of the longer more amyloidogenic form, Abeta 42, reportedly through the inhibition of Abeta degradation by NEP. NEP and ACE have been reported to reside predominantly on the cell surface, although a soluble form of NEP is also present in serum and cerebral spinal fluid (43-46). A recently identified thiorphan-sensitive NEP homologue SEP/NL1/NEPII is expressed both as a membrane-bound and secreted protease (47-49). We evaluated a role for NEP and ACE in H4 neuroglioma cells using more selective inhibitors of these enzymes, thiorphan and captopril, respectively. We have not yet been able to similarly analyze ECE, since a more selective inhibitor of ECE is not commercially available.

Treatment of H4 cells with phosphoramidon (34 µM) resulted in a greater than 2-fold elevation in Abeta 40 accumulation (Fig. 1), with a half-maximal effect occurring at a dose of ~7.5 µM (data not shown). However, treatment with thiorphan or captopril at concentrations greater than 1000 times the reported IC50 for the target enzymes in in vitro studies (50, 51), but less than that required to inhibit ECE, failed to result in increases in extracellular Abeta (Fig. 1), indicating that the phosphoramidon-induced effect in H4 cells is not likely to be due to inhibition of NEP or ACE. Similar results were obtained for Abeta 42 (data not shown). Since NEP and ACE are localized mainly on the cell surface, the membrane permeability of these compounds is not relevant to the inhibition of the known protease, although we cannot rule out the possible presence of an intracellular form of NEP or ACE based on these results. Similarly, we cannot rule out the possibility that the phosphoramidon-induced effect in these cells is due to an as yet unidentified enzyme that is insensitive to treatment with thiorphan and captopril. We did, however, find endogenous ECE activity in solubilized membranes of H4 cells using a big ET conversion assay (17, 36) (data not shown). Collectively, these data led us to investigate ECE more closely.


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Fig. 1.   Effect of metalloprotease inhibitors on Abeta concentration in the culture medium of H4 cells. Confluent wells of H4 cells were treated with phosphoramidon, captopril (selective inhibitor of ACE), or thiorphan (selective inhibitor of NEP) for 24 h at the indicated concentrations. Abeta 40 concentration in the conditioned medium was determined using the BAN50/BA27 sandwich ELISA. Data are plotted as mean ± S.E. of triplicate wells. MTS cell proliferation assays on the treated cells did not reveal cellular toxicity at any of the doses.

Overexpression of Endothelin-converting Enzyme-1 Results in a Significant Decrease in Extracellular Abeta Concentration That Is Completely Reversed by Treatment with Phosphoramidon-- Evidence implicating a potential role for ECE in modulating Abeta concentration came further from the casual observation that CHO cells, which have no endogenous ECE activity (17), produce very high levels of Abeta when compared with most other cell types2 and fail to respond to phosphoramidon (see Fig. 2). Conversely, HUVECs, which have high levels of endogenous ECE (52), accumulate very little Abeta unless treated with high concentrations of phosphoramidon (data not shown). To further investigate the role of ECE in Abeta accumulation, we cloned and stably transfected CHO cells with human ECE-1b and ECE-1a. ECE activity, determined using a big ET-1 conversion assay (17, 36), was confirmed to be present in solubilized membranes from the ECE-1-transfected cells and absent in vector-transfected cells (data not shown). The amount of ECE-1 activity in each of our stable ECE-1 lines was similar. Overexpression of either ECE-1a or ECE-1b in CHO cells, which lack endogenous ECE activity, resulted in a striking 75-90% reduction in Abeta 40 and a 45-60% reduction in Abeta 42 (Fig. 2). No significant changes were observed in the amount of sAPP accumulation in ECE-1-transfected cells compared with the vector controls, indicating that the cells were similarly viable and that general secretion is not affected by ECE-1 overexpression. The reduction in Abeta concentration in ECE-1a- and ECE-1b-transfected cells was completely reversed by treatment with phosphoramidon, indicating that the observed phenotype was probably due to the enzymatic activity of the overexpressed ECE-1.


