From the Mayo Clinic Jacksonville, Jacksonville, Florida 32224
Received for publication, August 19, 2000, and in revised form, May 2, 2001
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ABSTRACT |
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Deposition of Alzheimer's disease
(AD)1 is the most common
cause of dementia in the elderly and is characterized pathologically by
the accumulation of A Recent reports suggest a role for both insulin-degrading enzyme
and neprilysin (NEP) in the degradation of extracellular A 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 A Analysis of A 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
A 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 ELISA Analysis of solECE-1-mediated A HPLC Analysis of solECE-1-mediated Degradation of
3H-Labeled A Characterization of A Kinetics of A
We next determined the second order rate constant,
kcat/Km, for A Phosphoramidon, but Not Thiorphan or Captopril, Increases A
Treatment of H4 cells with phosphoramidon (34 µM)
resulted in a greater than 2-fold elevation in A Overexpression of Endothelin-converting Enzyme-1 Results in a
Significant Decrease in Extracellular A Increased Removal of Exogenous A
To determine whether extracellular A
Following a 24-h incubation, we did observe a significant increase
(p = 0.0495) in the removal of the spiked-in A Partially Purified solECE-1 Degrades A Determination of Cleavage Sites of A Kinetic Analysis of A
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 A Recently, there has been considerable debate over the enzyme or
enzymes that contribute most to A Taken together, the data presented in this report indicate that ECE-1
activity can dramatically affect A 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 A While detailed co-localization studies have not been performed,
separate studies indicate that ECE and A 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
A The 2-3-fold increase in endogenous A Regardless of the extent of the in vivo role of ECE activity
in the amount of A 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.
-amyloid (A
) 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 A
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 A
.
Much less, however, is known about the mechanisms responsible for A
removal in the brain. In this report, we describe the identification of
endothelin-converting enzyme-1 (ECE-1) as a novel A
-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 A
concentration that appears to be due to
inhibition of intracellular A
degradation. Furthermore, we show that
overexpression of ECE-1 in Chinese hamster ovary cells, which lack
endogenous ECE activity, reduces extracellular A
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 A
40 and A
42 in
vitro at multiple sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid peptides (A
) in the brain in the
form of senile plaques. A
is normally produced from the
-amyloid
precursor protein (
APP) through the combined proteolytic actions of
- and
-secretase and is then secreted into the extracellular
milieu (1, 2). The degree of A
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 A
, much less is known
regarding A
catabolism.
catabolism is likely to involve proteases at multiple sites, both
intracellular and extracellular. Proteases acting at the site of A
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 A
may be further regulated
by direct degradation by extracellular proteases and by
receptor-mediated endocytosis or phagocytosis followed by lysosomal
degradation. Catabolism of A
peptides at each of these steps would
limit the accumulation of extracellular A
, and disruption of this
catabolism may be a risk factor for AD. Additionally, the
identification of enzymes that degrade A
intracellularly and
extracellularly may lead to development of novel therapeutics aimed at
reducing A
concentration by enhancing its removal.
(3-10).
Matrix metalloproteinase-9, EC 3.4.24.15, and
2-macroglobulin complexes have also been reported to
play a role in A
degradation (11-13). In this report, we describe
the identification of endothelin-converting enzyme-1 (ECE-1) as a novel
A
-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).
both in
vitro and in vivo. These data indicate a potential role
for this enzyme family in A
catabolism.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Concentration by Sandwich ELISA--
Human A
was measured by sandwich ELISA as previously described (34), using the
BAN50/BA27 and BAN50/BC05 antibody systems (Takeda) to detect A
40
and A
42, respectively. Hamster A
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).
A
concentration was determined by comparing values obtained for
samples with those obtained for synthetic A
40 and A
42 standards (Bachem).
40 and A
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.
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,
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.
40 and A
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 A
40 or
A
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, A
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. A
concentration was then analyzed using a highly specific sandwich ELISA,
which captures A
via binding of antibody BAN50 to the N terminus and
detects full-length peptides ending at position 40 (BA27) or 42 (BC05). Thus, A
that has been cleaved will not be detected in this assay.
40--
To further analyze the degradation
of A
by solECE-1, the enzyme was incubated as above with
3H-radiomethylated A
40 (0.56 µM, 2 × 104 dpm), a gift from Dr. T. L. Rosenberry. A
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.
