From the Department of Molecular Cardiology, Lerner
Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio
44195, and ¶ Immunex Corporation, Seattle, Washington 98101
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ABSTRACT |
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Mammalian angiotensin-converting enzyme (ACE) is
one of several biologically important ectoproteins that exist in both
membrane-bound and soluble forms as a result of a post-translational
proteolytic cleavage. It has been suggested that a common proteolytic
system is responsible for the cleavage of a diverse group of membrane ectoproteins, and tumor necrosis factor- Many proteins exist in both membrane-bound and soluble forms as a
result of post-translational proteolytic processing. The responsible
proteases, variously called secretases, sheddases, or convertases,
cleave the membrane-bound form at sites near the plasma membrane on the
extracellular side of the single membrane-spanning domain. The entire
extracellular domain, often physiologically active, is released into
the medium or the circulation. This process of cleavage and secretion
appears to be an important and widely used cellular post-translational
regulatory process because a variety of structurally and functionally
unrelated cell-surface proteins undergo this process. They include
tumor necrosis factor Despite the biological importance of this process, little is known
about the identity of the responsible proteases. We have been studying
the characteristics of one such protease, ACE-secretase, the enzyme
involved in cleavage and secretion of ACE (3-7). Although ACE is
primarily a cell-associated protein, under normal physiological conditions soluble ACE is found in serum and other body fluids (8). The
cleavage and secretion of ACE is particularly significant because
tissue-bound ACE and soluble ACE in circulation may have different
physiological roles. ACE has two structurally related isozymes:
testicular ACE (ACET) and pulmonary ACE (ACEP),
involved in male fertility and blood pressure regulation, respectively (9, 10). We and others have shown that both ectoproteins, ACET and ACEP, undergo specific cleavage and
secretion (3, 11-13). The nature of the ACE-secretase activity has
been studied in vitro using cell lines that do not express
ACE, as well as primary cultures of cells that generate ACE in
vivo. The results obtained from these studies are corroborated by
observations made with human and animal tissue expressing natural ACE
(6, 13). ACE89, a mouse epithelial cell line permanently transfected
with a rabbit ACET expression vector, synthesizes,
glycosylates, and secretes enzymatically active soluble
ACET (3). The secretion process is slow, and
ACET accumulates on the cell surface. However, secretion
can be enhanced by treatment of cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, indicating the
existence of cellular mechanisms that regulate the conversion of
cell-bound ACET to its soluble form. The secreted protein
has a molecular mass lower by 8 kDa than the corresponding cellular form, suggesting that a proteolytic cleavage is involved. The exact
site of cleavage in this system has been determined. The cleavage
occurs in the ectodomain near the transmembrane domain between
Arg663 and Ser664, generating the short form,
soluble ACET, which lacks the membrane-anchoring C-terminal
tail of the cell-associated ACET (4).
Recently we have developed a cell-free in vitro assay system
to quantitate and characterize ACE-secretase, utilizing membranes prepared from ACE89 cells and rabbit lung. The in vitro
secretase activity was resistant to high salt extraction and to
inhibitors of serine, chymotrypsin, trypsin, cysteine, aspartate, and
elastase type proteases, but it is susceptible to Compound 3, a
hydroxamic acid-based inhibitor of certain metalloproteases (5). These results indicate that ACE-secretase is an integral membrane
metalloprotease. We extended the observation made with ACET
in a transfected system to explain the mechanism of ACEP
secretion in vivo. A similar pattern of inhibition of
ACEP production was observed in natural and transfected
vascular endothelial cells and freshly isolated kidney epithelial
cells, the two major sources of ACEP in the body. These
observations, together with analysis of ACEP proteins present in rabbit serum, lung, and kidney, established that
ACEP secreted in vivo is also caused by the
cleavage removal of the C-terminal region of the cell-associated
protein (6). In this article, we report the use of the same in
vitro assay to characterize, for the first time, detergent-soluble
ACE-secretase from both ACE89 cells and rabbit lung membranes.