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Fig. 2.   Overexpression of ECE-1a or ECE-1b in CHO cells reduces extracellular Abeta concentration without affecting secretion of sAPP. Abeta 40 (A) and Abeta 42 (B) concentration in the conditioned media of stable ECE-1a- and ECE-1b-transfected CHO cell lines was determined by sandwich ELISA (BNT77/BA27 and BNT77/BC05, respectively) following a 24-h incubation with or without 100 µM phosphoramidon. Data are plotted as mean ± S.E. of triplicate wells. Western blot analysis (C) was also performed on the conditioned media of cells incubated with or without phosphoramidon (phos), using 22C11 antibody to detect sAPP.

Increased Removal of Exogenous Abeta Is Apparent Only in ECE-1a-transfected Cells-- While the exact mechanism of the phosphoramidon-induced increase in Abeta concentration in neuronal cells was unknown, it has been suggested that it may be the result of inhibition of intracellular degradation of the peptide (40). Consistent with this hypothesis, treatment with phosphoramidon is reported to result in a 2-fold increase in extracellular Abeta concentration and an increase in cell-associated Abeta without affecting sAPP levels in SY5Y cells treated with the compound (40). As we have shown, treatment of H4 neuroglioma cells with phosphoramidon also results in a significant increase in extracellular Abeta concentration (see Fig. 1). To examine whether the phosphoramidon-induced increases in Abeta were likely to be due to inhibition of a cell-surface or secreted protease, we performed a spike experiment in which exogenous Abeta was added to the medium bathing H4 cells in the absence or presence of phosphoramidon. As shown in Fig. 3, exogenous Abeta 42 was removed equally well in the presence or absence of phosphoramidon. The inset shows the results from a sister set of culture wells where synthetic Abeta was not added, indicating that phosphoramidon was indeed promoting endogenous Abeta accumulation in this experiment. Similar data were obtained using synthetic Abeta 40 (data not shown).


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Fig. 3.   Effect of phosphoramidon on removal of exogenous Abeta by H4 cells. Synthetic Abeta 42 (150 pM) was added to confluent H4 cells in the presence (closed symbols) or absence (open symbols) of 34 µM phosphoramidon and incubated for 6 and 24 h. A second set of control H4 cells was incubated with or without phosphoramidon for the same time period. Abeta 42 was measured in both sets at the indicated time points using the BAN50/BA27 sandwich ELISA, and remaining synthetic Abeta concentration was determined by subtracting values obtained from control (endogenous) cells from those obtained from the cells incubated with spiked-in Abeta . The inset graph shows the accumulation of endogenous Abeta 42 in the conditioned medium of H4 cells after 24 h of treatment with phosphoramidon. Data are plotted as mean ± S.E. of triplicate wells.

To determine whether extracellular Abeta removal could account, at least in part, for the dramatic decrease in extracellular Abeta concentration in the ECE-1-transfected cell lines, we next spiked synthetic Abeta 40 into the culture medium in the presence or absence of phosphoramidon and determined the percentage of removal by sandwich ELISA at 6 and 24 h. After a 6-h incubation, removal of Abeta was similar in the culture medium of vector- and ECE-1-transfected CHO cells (Fig. 4A) and was not affected by phosphoramidon treatment (data not shown), although endogenous Abeta accumulation by phosphoramidon-treated ECE-1-transfected cells was increased 1.5-2-fold during the same time period (Fig. 4B).