40 Proteolytic Fragments by HPLC, Mass
Spectrometry, and Edman Sequencing--
solECE-1 (~20
nM) was incubated as above with synthetic A
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
-cyano-4-hydroxycinnamic acid. The first two amino acids of
each peptide were determined using an Applied Biosystems Procise 492 sequencer.
Hydrolysis by solECE-1--
In an attempt to
determine the Km of A
40 hydrolysis by solECE-1,
we incubated the enzyme (~17 nM) with synthetic A
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 A
40 concentration was
determined by sandwich ELISA using the BAN50/BA27 system. The rate of
A
hydrolysis in these assays was linear with respect to substrate concentration, precluding a determination of Km and
Vmax.
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, A
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 A
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 A
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 A
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
A
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
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 A
40 and A
42 in the conditioned medium of
neuronal cell lines without affecting the concentration of secreted APP
(sAPP) (39, 40). Importantly, this increase in A
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 A
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
A
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 A
and in the deposition of the longer more amyloidogenic
form, A
42, reportedly through the inhibition of A
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.
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
A
(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 A
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
A 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. A
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.
Concentration That Is
Completely Reversed by Treatment with Phosphoramidon--
Evidence
implicating a potential role for ECE in modulating A
concentration
came further from the casual observation that CHO cells, which have no
endogenous ECE activity (17), produce very high levels of A
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 A
unless treated with high concentrations of phosphoramidon (data not shown). To further investigate the role of ECE in A
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 A
40 and a 45-60% reduction in A
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 A
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.
View larger version (20K):
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Fig. 2.
Overexpression of ECE-1a or ECE-1b in CHO
cells reduces extracellular A concentration
without affecting secretion of sAPP. A
40 (A) and
A
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.
Is Apparent Only in
ECE-1a-transfected Cells--
While the exact mechanism of the
phosphoramidon-induced increase in A
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 A
concentration and an
increase in cell-associated A
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 A
concentration (see Fig. 1). To examine
whether the phosphoramidon-induced increases in A
were likely to be
due to inhibition of a cell-surface or secreted protease, we performed
a spike experiment in which exogenous A
was added to the medium
bathing H4 cells in the absence or presence of phosphoramidon. As shown
in Fig. 3, exogenous A
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 A
was not added, indicating that phosphoramidon was indeed promoting endogenous A
accumulation in this experiment. Similar data were obtained using synthetic A
40 (data not shown).
View larger version (11K):
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Fig. 3.
Effect of phosphoramidon on removal of
exogenous A by H4 cells. Synthetic A
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. A
42 was
measured in both sets at the indicated time points using the BAN50/BA27
sandwich ELISA, and remaining synthetic A
concentration was
determined by subtracting values obtained from control (endogenous)
cells from those obtained from the cells incubated with spiked-in A
.
The inset graph shows the accumulation of
endogenous A
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.
removal could account, at least
in part, for the dramatic decrease in extracellular A
concentration
in the ECE-1-transfected cell lines, we next spiked synthetic A
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 A
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 A
accumulation by phosphoramidon-treated ECE-1-transfected cells was
increased 1.5-2-fold during the same time period (Fig.
4B).
View larger version (16K):
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Fig. 4.
Removal of exogenous synthetic
A by ECE-overexpressing CHO cells.
A, synthetic human A
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 A
40 was
measured at the indicated time points using the BAN50/BA27 sandwich
ELISA, which does not detect endogenous CHO A
. Data shown represent
the mean ± S.E. of triplicate wells that were incubated with
synthetic A
40. The concentration of A
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 A
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 A
. Endogenous A
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 A
, this assay can also detect
amino truncated peptides and may lead to an overestimation of
full-length A
40.
in
the medium of ECE-1a-transfected cells compared with the vector
controls (Fig. 4A). No significant change in exogenous A
removal was observed in cells expressing ECE-1b. The ECE-1a-induced
increase in A
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 A
accumulation at the
24-h time point (Fig. 4B).
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 A
,
we generated a soluble ECE-1 similar to those previously described.
