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INTRODUCTION |
Alzheimer's disease
(AD)1 is characterized by the
accumulation of amyloid
peptide (A
) in the brain. A
is
composed of 39-43 amino acids and is constitutively produced by
proteolysis of the
-amyloid precursor protein (APP). Alterations in
either synthesis or clearance of A
may potentially contribute to
increased levels of A
and amyloid deposits. Although much attention
has been focused on the production of A
, little is known about how
A
is degraded and cleared, especially in the brain. Identification
of the peptidases involved in A
catabolism in vivo is
important for the development of therapeutics designed to prevent or
treat AD.
Proteases, including cathepsin D (1), serine
protease-
2-macroglobulin complex (2) and
insulin-degrading enzyme (3), were purified and identified as
A
-degrading enzymes in vitro. Several other recombinant
or purified peptidases have also been shown to degrade A
in
vitro (4-14). We focused on the in vivo catabolism of
A
42, because this specific form, rather than A
40, is considered
to be the primary pathogenic agent in AD. Recently, we demonstrated
that an endopeptidase(s) similar or identical to neprilysin (NEP),
whose activity is sensitive to thiorphan and phosphoramidon, is
involved in the catabolism of A
1-42 in vivo (15,
16).
NEP is a type II membrane protein on the cell surface and is classified
as a member of the M13 family. NEP hydrolyzes and inactivates several
circulating peptides, such as enkephalin, atrial natriuretic peptide,
endothelin, and substance P, and has wide tissue distribution and
substrate specificity (17). The M13 family comprises six
zinc-dependent metalloproteases, NEP, endothelin-converting
enzyme (ECE-1) (18), ECE-2 (19), KELL antigen (20), phosphate
regulating gene with homologies to endopeptidases on the X chromosome
(PEX) (21), and the recently identified damage-induced neuronal
endopeptidase (DINE)/X-converting enzyme (XCE) (22, 23). Among these,
only NEP and DINE (22) have been shown to be sensitive to both
thiorphan and phosphoramidon. Because ECE-1 and ECE-2 are not inhibited
by thiorphan (18, 19), they can be excluded as candidates for
A
1-42-degrading enzymes in vivo. KELL is only partially
inhibited by phosphoramidon (24) and is mainly present in erythroid
tissues. Although PEX degrades parathyroid hormone-derived peptides
(25), its sensitivity to thiorphan or phosphoramidon is unknown. These
facts indicate that NEP, DINE, and PEX are candidates for
A
1-42-degrading enzymes in vivo. We also need to
consider the possible presence of an unidentified protease(s), whose
activity is sensitive to thiorphan and phosphoramidon and responsible
for A
1-42 degradation.
In this study, considering the redundancy of peptidases, we
investigated the presence of unidentified peptidase(s), which is
homologous to NEP, DINE, or PEX. We cloned novel cDNAs, which were
termed neprilysin-like peptidases
(NEPLPs) and examined their sensitivities to thiorphan and
phosphoramidon. We compared the ability to proteolyse A
among NEP,
PEX, DINE, and NEPLPs and found that NEP was the most potent
A
-degrading enzyme.
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EXPERIMENTAL PROCEDURES |
cDNA Cloning--
To obtain cDNAs homologous to
those of NEP and PEX, two degenerate oligonucleotides corresponding to
amino acid residues 646-654 (GENIADNGG) and 685-696 (QLFFLNFAQVWC) in
the mouse NEP were used; 5'-GGAGAAAA(C/T)ATTGCTGATAATGG(A/G)GG-3' and
5'-CACC(A/T)(C/T)AC(G/C/A)TG(A/G)GCA(A/T)A(G/ A)(T/C)TCA(G/A)GAA(GA)AA(G/T)AG(T/C)TG-3'.
PCR was performed in a 50-µl 1× EX-Taq buffer (Takara,
Kyoto, Japan) containing 2 mM MgCl2, 250 µM each of dNTPs, 2 µl of ICR mouse brain Marathon Ready cDNA (CLONTECH, Palo Alto, CA), 2.5 units
of EX-Taq polymerase (Takara), and 2 µM of
each primer. Amplifications were carried out according to initial
denaturation of 1 min at 94 °C, 30 cycles of 30 s at 94 °C,
1 min at 50 °C, and 1 min at 72 °C and a final extension of 2 min
at 72 °C. PCR products were analyzed by 6% acrylamide gel
electrophoresis. Products ~150 bp in length were excised from the
gel, cloned into a pCR-TOPO vector (Invitrogen, San Diego, CA), and
sequenced. A novel cDNA was named NEPLP. 5' and 3' regions of the
NEPLP gene were amplified by nested PCR with an Advantage cDNA PCR
kit (CLONTECH) using AP-1/AP-2 as sense primers at
the cDNA adaptor and
5'-TGGGCATAGGTCAGGTTCAGTCCCG-3'/5'-CAGTCGCTGATCTTTGCCGCCATCA-3' as
antisense primers and
5'-GGTGTGCGACAGGCATACAAGG-3'/5'-CCTACGGTGGCTGGCTGATGGCGGC-3' as sense
primers and AP-1/AP-2 as antisense primers, respectively. Both products
of the 5' and 3' regions were cloned into a pCR- TOPO vector and
sequenced. Full-length NEPLP cDNAs were amplified using the primer
pairs 5'-GCGGGTACCGGAAAGTCTGAGAGCCCAGTGGGG-3' and
5'-TTGGCTACCAGATGCGACATCGCTT-3' and subcloned into the SmaI site of pBluescript II KS (+).