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Fig. 4.   Removal of exogenous synthetic Abeta by ECE-overexpressing CHO cells. A, synthetic human Abeta 40 (150 pM) was added to confluent CHO cells stably transfected with ECE-1a, ECE-1b, or the control vector and incubated for 6 and 24 h. Human Abeta 40 was measured at the indicated time points using the BAN50/BA27 sandwich ELISA, which does not detect endogenous CHO Abeta . Data shown represent the mean ± S.E. of triplicate wells that were incubated with synthetic Abeta 40. The concentration of Abeta remaining after 24 h is significantly lower in ECE-1a cells than in vector controls (p = 0.0495, Mann-Whitney). The inset graph shows the percentage of exogenous Abeta removed by ECE-1a and vector-transfected cells after 24 h in the presence of phosphoramidon (34 µM). B, a second set of cells was incubated with or without phosphoramidon (34 µM) for the same time period to determine the accumulation of endogenous Abeta . Endogenous Abeta 40 was measured at the indicated time points using the BNT77/BA27 sandwich ELISA. Data are plotted as mean ± S.E. of triplicate wells. Given that BNT77 was raised against amino acids 11-28 of Abeta , this assay can also detect amino truncated peptides and may lead to an overestimation of full-length Abeta 40.

Following a 24-h incubation, we did observe a significant increase (p = 0.0495) in the removal of the spiked-in Abeta in the medium of ECE-1a-transfected cells compared with the vector controls (Fig. 4A). No significant change in exogenous Abeta removal was observed in cells expressing ECE-1b. The ECE-1a-induced increase in Abeta removal could be completely attenuated by phosphoramidon treatment, indicating that the effect was probably due to the enzymatic activity of ECE-1a (Fig. 4A, inset). In the same ECE-1a cells, phosphoramidon treatment resulted in an ~600% increase in endogenous Abeta accumulation at the 24-h time point (Fig. 4B).

Partially Purified solECE-1 Degrades Abeta in Vitro-- Recombinant, soluble forms of ECE-1 (solECE-1) lacking the intracellular and transmembrane domains have been reported to hydrolyze big ET-1 with activity comparable with that of membrane-bound ECE-1a (16, 36, 37). The soluble ECE-1 preparation described by Korth et al. (37) was active in a broad pH range from 5 to 7, with an optimum of pH 6.6-6.8 for big ET-1. Under the conditions assayed, Ahn et al. (36, 53) found that their soluble ECE-1 preparation had a somewhat narrower pH optimum, with an optimal pH for big ET-1 of 6.5. To examine whether ECE-1 is capable of direct catabolism of Abeta , we generated a soluble ECE-1 similar to those previously described. Incubation of synthetic Abeta 40 and Abeta 42 with this enzyme resulted in a nearly complete loss of the full-length peptides as detected by sandwich ELISA (Fig. 5). This reduction was completely blocked by incubation with phosphoramidon and also with a more selective ECE-1 inhibitor, PD069185. (This inhibitor, while very useful for in vitro studies, is not informative in cell-based studies due to its toxicity (38)). To confirm that the loss of Abeta was indeed due to Abeta catabolism and not to Abeta binding or some other phenomenon, we analyzed the effect of solECE-1 on Abeta by HPLC with a radiolabeled Abeta reporter molecule. Incubation of 3H-labeled Abeta 40 with solECE-1 resulted in loss of the full-length peptide and formation of at least three novel peaks detected by reversed-phase chromatography (Fig. 6). The formation of these peaks was completely blocked by treatment with PD069185.


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Fig. 5.   Degradation of synthetic Abeta 40 and Abeta 42 by soluble ECE-1. Soluble ECE-1 (~6 nM), partially purified from transfected CHO cell medium, was incubated for 24 h at 37 °C with 0.01 µM Abeta 40 (A) or Abeta 42 (B). As a control, the enzyme was preincubated for 15 min with the ECE inhibitor PD069185 (150 µM) or phosphoramidon (150 µM) prior to the addition of Abeta 40 or Abeta 42, respectively. Closed bars, ECE inhibitor; open bars, no inhibitor. Identical reactions were carried out with co-purifying proteins isolated from nontransfected CHO cells (Control). After the incubation, the remaining Abeta was detected using the BAN50/BA27 and BAN50/BC05 sandwich ELISA systems, which detect full-length Abeta 40 or Abeta 42 peptides, respectively. Data are plotted as mean ± S.E. of triplicate reactions.