The secretases studied so far share certain general properties, such as
stimulation by PMA and sensitivity to hydroxamic acid-based metalloprotease inhibitors. These common properties suggest that the
shedding phenomena may be mediated by the same cellular components since mutant cell lines have been isolated that are defective in
hydroxamic acid inhibitor-susceptible, PMA-stimulated cleavage and
secretion of many proteins, including TGF- In this report, we show that, unlike the release of several other
ectoproteins mentioned above, the cleavage and secretion of either
isozyme of ACE does not require TACE. Also, the process of cleavage and
secretion of ACE in vivo in mice is not affected by the
absence of TACE. These results indicate that ACE-secretase is a unique
enzyme, different from TACE. Here, for the first time, we also report
solubilization of ACE-secretase and characterization of some of the
unusual properties of the solubilized enzyme.
Materials--
Lisinopril
(N Cells, Transient Transfections, Pulse-Chase Analysis,
Immunoprecipitation, and Quantitation of Secretion--
TACE-deficient
ear fibroblasts were isolated from TACE-deficient mouse, as described
(19). Wild-type or TACE-deficient fibroblasts were transiently
transfected with ACET cDNA using the vaccinia virus-T7
RNA polymerase system, as described previously for HeLa and Chinese
hamster ovary cells (20, 21). Labeling with
[35S]methionine, pulse-chase analysis,
immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and autoradiography have been described
previously (21). For the co-transfection experiments (Fig.
2B and Table III), ACET and TACE were subcloned
into pcDNA3zeo expression vector from Invitrogen and transiently
transfected using DEAE-dextran (22). For quantitating cleavage and
secretion, the dried gels were subjected to PhosphorImager analysis by
using ImageQuant software (both from Molecular Dynamics, Sunntvale,
CA). The amount of mature ACET or chimeric proteins (measured in arbitrary PhosphorImager units) present in the cell extract and medium combined after chase, is taken as 100%, and the
amount present in the culture medium at that time is represented as
cleaved and secreted (7).
Western Analysis and Enzyme Activity Measurement--
Two
different goat polyclonal antibodies were used for Western analysis.
The first antibody was generated against purified rabbit lung ACE
(anti-ACE antibody). The second antibody was generated against a
synthetic peptide corresponding to the 14 amino acids of the C-terminal
sequence of rabbit ACE (anti-C-terminal peptide antibody). Both of
these antibodies have been used previously (4, 6). ACE enzyme activity
was assayed by using
hippuryl-L-histidyl-L-leucine (Hip-His-Leu) as
substrate (21).
Preparation of the Membrane Fractions and Solubilization of
ACE-secretase Activity--
Confluent ACE89 cells (3), labeled
overnight with [35S]methionine (100 µCi/150-mm plate)
were scraped and suspended in 0.02 M HEPES buffer, pH 7.0, containing 0.2 M sucrose, protease inhibitor mixture
(Boehringer Mannheim, prepared according to manufacturer's instructions), phenylmethylsulfonyl fluoride (1 mM),
leupeptin (1 µM), and phosphoramidon (50 µM). The cell suspension was homogenized in a Polytron
and centrifuged at 700 × g for 10 min. The pellet was
discarded, and the supernatant was centrifuged again at 36,000 × g for 40 min. The sedimented membranes were washed and used as the membrane preparation, as described previously (5). For solubilization, the membrane pellet was suspended in 0.05 M
Tris-HCl, pH 8.0, containing 0.3 M NaCl, the protease
inhibitors and 0.1% Triton X-100 (0.25 ml/150-mm plate). The
suspension was stirred at 4 °C for 1 h and centrifuged at
36,000 × g for 45 min. The supernatant was used as the
source of solubilized ACE-secretase.
Rabbit lung membranes were prepared as described previously (5), and
the ACE-secretase activity was solubilized from these membranes by
detergent extraction, as described above for ACE89 cell membranes.