A similar procedure was performed to obtain cDNA homologous to
NEP and DINE/XCE. Briefly, two oligonucleotides,
5'-AA(T/C)CA(A/G)AT(A/C/G/T)GT(A/C/G/T)TT(C/T)CC(A/C/G/T)GC-3' and
5'-CC(A/C/G)(A/T)T(A/G)TC(A/C/G/T)GC(A/G/T)AT(A/G)TT(C/T)TC-3' corresponding to amino acid residues 551-557 (NQIVFPA) and
647-653 (ENIADNG) of mouse NEP, respectively, were prepared, and PCR
was performed under the same conditions described above. Products ~300 bp in length were sequenced.
Human NEP and PEX cDNAs were amplified from human hippocampus
Marathon Ready cDNA (CLONTECH). Rat DINE
cDNA was obtained as described previously (22). FLAG sequences
(MDYKDDDDK) were introduced at the C-terminal end of each cDNA by
PCR using appropriate primers containing FLAG nucleotides. All
the cDNAs were subcloned into the mammalian expression vector
pcDNA3.1 (+) (Invitrogen).
Cell Culture and Transfection--
293 human embryonic kidney
cell lines were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin (Life Technologies, Inc.). The 293 cells were
transfected with either an empty vector, NEP, PEX, NEPLP
, NEPLP
, NEPLP
, or DINE cDNAs using a calcium phosphate method.
Stable 293 cell lines were obtained by selection with 600 µg/ml G418.
Stable mouse neuroblastoma N2a cells expressing both human APP695 with
the Swedish mutation and presenilin 2 (PS2) with the N141I mutation
were obtained by selection with 150 µg/ml hygromycin and 500 µg/ml
G418, respectively.
Antibodies--
The rabbit polyclonal anti-NEPLPC (named as 065P
and M97P) and anti-PEXC (M96G) antibodies were raised against a
synthetic peptide, CCPRGSPMHPMKRCRIW corresponding to the C-terminal
amino acid residues of the NEPLP, glutathione S-transferase
fusion protein encompassing residues 534-646 of the NEPLP
, and
glutathione S-transferase fusion protein encompassing
residues 700-749 of human PEX, respectively. 56C6 (Novocastra
Laboratory, Tyne, UK) and M2 (Stratagene, La Jolla, CA) are mouse
monoclonal antibodies against the extracellular domain of human NEP and
FLAG, respectively. DINE antibody was characterized previously
(22).
Western Blot Analysis--
Cells were homogenized in solution A
(0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl, 1 µg/ml
leupeptin, and 1 µg/ml pepstatin A) and centrifuged at 500 × g for 5 min. Membranes were prepared by precipitation of the
postnuclear supernatant at 100,000 × g for 30 min. The supernatant was used as a cytosolic fraction. The resulting pellet was
lysed in solution A containing 1% Triton X-100 for 1 h at 4 °C
and centrifuged at 100,000 × g for 15 min. The
supernatant was used as a membrane fraction. Aliquots of the membrane
fraction (3 µg) or 10 µl of culture medium (CM) were separated on
7.5% SDS-polyacrylamide gels and transferred to polyvinylidene
difluoride membrane. The blotted membrane was blocked with 5% skim
milk in a buffer containing 10 mM Tris-HCl, pH 8.0, and
0.15 M NaCl and sequentially incubated with the primary
antibody in the buffer with 0.05% Tween 20 and with horseradish
peroxidase-goat anti-rabbit or anti-mouse IgG (Amersham Pharmacia
Biotech, Buckinghamshire, UK) and visualized using an ECL-plus kit
(Amersham Pharmacia Biotech) according to the manufacturer's
directions. Cell lysates were also prepared using the solution A
containing 1% Triton X-100.