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Fig. 6.   HPLC analysis of 3H-labeled Abeta 40 degradation by soluble ECE-1. Soluble ECE-1 (~6 nM) was incubated for 24 h at 37 °C with 3H-labeled Abeta 40 (0.56 µM, 2 × 104 dpm). As a control, the enzyme was preincubated for 15 min with the ECE inhibitor PD069185 (150 µM) prior to the addition of Abeta . The resulting peptides were separated by reversed-phase chromatography (C4 column). 1-min fractions were collected, and 3H dpm was determined by liquid scintillation counting. For comparative purposes, synthetic Abeta alone elutes as a single peak at fraction 21, identical to that observed for Abeta incubated with solECE-1 in the presence of PD069185 (data not shown).

Determination of Cleavage Sites of Abeta 40 by solECE-1-- Since ECE-1 has been shown to cleave a number of biologically active peptides on the amino side of hydrophobic residues (16), there are multiple potential ECE-1 cleavage sites within the Abeta peptide. To determine the sites of Abeta 40 cleavage, synthetic unlabeled peptide was digested with solECE-1, and the cleavage products were separated by reversed-phase HPLC. Digestion of Abeta 40 in this experiment resulted in the formation of four major product peaks, similar to that which was observed with the 3H-labeled peptide. The major peaks were further analyzed by mass spectrometry and NH2-terminal sequencing, leading to the identification of three NH2-terminal fragments: Abeta -(1-16), Abeta -(1-17), and Abeta -(1-19). Only one C-terminal fragment was observed, which corresponds to Abeta -(20-40) (Fig. 7 and Table I).


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Fig. 7.   Determination of sites of Abeta 40 cleavage by solECE-1. A, unlabeled synthetic Abeta 40 (100 µM) was digested with solECE-1 (~20 nM) overnight at 37 °C. The cleavage products were separated by reversed-phase HPLC using a C4 column. Half-minute fractions were collected, and absorbance was monitored at 215 nm. B, amino acid sequence of Abeta 40 showing principal solECE-1 cleavage sites. The identities of HPLC peaks 1-5 were determined by mass spectrometry and NH2-terminal sequencing (see Table I). The open arrows under the sequence indicate previously determined NEP cleavage sites (8).

                              
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Table I
Identification of cleavage sites of Abeta 40 by solECE-1
Synthetic Abeta 40 was digested with solECE-1, and the resulting peptides were separated by HPLC as shown in Fig. 7A. Peaks 1-5 were collected and further analyzed by mass spectrometry and NH2-terminal sequencing.

Kinetic Analysis of Abeta 40 Cleavage by solECE-1-- SolECE-1 has been reported to hydrolyze big ET-1 with a Km of ~2-4 µM (16, 36, 37) and bradykinin with a Km of 340 µM (16). Despite a much higher Km, the catalytic efficiency of bradykinin hydrolysis by solECE-1 actually exceeds that of big ET-1, with a second order rate constant of 6.6 × 104 M-1 s-1 for bradykinin compared with 2.5 × 104 M-1 s-1 for big ET-1 at pH 6.5 (16). To attempt to determine the Km for Abeta 40 hydrolysis by solECE-1, we initially examined the rate of hydrolysis of Abeta at various substrate concentrations up to 20 µM at the reported pH optimum for big ET-1 cleavage. The rate of Abeta hydrolysis was linear with respect to substrate concentration up to 20 µM (data not shown). We were concerned that the use of significantly higher concentrations of Abeta in these experiments would complicate the kinetic measurements, since Abeta 40 peptide is prone to aggregate and precipitate in the high micromolar range. Thus, we were unable to determine the Km and Vmax. We were able, however, to calculate the kcat/Km by measuring the rate of Abeta hydrolysis by solECE under second-order conditions. Under these conditions, when the substrate concentration is well below Km, the rate of substrate hydrolysis is equal to the kcat/Km multiplied by the enzyme concentration (see "Experimental Procedures"). Using this method, the kcat/Km for Abeta 40 hydrolysis by solECE-1 was determined to be (1.7 ± 0.6) × 103 M-1 s-1 at pH 6.5. This value is 15-fold lower than that for big ET-1 hydrolysis under the same conditions.