Assay for ACE-secretase Activity--
In the
deglycosylation-PAGE assay (Fig. 3A), detergent extracts of
ACE89 cell membranes (0.25 ml) were incubated with or without 300 µl
of lisinopril-Sepharose (6) or concanavalin A (ConA)-agarose (Sigma,
200 µl) in a final volume of 1.5 ml for 1 h at 37 °C. For
incubations using lisinopril-Sepharose, 0.02 M HEPES, pH
7.0, 0.3 M NaCl, and 100 µM ZnCl2
were included, whereas for incubations using ConA-agarose, 0.05 M Tris-HCl, pH 7.5, and 0.2 M NaCl were included. Protease inhibitors were present in all incubations. The
reaction was stopped by boiling with SDS (1%) for 3 min; the incubation mixture was then centrifuged, and ACE-related proteins were
immunoprecipitated from the supernatant and deglycosylated using
N-glycosidase F, neuraminidase, and O-glycosidase
before being analyzed by PAGE (5). ACE-secretase activity in the ACE89 cell membranes was assayed, as described previously (5).
For the detergent-extraction assay (Table IV), membranes or Triton
X-100 extract of the membranes prepared from rabbit lung were incubated
with or without lisinopril-Sepharose, as described above, at 4 °C or
37 °C for 1 h. Uncleaved and cleaved ACEP proteins were physically separated by the temperature-induced phase-separation method utilizing Triton X-114, as described previously (5), and
ACEP enzyme activity was measured in both the detergent and the aqueous phase. In the incubations containing lisinopril-Sepharose, phase separation was performed after ACE proteins were eluted from the
lisinopril-Sepharose by borate buffer (6). As determined previously
(5), cleaved ACE partitions to the aqueous phase almost exclusively
(more than 98%), whereas the majority (more than 66%) of the
uncleaved ACE is present in the detergent phase. A considerable amount
(34%) of uncleaved ACE also partitions to the aqueous phase. Thus, the
corrected value for cleaved ACEP was calculated by
subtracting this latter value from the total ACEP activity
determined in the aqueous phase.
ACET Is Cleaved and Secreted from TACE-deficient
Cells--
To determine if TACE is required for the cleavage and
secretion of ACE, we investigated the ability of TACE-deficient
fibroblasts to secrete ACE. These mutant cells were generated by
immortalizing fibroblasts derived from TACE-deficient mice and
comparing them with similarly immortalized cells from wild-type mice.
As expected, the mutant cells were defective in secretion of TNF- ACE-secretase Activity in TACE-deficient Cells Is Stimulated by
Phorbol Ester and Inhibited by Compound 3--
We have shown
previously that ACE-secretase activity present in ACE89 cells, as well
as in the primary cultures of rabbit renal proximal tubular epithelial
cells, is resistant to inhibitors of various known classes of
proteases. None of the inhibitors of trypsin, chymotrypsin, elastase,
cysteine, serine, aspartate, and certain metalloprotease inhibitors had
any effect on cleavage and secretion of ACET by ACE89 cells
(4) or ACEP by renal epithelial cells (6). In contrast, a
hydroxamic acid-based metalloprotease inhibitor, Compound 3, significantly inhibited secretion of both ACET and
ACEP. In addition, cleavage and secretion of both isozymes of ACE was enhanced by treatment of cells with PMA. Thus, to further characterize the properties of ACE-secretase present in TACE-deficient cells, the effects of Compound 3 and PMA on ACE-secretase activity in
TACE-deficient cells were studied. The wild-type and the mutant cells
were transfected with ACET cDNA, pulse-labeled with
[35S]methionine, and chased in the absence (Fig.
1B, lanes 3 and 4) or in
the presence of Compound 3 (lanes 5 and
6) or PMA (lanes 7 and 8),
added to the culture media. PMA increased secretion moderately, but
significantly (p < 0.05), whereas Compound 3 inhibited secretion almost completely (Fig. 1B and Table
I). Thus, the ACE-secretase present in
TACE-deficient cells exhibited biochemical characteristics similar to
those of wild-type cells. It should be noted that, unlike in ACE89
cells (4), the enhancement of secretion by PMA in fibroblasts is less
pronounced.