Peptidase Assay--
Membrane fractions (0.5 µg) from the 293 cells transfected with the various cDNAs were incubated with 50 µM Z-Ala-Ala-Leu-p-nitroanilide (ZAAL-pNA) (Peptide Institute, Osaka, Japan) in 100 µl of
100 mM MES (pH 6.5) or 50 mM HEPES (pH 7.2) for
1 h at 37 °C. The reaction mixture was added with 0.4 milliunit
of leucine aminopeptidase (Sigma Chemical Co., St. Louis, MO), further
incubated for 20 min at 37 °C and measured at 405-nm absorption. For
the inhibition study the membrane fractions were preincubated with 10 µM thiorphan or 10 µM phosphoramidon for 5 min before the addition of ZAAL-pNA.
Digestion Assay of 3H/14C-radiolabeled
A
1-42--
Membrane fractions (0.5 µg) were incubated with
3H/14C-radiolabeled A
1-42 (20,000 dpm, 1.3 µM) in 50 µl of 50 mM HEPES (pH 7.2) containing 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1× complete without EDTA (Roche Molecular Biochemicals) at 37 °C for 1 h. The reaction was stopped by adding 250 µl of a solution containing 0.05% triethylamine, 10 mM betaine, and 0.1 mM
EDTA, followed by boiling for 5 min, and was analysed using HPLC as
described (15). For the inhibition studies, membrane fractions were
preincubated with 10 µM thiorphan for 5 min before adding
the 3H/14C-radiolabeled A
1-42.
Digestion Assay of Cold A
1-40 and A
1-42--
A
(Bachem, Torrance, CA) was dissolved in dimethyl sulfoxide at 5 mg/ml
as a stock solution. 2 µg of A
1-40 (4.6 µM) or 5 µg of A
1-42 (11 µM) was incubated with 10 µg of
the membrane fractions in 100 µl of 50 mM HEPES (pH 7.2)
containing 1 µg/ml leupeptin and 1 µg/ml pepstatin A at 37 °C
for the times indicated. The reaction was stopped by adding 200 µl of buffer A (0.1% trifluoroacetic acid), and the solution was
injected into a reverse-phase HPLC (CAPCELL PAK C18 UG 120, particle
size 5 µm, column dimensions 4.6 × 250 mm) (Shiseido, Tokyo,
Japan) equilibrated with the buffer A. The HPLC protocol used to
analyze 3H/14C-radiolabeled A
1-42 was not
suitable for analyzing cold A
peptides, because it produced high
background noise when analyzed at the absorbance of 210 nm. A
and
its proteolytic products were eluted with a linear solvent gradient of
0-70% buffer B (0.1% trifluoroacetic acid in acetonitrile) over
10-40 min at a flow rate of 0.5 ml/min at 50 °C. Peptides were
detected by absorbance at 210 nm. 10 µM thiorphan was
preincubated for the inhibition.
Mass Spectrometric Analysis and Protein Sequencing--
HPLC
peaks in digestion assays of cold A
peptides were collected, dried
under vacuum, and analyzed by a mass spectrometer and a protein
sequencer as described previously (15).
Kinetic Analysis--
Various amounts of cold A
peptides were
incubated with membrane fractions of pcDNA-transfected cells or
NEP-transfected cells, and the remaining peaks of the A
peptides on
the HPLC chart were used to determine the initial A
concentrations
(S0) or A
concentrations at time t
(St), respectively. Note that membrane fractions
of pcDNA-transfected cells showed no detectable degradation to
A
. Km and Vmax were
determined according to the following first degree equation (26),
Digestion Assay of Secreted A
from N2a Cells--
CM from the
N2a cells in the absence of fetal bovine serum was collected, diluted
5-fold with medium, and used as a substrate. The CM was incubated with
4 µg of membrane fractions in 100 µl of 50 mM HEPES (pH
7.2) at 37 °C for 16 h. The reaction was stopped by boiling and
the samples were subjected to BAN50/BA27 and BAN50/BC05 sandwich ELISAs
to quantify A
1-40 and A
1-42, respectively (27).
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RESULTS |
cDNA Cloning of NEPLPs--
To clone peptidases homologous to
NEP and PEX, we designed degenerate oligonucleotide primers. With mouse
brain cDNA as a template, the PCR resulted in the amplification of
DNA with the predicted size (150 bp). After subcloning the PCR products
and sequencing individual clones, we found that 11 of the 42 clones had
a novel identical sequence that was similar to but distinct from
members of the M13 family. The other 16 clones were identical to ECE-1,
11 clones were NEP, 2 clones were ECE-2, and 2 clones were PEX. During
sequencing the full-length of the novel gene, we noted the presence of
three isoforms, which probably resulted from alternative splicing. We
termed them NEPLP
, NEPLP
, and NEPLP
, and their structural
characteristics are outlined in Fig. 1.