Intrigued by a recent report indicating that solECE-1 cleaves bradykinin and substance P with an acidic pH optimum of ~5.6 compared with the optimum of pH 6.5 for big ET-1 (53), we next compared the efficiency of solECE-1 hydrolysis of Abeta 40 and big ET-1 at pH 5.6. Similar to bradykinin and substance P, solECE-1 cleaves Abeta 40 more efficiently at pH 5.6, with a kcat/Km determined to be (2.0 ± 0.5) × 104 M-1 s-1, ~12 times greater than the value determined at pH 6.5. The kcat/Km for big ET-1 hydrolysis by our preparation of solECE-1 at pH 5.6 under these conditions was determined to be ~6.1 × 104 M-1 s-1 at pH 5.6, slightly greater than that determined at pH 6.5. This value of kcat/Km for big ET-1 hydrolysis at pH 5.6 is only 3-fold greater than that for Abeta 40 at the same pH. It is important to note that the kcat/Km values reported here rely on a determination of enzyme concentration based on the reported kcat/Km of big ET-1 hydrolysis by solECE-1 at pH 6.5 (see "Experimental Procedures"), and thus the absolute values may differ based on any differences in actual enzyme concentration from our estimates. However, the relative kcat/Km for Abeta 40 hydrolysis versus big ET-1 hydrolysis was determined in parallel experiments with the same enzyme and would not change even if the estimated concentration of enzyme were slightly different.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recently, there has been considerable debate over the enzyme or enzymes that contribute most to Abeta catabolism in the human brain. Both insulin-degrading enzyme and neprilysin have been argued to be major proteases involved in the degradation of secreted Abeta (5, 9). However, with the data available at this time, it is impossible to determine which, if any, of the identified proteases contributes most to Abeta degradation in the intact brain. It is likely that multiple proteases, both intracellular and extracellular, may play a role in determining Abeta concentration. The relative contribution of Abeta -degrading enzymes and other mechanisms of Abeta removal may vary in different regions of the brain and may also differ for Abeta 40 and Abeta 42. A decrease in the activity of any of these mechanisms, whether they are major or minor, may potentially result in increased Abeta accumulation and the development of AD pathology. Conversely, an increase in the activity of any enzyme capable of degrading Abeta may result in decreased accumulation of the peptide, potentially reducing the risk for AD.

Taken together, the data presented in this report indicate that ECE-1 activity can dramatically affect Abeta concentration, probably by direct degradation of the peptide. Recombinant soluble ECE-1 has a substrate specificity in non-ET peptides similar to that of NEP, with preferential cleavage on the amino side of hydrophobic residues (16). In contrast to NEP, however, ECE-1 appears not to cleave peptides smaller than 6 amino acids in length. Given this specificity, there are ~13 potential ECE/NEP cleavage sites in the Abeta 40 peptide. At least five of these sites are reported to be cleaved by recombinant NEP in vitro (8). Using HPLC, mass spectrometry, and NH2-sequence analysis, we have determined that soluble ECE-1 cleaves synthetic Abeta 40 at least three sites, resulting in the formation of Abeta fragments 1-16, 1-17, 1-19, and 20-40. Consistent with the known substrate specificity of ECE-1, each of these observed cleavages by solECE-1 occurred on the amino side of hydrophobic residues (Leu17, Val18, and Phe20). Given that ECE-2 is highly homologous to ECE-1 and shares similar catalytic activity (29), we hypothesize that ECE-2 may also be capable of degrading Abeta . Since ECE-2 is abundantly expressed in the central nervous system, this activity may also potentially be relevant to the accumulation of Abeta in the brain.