Incubation of synthetic A
40 and A
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 A
was indeed due to A
catabolism and not to A
binding or
some other phenomenon, we analyzed the effect of solECE-1 on A
by
HPLC with a radiolabeled A
reporter molecule. Incubation of
3H-labeled A
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.
View larger version (13K):
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Fig. 5.
Degradation of synthetic
A 40 and A
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 A
40 (A)
or A
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 A
40
or A
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 A
was detected using the BAN50/BA27 and BAN50/BC05
sandwich ELISA systems, which detect full-length A
40 or A
42
peptides, respectively. Data are plotted as mean ± S.E. of
triplicate reactions.
View larger version (13K):
[in a new window]
Fig. 6.
HPLC analysis of 3H-labeled
A 40 degradation by soluble ECE-1. Soluble
ECE-1 (~6 nM) was incubated for 24 h at 37 °C
with 3H-labeled A
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 A
. 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 A
alone
elutes as a single peak at fraction 21, identical to that observed for
A
incubated with solECE-1 in the presence of PD069185 (data not
shown).
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 A
peptide. To determine
the sites of A
40 cleavage, synthetic unlabeled peptide was digested
with solECE-1, and the cleavage products were separated by
reversed-phase HPLC. Digestion of A
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: A
-(1-16), A
-(1-17),
and A
-(1-19). Only one C-terminal fragment was observed, which
corresponds to A
-(20-40) (Fig. 7 and
Table I).
View larger version (15K):
[in a new window]
Fig. 7.
Determination of sites of
A 40 cleavage by solECE-1. A,
unlabeled synthetic A
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 A
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).
Identification of cleavage sites of A40 by solECE-1
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.
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 A
40 hydrolysis by solECE-1, we initially
examined the rate of hydrolysis of A
at various substrate
concentrations up to 20 µM at the reported pH optimum for
big ET-1 cleavage. The rate of A
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 A
in these experiments would complicate the kinetic measurements,
since A
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 A
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
A
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.
40 and big ET-1 at pH 5.6. Similar to bradykinin and substance P, solECE-1 cleaves A
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 A
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 A
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
catabolism in the human brain.
Both insulin-degrading enzyme and neprilysin have been argued to be
major proteases involved in the degradation of secreted A
(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 A
degradation in the intact brain. It is likely that multiple
proteases, both intracellular and extracellular, may play a role in
determining A
concentration. The relative contribution of
A
-degrading enzymes and other mechanisms of A
removal may vary in
different regions of the brain and may also differ for A
40 and
A
42. A decrease in the activity of any of these mechanisms, whether
they are major or minor, may potentially result in increased A
accumulation and the development of AD pathology. Conversely, an
increase in the activity of any enzyme capable of degrading A
may
result in decreased accumulation of the peptide, potentially reducing
the risk for AD.
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 A
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 A
40 at least three sites, resulting in the
formation of A
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 A
. Since ECE-2 is abundantly expressed in the central
nervous system, this activity may also potentially be relevant to the
accumulation of A
in the brain.
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 A
was not sensitive to phosphoramidon.
These results raise the possibility that the dramatic increase in A
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 A
concentration upon treatment with phosphoramidon does not appear to be
accounted for by the modest increase in exogenous A
degradation. We
cannot, however, rule out the possibility of a local event at the cell
surface upon secretion of endogenous A
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 A
intracellularly in CHO cells as it is being
trafficked to the cell surface.
are present in the same
cellular compartments. Human ECE-1b has been reported to be present in
the TGN, a proposed site of A
40 generation in neuronal cells (24,
55). Interestingly, we found that solECE-1 hydrolyzed A
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 A
, Fuller et al.
(40) have reported that phosphoramidon treatment of SY5Y neuronal cells
results in an increase in cell-associated A
. Unfortunately, we have
not been able to convincingly detect intracellular A
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 A
by H4 cells is insensitive to phosphoramidon suggests
that the compound may exert its effect on A
through an intracellular event.
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.
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 A
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 A
accumulation in vivo.
Nonetheless, these experiments are under way and may provide additional
insight into the role of ECE in endogenous A
accumulation in the brain.
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 A
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 A
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 A
catabolism.
![]() |
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-A 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.
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;
A,
-amyloid;
ECE, endothelin-converting enzyme;
CHO, Chinese hamster ovary;
APP, amyloid precursor protein;
APP,
-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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---|
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