Of 42 isolated full-length clones, 21 were NEPLP
, 20 were NEPLP
, and 1 was NEPLP
. Compared with NEPLP
, NEPLP
and NEPLP
had an insertion of 23 amino acids immediately following the
putative transmembrane region and 37 amino acids near the center of the
protein, respectively. All the isoforms were predicted to be type II
transmembrane proteins with a putative zinc-binding domain
(HEXXH) in the extracellular portion. NEPLPs showed the highest homology (~54%) to NEP among the members of the M13 family. The degenerate primers, which were designed to amplify the peptidases homologous to NEP and DINE/XCE, did not amplify a novel gene. Recently,
three research groups independently reported a novel member of the M13
family (28-30). NEPLP
and NEPLP
were identical to
SEP
/splice 1 and SEP/NL1/NEPII, respectively. NEPLP
was a novel isoform.

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Fig. 1.
Schematic representation of the primary
sequences of the NEPLP , NEPLP
, and NEPLP proteins. The black rectangles represent the
inserted 23 and 37 amino acids in the NEPLP and proteins,
respectively. The inserted nucleotide and amino acid sequences in NEPLP
are shown by underlined letters. TM and
HEITH represent the predicted transmembrane domain and the
HEXXH zinc-binding motif, respectively.
Arrowheads indicate the positions of the degenerate primers
to amplify the NEPLP gene.
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Expression of NEPLPs, NEP, PEX, and DINE in the 293 Cell
Line--
We stably transfected NEPLP cDNAs in 293 cells and
determined the expression using the 065P antibody, which was raised
against the C-terminal portion of NEPLP. In the 293 cells transfected with pcDNA vector alone (referred to as pcDNA cells), no
endogenous NEPLP protein was detected (Fig.
2A, lane 5). In
membrane fractions of 293 cells stably transfected with NEPLP
(NEPLP
cells), 100- and 120-kDa broad bands were detected (Fig.
2A, lane 6). Because NEPLP
was expressed as a
single 100-kDa band in COS cells (not shown) and Chinese hamster ovary
cells (28), the 120-kDa band probably represented a highly glycosylated
or modified protein specific for the 293 cells. NEPLP
and NEPLP
proteins were expressed in membrane fractions as 110- and 115-kDa
bands, respectively (Fig. 2A, lanes 7 and
8). This corresponded to the increasing sizes of amino
acids, although proteins with higher molecular mass did not accumulate
as seen in NEPLP
cells. The amounts of NEPLP
and NEPLP
proteins were lower than that of NEPLP
. NEPLP
, NEPLP
, and
NEPLP
were not detected in the cytosolic fractions (not shown). In
the culture medium, we could detect only NEPLP
(Fig. 2C,
lanes 13) with a molecular mass of 125 kDa, which was larger
than that of membrane-associated NEPLP
. This result suggested that
the 23-amino acid sequences inserted in NEPLP
possessed a secretion
signal and that NEPLP
is much more glycosylated or modified in the
secretory pathway than in the membrane. Indeed Ikeda et al.
(28) reported that the molecular mass of membrane-associated and
secreted NEPLP
/SEP were reduced to the same mass (89 kDa) by
peptide-N-glycanase F treatment.

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Fig. 2.
Western analysis of NEP, PEX, NEPLP
, NEPLP , NEPLP
, and DINE expressed in the 293 cell line.
A, equal amounts (3 µg) of membrane fractions of 293 cells
stably transfected with cDNAs indicated below each lane
were subjected to Western analysis using the antibodies 56C6
(lanes 1 and 2), M96G (lanes 3 and
4), and 065P (lanes 5-8). B, membrane
fractions of 293 cells transiently transfected with pcDNA and DINE
(lanes 9 and 10, respectively) were subjected to
Western analysis using the DINE antibody. C, culture medium
(CM) of 293 cells stably transfected with pcDNA, NEPLP
, NEPLP , and NEPLP were subjected to Western analysis using
065P (lanes 11-14). The 66-kDa bands indicated with an
asterisk may be nonspecific staining of albumin derived from
the serum. D and E, estimation of relative
amounts of each protein expressed in 293 cells. Cell lysates of 293 cells transiently transfected with cDNAs tagged with FLAG were
subjected to Western analysis using the anti-FLAG antibody M2
(D). The amounts of lysates transfected with NEP, PEX, NEPLP
, and DINE without FLAG were 0.5, 0.5, 0.5, and 1.5 µg,
respectively (E). Each protein was blotted with the same
antibodies as in A and B with the exception of
NEPLP . NEPLP was blotted with the M97P antibody. Note that
almost equal amounts of peptidases were loaded.