ECE-1 activity is localized both intracellularly and on the cell surface in many cell types, with ECE-2 appearing to reside exclusively within the cell. In CHO cells overexpressing human ECE-1a, we observed increased phosphoramidon-sensitive removal of exogenous Abeta after 24 h of incubation, indicating that ECE-1a may also contribute slightly to the extracellular degradation of the peptide. In CHO cells overexpressing ECE-1b and in H4 neuroglioma cells expressing endogenous ECE, degradation of exogenous Abeta was not sensitive to phosphoramidon. These results raise the possibility that the dramatic increase in Abeta accumulation by these cells in the presence of phosphoramidon may be due to inhibition of intracellular degradation of the peptide. Even in ECE-1a-expressing CHO cells, the dramatic increase in Abeta concentration upon treatment with phosphoramidon does not appear to be accounted for by the modest increase in exogenous Abeta degradation. We cannot, however, rule out the possibility of a local event at the cell surface upon secretion of endogenous Abeta that might not be evident in our spike experiments, where the peptide is diluted directly into the culture medium. While ECE-1a has been reported to be localized predominantly to the cell surface, this isoform has been shown to process big ET-1 intracellularly in CHO cells, most likely in secretory vesicles, as well as at the cell surface (54). Therefore, ECE-1a may similarly degrade Abeta intracellularly in CHO cells as it is being trafficked to the cell surface.

While detailed co-localization studies have not been performed, separate studies indicate that ECE and Abeta are present in the same cellular compartments. Human ECE-1b has been reported to be present in the TGN, a proposed site of Abeta 40 generation in neuronal cells (24, 55). Interestingly, we found that solECE-1 hydrolyzed Abeta 40 more efficiently at pH 5.6 than at pH 6.5, with a kcat/Km at pH 5.6 only 3-fold lower than that for a known physiological substrate, big ET-1. This result may be particularly relevant, since the TGN, where ECE-1b appears to be expressed, has an acidic pH. ECE-2 is also likely to be present in the TGN and vesicles of the constitutive secretory pathway (29, 31). Consistent with the hypothesis that phosphoramidon may inhibit intracellular degradation of Abeta , Fuller et al. (40) have reported that phosphoramidon treatment of SY5Y neuronal cells results in an increase in cell-associated Abeta . Unfortunately, we have not been able to convincingly detect intracellular Abeta in H4 cells either in the presence or absence of phosphoramidon, presumably due to rapid secretion of the peptide. However, the fact that removal of exogenous Abeta by H4 cells is insensitive to phosphoramidon suggests that the compound may exert its effect on Abeta through an intracellular event.

Phosphoramidon itself has a polar structure that would appear to make it poorly cell-penetrant (56). However, this compound has been shown to be able to inhibit the intracellular conversion of big ET-1 to mature endothelin (17, 29, 57). It is possible that phosphoramidon may elevate Abeta accumulation through inhibition of intracellular ECE, cell surface ECE, or both, depending on the expression of cell surface and intracellular ECE in various cell types.

The 2-3-fold increase in endogenous Abeta accumulation by cell lines treated with phosphoramidon is as large as or larger than that seen in cells expressing most AD-causing mutations (2), suggesting that disruption of phosphoramidon-sensitive Abeta degradation may be a significant risk factor for AD. While many factors in addition to ECE probably contribute to endothelin levels, and the results are controversial, it is noteworthy that decreases in endothelin levels have been reported in the cerebral spinal fluid of AD patients when compared with nondemented control individuals (58). A reasonable test for the degree of involvement of ECE or any protease in the brain is to examine the effect of animals null for these enzymes. ECE-1 knockout mice have craniofacial and cardiac abnormalities resulting in embryonic lethality, while ECE-2 knockout mice develop normally and are viable (32, 59). Surprisingly, large amounts of mature ET-1 peptide are detected in both lines. In ECE-1/ECE-2 double knockout mice, the cardiac defects are even more severe; however, mature ET-1 peptides are still detected, indicating compensation by as yet unidentified proteases (32, 59). The apparent redundancy of endothelin-converting enzyme activity may limit the utility of the knockouts to examine the contribution of ECE in Abeta accumulation in vivo. Nonetheless, these experiments are under way and may provide additional insight into the role of ECE in endogenous Abeta accumulation in the brain.