We have generated (7) a chimeric type 1 ectoprotein
(ACET/CD4-5F) containing the distal extracellular domain
of ACET and the membrane-proximal, transmembrane, and
cytoplasmic domains of CD4, another type 1 ectoprotein that is not
cleavage-secreted. This chimera was cleaved extremely efficiently, with
a rate of secretion much higher than the natural substrate,
ACET. The cleavage occurred in the membrane-proximal CD4
sequences. We transfected TACE-deficient and wild-type cells with
ACET/CD4-5F construct to study its secretion in the
absence of TACE. As shown in Fig. 2A, ACET/CD4-5F
was also cleavage-secreted in a similar fashion (lanes
3 and 4) from wild-type and TACE-deficient cells.
We have shown previously that transfected HeLa cells secreted
ACET/CD4-5F more efficiently than ACET.
ACE-secretase present in either TACE-deficient or wild-type fibroblasts
exhibited similar characteristics, as 45-55% ACET/CD4-5F
as compared with 18-16% of ACET was secreted by these
cells (Table I). Cleavage and secretion was stimulated by PMA (Fig.
2A, lanes 7 and 8) and
inhibited by Compound 3 (lanes 5 and
6). Thus, the TACE-deficient cells can cleave and secrete two different substrates of ACE-secretase, ACET and the
chimeric protein ACET/CD4-5F. In addition, the cleavage
and secretion of both substrates was stimulated by PMA and inhibited by
Compound 3 to the same extent in both wild-type and TACE-deficient
cells (Table I).
Soluble ACEP Is Generated in Vivo in TACE-deficient
Mice--
ACEP is present in the body both as a
cell-associated protein in endothelial, epithelial, and monocytic cells
and as a soluble protein in various body fluids, including serum. To
delineate the mechanism by which soluble ACEP is produced
in vivo, we have previously analyzed soluble
ACEP present in rabbit serum and cell-associated ACEP in tissues such as lung and kidney. From these, we
have established that ACEP secretion in vivo is
caused by the cleavage removal of the C-terminal region of the
cell-associated protein (6). To characterize the proteolytic enzyme
responsible for ACEP secretion, we employed rabbit renal
proximal tubular epithelial cells and demonstrated significant
inhibition of secretion by Compound 3. These and other (13) results
suggest that soluble serum ACE is generated by an ACE-secretase-like
enzyme. Thus, if TACE is the major protease in the body that generates
soluble ACEP, then serum obtained from a TACE-deficient
mouse should have little or no soluble ACEP. But if soluble
ACEP levels are comparable in wild-type and mutant mice, it
would indicate that ACE cleavage and secretion was unaffected by the
absence of TACE and was carried out instead by a different enzyme,
ACE-secretase.
To test this hypothesis, we quantitated, by enzyme activity assay, the
soluble ACEP present in serum samples from TACE-deficient mice and their normal littermates. Although most TACE-deficient mice
die at birth, we were able to collect serum from three such mutant
mice, and as shown in Table II,
comparable amounts of soluble ACEP were present in the
serum of normal and TACE-deficient mice (390 and 302 units/ml of serum,
respectively). These results indicate that TACE is not the major enzyme
responsible for solubilization of ACEP in vivo.
The slightly lower level of soluble ACEP consistently observed in mutant mice might indicate that in vivo TACE
could play a minor role in ACEP production. This
possibility was further explored in the experiments described
below.
Co-expression of ACET and TACE in TACE-deficient
Cells--
Experiments described above using TACE-deficient cells, and
serum from mice devoid of TACE suggest that TACE is not required, in vitro or in vivo, for cleavage and secretion
of either isozyme of ACE. The next experiment was designed to determine
if a TACE-like protein might participate in solubilization of ACE. For
this purpose, the TACE-deficient cells were transfected with
ACET cDNA and cleavage and secretion of
ACET by these cells was compared with those that were
co-transfected with TACE cDNA as well. The cell extracts and the
media were immunoprecipitated and analyzed by SDS-PAGE (Fig.