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When we expressed other members of the M13 family (NEP, PEX, and DINE)
in the 293 cells, these proteins were detected as 110-, 110-, and
95-kDa bands in membrane fractions (Fig. 2A, lanes
2, 4, and 10), whereas they were not
detected in the pcDNA cells (Fig. 2A, lanes
1, 3, and 9). NEP, PEX, and DINE were not
detected in cytosolic fractions or CM (not shown). To estimate the
relative amounts of the peptidases, we expressed each cDNA to which
FLAG sequence was added at its C-terminal end. Because nearly identical intensities of bands were detected with the anti-FLAG antibody (Fig.
2D), equal amounts of peptidases with FLAG were loaded onto the gels. Fig. 2E shows the results of loading the same
amounts of FLAG-tagged peptidases as those in Fig. 2D. The
gel shown in Fig. 2E was also loaded with 0.5 µg of
lysates from NEP, PEX, and NEPLP
cells and 1.5 µg from DINE cells
and was then stained with the corresponding antibodies. Almost
identical band intensities resulted regardless of whether or not the
peptidases contained FLAG. We estimated from the results that the
relative amounts of peptidases were 1:1:1:1/3 for NEP:PEX:NEPLP
:DINE. The addition of FLAG to PEX and NEPLP
reduced their
molecular weight compared with their weights in the absence of FLAG
(Fig. 2E), suggesting immature glycosylation or modification
resulting from the FLAG sequence at the C terminus. We also observed
reduced proteolytic activity of NEP-FLAG than that of NEP (not shown).
Therefore, we used each peptidase without FLAG in the following
proteolytic analyses.
Proteolytic Activity to Synthetic Peptide (ZAAL-pNA)--
We
investigated the peptidase activity of NEPLPs, and their sensitivities
to thiorphan and phosphoramidon. Membrane fractions of NEPLPs and other
peptidases expressed in the 293 cells were incubated with
ZAAL-pNA at pH 6.5 (Fig. 3,
A and B). Membrane fractions of pcDNA cells
showed a very low level of thiorphan- and phosphoramidon-sensitive
activity due to the presence of an endogenous enzyme. A high degree of
proteolytic activity was detected in the membrane fractions of the NEP
cells (Fig. 3, A and B). PEX showed a low but
significant proteolytic activity compared with that of the control
(pcDNA) (Fig. 3A). Both NEPLP
and NEPLP
proteolyzed the peptide, and their activities were almost the same and
one-third compared with that of NEP, respectively. In contrast, NEPLP
had no proteolytic activity compared with the control, although the
level of the NEPLP
protein was similar to that of NEPLP
(Fig.
2A). This suggested that NEPLP
protein has different
substrate specificity or that it is a zymogen that requires proteolytic
activation. It is also possible that the 37-amino acid sequence
inserted into NEPLP
inhibits its activity by interfering with
proper folding. DINE had no proteolytic activity in this system (Fig.
3B), although its endopeptidase activity and sensitivity to
thiorphan and phosphoramidon were demonstrated previously in a
baculovirus expression system (22). Proteolytic activities of NEP, PEX,
NEPLP
, and NEPLP
were inhibited by thiorphan and phosphoramidon
(Fig. 3A), suggesting that they could all be candidates for
A
-degrading enzymes in vivo. The secreted form of NEPLP
also had the proteolytic activity (not shown). We obtained
identical results at pH 7.2, too.

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Fig. 3.
Proteolytic activity of the peptidases
expressed in the 293 cell line. A, membrane fractions
of 293 cells stably transfected with cDNAs indicated below each
column were analyzed for peptidase activity using ZAAL-pNA
as a substrate in the absence (white columns) or presence of
thiorphan (black columns) and phosphoramidon (hatched
columns). Data are representative of at least three independent
experiments and shown with means ± S.D. B, membrane
fractions of 293 cells transiently transfected with pcDNA, NEP, and
DINE were analyzed for peptidase activity.
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Degradation of Radiolabeled A
1-42--
We characterized the
A
-degrading activity of the peptidases using
3H/14C-radiolabeled A
1-42, which we had
synthesized previously (15). To exclude the effects of endogenous
peptidases, equal amounts of membrane fractions (0.5 µg) were
analyzed for their ability to proteolyze A
. Membrane fractions of
the pcDNA cells exhibited almost no proteolytic activity to the
radiolabeled A
1-42 (peak at 42 min in Fig.