Regardless of the extent of the in vivo role of ECE activity in the amount of Abeta accumulation in the brain, these results are highly significant for several reasons. First, ECE inhibitors have received a large amount of pharmaceutical interest for their potential as anti-hypertension drugs (60). If degradation of Abeta by ECE does, as we hypothesize, contribute to the extracellular concentration of the plaque-forming peptide in the brain, inhibiting this activity may lead to the development of AD and/or accelerate the disease in susceptible individuals. Because most ECE and NEP inhibitors will inhibit both enzymes at certain concentrations, the use of these drugs may be particularly risky if ECE and NEP both play physiological roles in the degradation of Abeta in the brain. ET receptors may be a safer target for pharmacological interference with the endothelin system to reduce hypertension without the side effect of decreased Abeta catabolism.

Second, up-regulation of ECE activity may be useful therapeutically for the treatment of AD. One obvious concern with this treatment method is that patients may become hypertensive. Up-regulation of ECE activity in the periphery of mice injected with a construct to increase ECE expression, however, does not appear to result in increased circulating endothelin levels, indicating that ECE is not likely to be rate-limiting in the conversion of big ET to ET (61). Further, even if increased ECE activity does augment endothelin levels, endothelin receptor antagonists could be given in parallel to reduce or block any effect of increased endothelin levels.

Third, mutations in ECE may be identified that are causative of Alzheimer's disease in certain individuals. In this regard, it is worthwhile to note that the sib-pair analyses of genetic factors contributing to late onset AD have not excluded the region on chromosome 1 where the ECE-1 gene is located (62). Equally important, however, is the possibility that there may be individuals with normally high levels of ECE activity who are at a reduced risk for the disease. A careful analysis of ECE activity in AD and control individuals is necessary to determine the extent of the involvement of this enzyme family in the development of AD.

    ACKNOWLEDGEMENTS

We thank Dr. Steven Younkin for continued support and critical evaluation of the manuscript. We thank Dr. Terrone Rosenberry for help with kinetic analyses and for critical evaluation of the manuscript. We also thank Debra Yager and Dr. Cristian-Mihail Prada for expert technical assistance, Takeda Industries for anti-Abeta antibodies, Dr. Abdul Fauq for synthesis of PD069185, and Dr. Kumar Sambamurti for critical evaluation of the manuscript. We thank Dr. Masashi Yanagisawa for helpful discussions on the localization of ECE-1 isoforms.

    FOOTNOTES

* This work was supported by a Smith Fellowship (to C. E.), by a Bursak fellowship (to E. E.), and by the Mayo Foundation for Medical Education and Research.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.

Dagger To whom correspondence should be addressed: Mayo Clinic Jacksonville, Birdsall Bldg. Rm. 253, 4500 San Pablo Rd., Jacksonville, FL 32224. Tel.: 904-953-2979; Fax: 904-953-7370; E-mail: Eckman@mayo.edu.

Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M007579200

2 C. B. Eckman, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , beta -amyloid; ECE, endothelin-converting enzyme; CHO, Chinese hamster ovary; APP, amyloid precursor protein; beta APP, beta -amyloid precursor protein; sAPP, secreted amyloid precursor protein; NEP, neprilysin; ET, endothelin; TGN, trans-Golgi network; HUVEC, human umbilical vein endothelial cell; solECE-1, soluble ECE-1; ACE, angiotensin-converting enzyme; ELISA, enzyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl fluoride; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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