2B). In these experiments, secretion was quantitated in two
different ways. In addition to the PhosphorImager analysis of
immunoprecipitated ACE, the cell extracts and the culture media were
also assayed for ACE enzyme activity. Both methods of estimating secretion indicated (Table III) that
co-expression of TACE with ACE in these cells increased ACE secretion
to some extent (from 49-58% to 71%). Thus, although TACE is not
required for ACE secretion, under certain circumstances ACE could be a
substrate for TACE. Indeed, TACE was able to cleave a synthetic peptide
spanning the cleavage site of ACET when used in large
excess (1:100 relative to ACE; data not shown). It should be noted that
the rate of PMA-stimulated ACET secretion in the
experiments described in Table III was significantly higher than rates
observed in the experiments shown in Fig. 1B and Table I
(28% versus 49-58%). The reason for this discrepancy is
not yet clear. One possible explanation is that a cytomegalovirus promoter-driven system (Fig. 2B and Table III) instead of a
vaccinia virus T7 polymerase-based system (Fig. 1, A and
B, and Table I) was used for the co-transfection experiment,
leading to different levels of protein expression, which in turn might
affect secretion.
Solubilization of ACE-secretase Activity--
The next series of
experiments were designed to solubilize ACE-secretase activity from
membranes, the first step toward its purification. Previously, we have
developed an in vitro cell-free assay system for the
measurement of ACE-secretase activity in membranes. Two different
methods were used by which uncleaved and cleaved ACET (the
substrate and the product, respectively, of the putative enzyme) were
distinguished and quantitated. In the first method, the two
[35S]methionine-labeled ACE proteins were quantitated by
PhosphorImager analysis after separation by PAGE. In the second method,
the two forms were physically separated and quantitated by ACE enzyme activity measurements. Here we utilized both these methods to assay
ACE-secretase activity solubilized from membranes isolated from either
ACE89 cells or rabbit lung by detergent extraction.
In ACE89 cells, the enzyme ACE-secretase cleaves cell-bound
ACET near its C terminus (between Arg663 and
Ser664 to generate the soluble, C-terminally truncated form
of ACE. Cell-bound uncleaved ACET and secreted, cleaved
ACET differ in molecular mass by 8 kDa. Being glycosylated,
both forms appeared as broad diffuse bands and could not be resolved
into separate bands by PAGE analysis after immunoprecipitation.
However, when these proteins were completely deglycosylated by
treatment with glycosidases after immunoprecipitation but
before PAGE analysis, the cleaved and the uncleaved forms
resolved clearly into two separate bands (5). We quantitated these
bands by PhosphorImager analysis and thus measured the rate of cleavage
and secretion. Membranes isolated from
[35S]methionine-labeled ACE89 cells, when analyzed by the
deglycosylation-PAGE analysis method, showed that almost all the ACE
proteins present in the membrane preparation were of the uncleaved,
84-kDa variety (5) (Fig. 3A,
lane 1). Much of the membrane-bound
ACET was cleaved when the membranes were incubated at
37 °C (lane 2), indicating the presence of
ACE-secretase activity. No such cleavage was observed if Compound 3 was
present during incubation, confirming its authenticity (lane
3). To solubilize ACE-secretase, these labeled membranes were extracted with the nonionic detergent Triton X-100, and the detergent extract was assayed for ACE-secretase activity in a similar
manner. Unlike incubation containing membranes (lane
2), generation of cleaved ACE was not observed when
detergent extracts of membranes were incubated at 37 °C
(lane 4). This indicated that ACE-secretase
either was not extracted from the membrane or was rendered inactive
during the extraction procedure. A third possibility is that the
interaction of ACE-secretase and ACE is hampered in the
detergent-solubilized state. Indeed, it has been shown that, although
solubilized TACE cleaves various peptide substrates in solution, it
failed to cleave any of its putative full-length protein substrates
(other than TNF-
To examine the cleavage and secretion of ACEP by
solubilized ACE-secretase, membranes were prepared from rabbit lung, an
organ rich in vascular tissue and thus in ACEP. Detergent
extracts of rabbit lung membranes were prepared and incubated, as
described above, to measure the ACE-secretase activity. Cleaved and
uncleaved ACEP proteins were physically separated by
temperature-induced phase separation using Triton X-114. This method
takes advantage of the fact that uncleaved ACE is hydrophobic in
nature, whereas cleaved ACE, having lost its transmembrane domain, is
hydrophilic. The separated ACEP proteins were quantitated
by ACE activity measurements. Assayed by this method (Table
IV), 55% of ACEP in rabbit
lung membrane was converted to the cleaved form when incubated at
37 °C. Very little cleaved ACE was generated (8%) at 4 °C. When
the detergent extracts of these membranes were incubated in a similar fashion at 37 °C, only 17% of ACEP was cleaved, but
inclusion of lisinopril-Sepharose in the incubation increased
conversion to the cleaved form to 80%. Incidentally, significant
conversion was observed even at 4 °C in the presence of
lisinopril.