4A, closed
triangle) in 1 h at 37 °C. NEP decreased the radioactive
peak at 42 min and increased the peaks at 7 and 37 min, suggesting that
NEP efficiently proteolyzed the A
1-42 into free amino acids/small
peptides and a catabolic intermediate (Fig. 4B, open
triangles). This degradation by NEP was very similar to the
in vivo proteolysis as described previously (15). The degradation was completely inhibited by thiorphan, suggesting that NEP
directly proteolyzed the peptide (Fig. 4H). In 4 h, NEP almost completely digested the A
1-42 (not shown). In contrast PEX,
NEPLP
, NEPLP
, NEPLP
, and DINE did not degrade the
A
1-42, because their HPLC profiles were almost the same as that of
the control (pcDNA) (Fig. 4, C-G). HPLC profiles in the
3H mode were essentially identical to the 14C
mode (not shown), as reported previously (15), suggesting that NEP
proteolyzed the A
1-42 from both the N and C termini.

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Fig. 4.
Proteolysis of synthetic
3H/14C-radiolabeled
A 1-42 by the peptidases. Radioactive
patterns in HPLC are shown in the 14C mode.
A-G, membrane fractions (0.5 µg) of 293 cells transfected
with the cDNAs indicated in each panel were analyzed for degrading
activity to 3H/14C-radiolabeled A 1-42.
H, thiorphan was preincubated with membrane fractions of
NEP-transfected cells. Black and white triangles
represent intact 3H/14C-radiolabeled A 1-42
and degrading products, respectively. Data are representative of at
least two independent experiments.
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Degradation of Cold A
1-40 and A
1-42--
We examined the
degradation of cold A
1-40 and A
1-42 using HPLC under different
conditions from those used for analyzing the degradation of
3H/14C-radiolabeled A
1-42 (see
"Experimental Procedures"). Membrane fractions of the pcDNA
cells were used as a negative control (Fig. 5A, panel 1). NEP
almost completely degraded the A
1-40 peptide corresponding to the peak at 35.7 min (Fig.
5A, panel 2) in 2 h at
37 °C. The degradation was accompanied by the appearance of multiple
peptides between 23 and 28 min (Fig. 5A, panel 2, open triangles). NEPLP
showed relatively low but
significant proteolytic activity to cold A
1-40 as discerned by the
decrease of the peak at 35.7 min and the appearance of new peaks
between 26 and 29 min (Fig. 5A, panel 4).
Thiorphan (Fig. 5A, panels 8 and 9)
and phosphoramidon (not shown) completely abolished the proteolytic
activity of NEP and NEPLP
. PEX, NEPLP
, NEPLP
, DINE (Fig.
5A, panels 3, 5-7) and secreted form
of NEPLP
(not shown) showed no proteolytic activity to A
1-40.
Although we assayed 3-fold amounts of membrane fractions of NEPLP
,
NEPLP
, and DINE cells to normalize the amounts of peptidases, they
showed no detectable proteolytic activity to cold A
1-40 in 2 h
(not shown). With a longer incubation time (Fig. 5C, 6-12
h), NEP degraded much larger amounts of A
1-40, although NEPLP
did not further degrade A
1-40. PEX showed very low proteolytic
activity, and NEPLP
, NEPLP
, and DINE showed no detectable
proteolytic activity to A
1-40 in 6-12 h (Fig. 5C).

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Fig. 5.
Proteolysis of synthetic cold
A 1-40 and A 1-42 by
the peptidases. A and B, membrane fractions
(10 µg) of 293 cells transfected with the cDNAs indicated in each
panel were analyzed using HPLC for degrading activity to cold A 1-40
for 2 h (A) or A 1-42 for 4 h (B).
Thiorphan was preincubated with membrane fractions of NEP-transfected
cells (A and B, panels 8) or with
those of NEPLP -transfected cells (A, panel
9). Black and white triangles indicate the
peaks of intact A and degrading products, respectively. An
arrow indicates the peak of thiorphan. Data are representative of at least two
independent experiments. C and D, time course of
degradation of cold A 1-40 (C) or A 1-42
(D) by membrane fractions (10 µg) of each peptidase. The
Y axis represents the remaining peak of the A peptides
compared with pcDNA cells (% of control) after indicated
incubation periods.
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When cold A
1-42 was used as a substrate, NEP degraded it in a
reproducible manner as demonstrated by the decrease of the original
peak at 36.4 min and the appearance of a new peak at 26.4 min (Fig.
5B, panel 2) compared with the control (Fig.
5B, panel 1) in 4 h at 37 °C. In
contrast, PEX, NEPLP
, NEPLP
, NEPLP
, and DINE (Fig.
5B, panels 3-7), and the secreted form of NEPLP
(not shown) showed almost no proteolytic activity. Degradation of
A
1-42 by NEP was completely inhibited by thiorphan (Fig.
5B, panel 8) and phosphoramidon (not shown).
Again, 3-fold amounts of membrane fractions of NEPLP
, NEPLP
,
and DINE cells showed no detectable proteolytic activity to cold
A
1-42 in 4 h (not shown). NEP degraded greater amounts of
A
1-42 with longer incubation times (Fig. 5D, 8-12 h).
Among the other peptidases, only NEPLP
degraded very small amounts
of A
1-42 in 8-12 h (Fig. 5D). N-terminal amino acid
sequence and the molecular mass of the peak at 26.4 min produced by NEP
(Fig. 5B, panel 2) were DAE--- and
1033.92, suggesting that the peak contained A
1-9 (calculated mass
MH+ = 1034.02). This result is consistent with the previous
study, which indicated that 3H/14C-radiolabeled
A
1-42 was cleaved at G9/Y10 by thiorphan-
and phosphoramidon-sensitive peptidase(s) in vivo (15). The
other degrading products by NEP could not be identified probably due to
further degradation.
Kinetic Analysis of A
Proteolysis by NEP--
We determined
Km and Vmax values for the
proteolysis by NEP using varying amounts of cold A
peptides (Table
I). The Km values were
11.2 and 6.95 µM and Vmax values
were 158 and 21.1 nM/min for A
1-40 and A
1-42,
respectively. It is difficult to determine the Km
and Vmax values for the other peptidases,
presumably because the Km values are too large
and/or the Vmax values are too small to
measure.
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Table I
Kinetic analysis of cold A 1-40 and A 1-42 proteolyzed by NEP
Various concentrations of A 1-40 or A 1-42 were incubated with
membrane fractions for 2 or 4 h, respectively.
Km and Vmax were calculated as
described under "Experimental Procedures."
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Degradation of Cell-secreted A
1-40 and A
1-42--
We
investigated the degradation of cell-secreted A
by the peptidases.
We chose the N2a cells as A
-producing cells because they were stably
co-transfected with APP with the Swedish mutation and PS2 with the
N141I mutation and secreted large amounts of both A
1-40 and
A
1-42. We tried to examine the effect of NEP expression by
co-culturing the NEP-expressing 293 cells with the N2a cells or by
culturing the NEP-expressing 293 cells in CM derived from the N2a
cells. However, the 293 cells used in the present study by themselves
possessed a potent activity that completely removed the A
in CM,
presumably through internalization and/or extracellular degradation
employed by endogenous proteins. To avoid cell-mediated removal of
A
, the membrane fractions of the 293 cells were incubated with CM
from the N2a cells at 37 °C for 16 h. NEP, which was expressed
either stably or transiently, reproducibly degraded both A
1-40 and
A
1-42 compared with the control (pcDNA) (Fig.
6, A and B). PEX,
NEPLP
, NEPLP
, NEPLP
, and DINE showed almost no proteolytic
activity to the cell-secreted A
1-40 or A
1-42 (Fig. 6,
A and B).

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Fig. 6.
Proteolysis of cell-secreted
A 1-40 and A 1-42 by
the peptidases. Membrane fractions of 293 cells stably
(A) or transiently (B) transfected with the
cDNAs indicated below each lane were analyzed for degrading
activity to A secreted from N2a cells stably transfected with both
the APP and PS2 mutants. After incubation with the membrane fractions
(4 µg) and A , quantitative ELISA analysis was performed to
determine the A 1-40 (white columns) and A 1-42
(black columns). Data (Exp. 1 and 2)
are representatives of at least three independent experiments and are
shown with means ± S.D.
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DISCUSSION |
We previously reported that an endopeptidase(s) similar or
identical to NEP is the most probable candidate for an
A
1-42-degrading enzyme in vivo (15). In the present
study we cloned NEPLP cDNAs and compared A
-degrading activity
among NEP, PEX, NEPLPs, and DINE whose activities are sensitive to
thiorphan and phosphoramidon. Using different methods (HPLC and ELISA)
and different substrates (synthetic and cell-secreted A
), we showed
that among the peptidases tested NEP most rapidly and efficiently
degraded not only A
1-42 but also A
1-40 at concentrations
ranging from picomolar to micromolar in vitro. Because
picomolar concentrations of A
peptides are present in human
cerebrospinal fluids and plasmas, NEP is most likely to be an
A
-degrading enzyme in vivo.
We first demonstrated that enzymatic activities of PEX and NEPLP
/SEP
/splice 1 were sensitive to thiorphan and
phosphoramidon and confirmed that that of NEPLP
/SEP/NL1/NEPII was
sensitive to these inhibitors (28, 29). PEX proteolyzed synthetic
A
1-40 more slowly and to an extremely weaker extent than NEP, but
it did not proteolyze synthetic A
1-42 or cell-secreted A
1-40
and A
1-42. Although NEPLP
had almost the same proteolytic
activity to ZAAL-pNA as NEP, it degraded cold A
1-40 and
A
1-42 more slowly and to a weaker extent than NEP and showed no
detectable degrading activity to cell-secreted A
1-40 and A
1-42.
These results suggest that PEX and NEPLP
have lower affinities
and/or smaller Vmax for A
than NEP, and
therefore, they could not be the major degrading enzymes of endogenous
A
1-40 or A
1-42 in vivo as compared with NEP.
However, PEX and NEPLP
might degrade A
in AD patients or APP
transgenic mice brains, where A
is accumulated in excess. It seems
unlikely that NEPLP
, NEPLP
, or DINE degrade A
in
vivo, because they showed almost no proteolytic activity to A
under any of the conditions we examined.
Our results suggest that NEP directly degrades both A
1-40 and
A
1-42 as determined by its inhibitor profiles, consistent with a
previous report using purified NEP and synthetic A
1-40 (7).
Alternatively, NEP may mediate the initial cleavage of A
and another
endogenous peptidase may further degrade A
. Although it is possible
that NEP is indirectly involved in A
degradation through, for
instance, the proteolytic activation of another peptidase, this is
unlikely because NEP is capable of proteolyzing peptides smaller than
4-5 kDa (31). In any case, it is noted that NEP is required for A
degradation.
We found that NEP degraded cold A
1-40 more rapidly than cold
A
1-42 in vitro (Fig. 5). This may be due to a higher
Vmax for A
1-40 than for A
1-42, although
NEP had a slightly higher affinity to A
1-42 than to A
1-40
(Table I). The rate of degradation by NEP was different among various
A
1-42 peptides (Fig. 4-6). The half-lives of radiolabeled
A
1-42, cold A
1-42, and cell-secreted A
1-42 were ~1, 8, and more than 16 h, respectively. Moreover, the degraded products
of radiolabeled 1-42 and cold A
1-42 may be different as evaluated
by their retention time in HPLC. These different degrees of degradation
are probably due to different conformations and concentrations of the
peptides and/or different concentrations of the peptidases under the
optimum conditions for each assay.
NEP is an ectoenzyme with a large extracellular domain containing a
catalytic site. This indicates the direct involvement of NEP in A
degradation, because NEP can interact with and degrade A
with the
correct topology on the cell surface. The fact that a soluble form of
NEP is found in human plasma and cerebrospinal fluid (32, 33) also
supports the possible interaction between A
and NEP in the
extracellular space. We could not determine whether NEP degraded
extracellular A
or intracellular A
in this study. To address this
issue a further study will be needed to measure intracellular A
as
well as extracellular A
in doubly transfected cells with APP and NEP cDNAs.
In that A
42 is preferentially accumulated in AD, it is important
that NEP is capable of degrading A
1-42. The high expression of NEP
protein in the brain is restricted to striatum, olfactory tubercle,
substantia nigra, choroid plexus, endopeduncular nucleus, and pontine
nuclei, and moderate expression in cerebellum and low expression in
hippocampus and cerebral cortex (34, 35). The relatively low levels of
NEP in hippocampus and cerebral cortex may explain the selective
deposition of A
42 in those areas. It is possible that altered
activity of NEP in brain upon aging might lead to accumulation of
A
42. Indeed NEP mRNA and protein levels in the hippocampus and
temporal gyrus were significantly lower in AD patients than those in
control cases (36). It would be interesting to examine possible
colocalization of NEP protein with senile plaques and
neurofibrillary tangles. Genetic analysis of NEP, which is located in
chromosome 3q21-27, in sporadic as well as familial AD cases may help
understanding of the pathogenesis.
An intriguing finding in this study is that NEP can degrade either
synthetic or cell-secreted A
1-40 and A
1-42 over a wide range of
concentrations, from picomolar to micromolar. These results suggested
not only that NEP degrades endogenous A
but also that NEP may be a
good therapeutic target for the treatment of AD. Because there is a
dynamic balance between monomer and polymer forms of A
(37-39),
reducing the amounts of monomer A
will lead to a reduction of
polymer A
. It is important to search for methods that would
up-regulate NEP activity or inactivate NEP inhibitors. Alternatively,
targeting endogenous NEP activity to the site(s) of A
production or
storage may reduce the A
levels in a specific and efficient manner
without altering the total NEP activity (40). The use of NEP is
advantageous in that NEP is non-destructive due to its limited ability
to act only on peptides smaller than 4-5 kDa. Although it remains to
be determined whether NEP degrades endogenous A
in vivo,
knockout and transgenic studies of the NEP gene should elucidate this
in future studies. The relative contribution of PEX and NEPLP in the
catabolism of A
in vivo will also be clarified by such
studies of each gene.