Immunological Characterization of the Product Generated by
Solubilized ACE-secretase--
To characterize the product generated
by the solubilized ACE-secretase, we took advantage of the two
different antibodies we used in experiments described in Fig.
1A. Detergent extract of membranes prepared from
metabolically labeled ACE89 cells were incubated at 37 °C for 1 h in the presence of lisinopril-Sepharose. The ACE proteins were
immunoprecipitated, deglycosylated, and analyzed by SDS-PAGE. The
resolved proteins were transferred to a nitrocellulose membrane and
autoradiographed to detect the labeled uncleaved and cleaved
ACET proteins. The same blot was subjected to Western
analysis using the two antibodies described above. The autoradiogram
(Fig. 3B, lane 1) showed that
approximately 40% of the initial uncleaved ACET had been
converted to the cleaved form, both of which reacted with the anti-ACE
antibody in the Western analysis (lane 2). In
contrast, the anti-C-terminal peptide antibody recognized only the
upper band, i.e. the uncleaved ACET, but not the
lower band, the cleaved ACET (lane 3),
indicating that the C-terminal tail is missing from the cleaved form.
Thus, similar to the ACET secreted by ACE89 cells in
culture or the cleaved ACET generated in the in
vitro assay system using ACE89 membranes (5), the ACET
generated by the solubilized ACE-secretase is also C-terminally truncated.
The results presented above, demonstrating the solubilization of
ACE-secretase, constitute a major technical advancement toward identification of this enzyme. The observed need for anchoring of the
substrate protein on a solid support for the cleavage to occur displays
a unique property of the catalysis process. The property of the
solubilized system mimics the physiological reaction between the enzyme
and the substrate, both of which are presumably anchored in the plasma
membrane. This matrix-reconstituted soluble system will be used in the
future to purify the ACE-secretase.
From our previous studies and the information in the literature, there
were strong indications that there are important differences in the
properties of different secretases. Cleavage sites are often different.
Moreover, for ACE-secretase, the distal extracellular domain of ACE was
shown to be the important determinant that governs cleavage and
secretion; a protein containing the distal extracellular domain of ACE
and the juxtamembrane, transmembrane, and cytoplasmic domains of CD4
(another type 1 ectoprotein that does not undergo cleavage and
secretion) was efficiently cleaved from the cell surface. The
reciprocal protein, which contained the extracellular domain of CD4
attached to a membrane-anchored ACE protein containing the authentic
cleavage site, was not at all cleaved (7). In contrast to these
results, for TGF--converting enzyme (TACE), a
recently purified disintegrin-metalloprotease, has been implicated in
the proteolytic cleavage of several cell surface proteins. Mice devoid
of TACE have been developed by gene targeting. Such mice could provide
a useful system to determine if TACE is responsible for the cleavage of
other ectoproteins. Cultured fibroblasts without TACE activity, when
transfected with cDNA encoding for the testicular isozyme of ACE
(ACET), synthesized and secreted ACET
normally after a proteolytic cleavage near the C terminus. In addition, similar quantities of the soluble, C-terminally truncated somatic isozyme of ACE (ACEP) were present in the serum of
wild-type and TACE-deficient mice. These results demonstrate that TACE
is not essential in the generation of soluble ACE under physiological conditions. Finally, we also report solubilization of ACE-secretase, the enzyme that cleaves ACE, from mouse ACE89 cells and from rabbit lung. We demonstrate that soluble ACE-secretase from both sources failed to cleave its substrate in solution, suggesting a requirement for anchoring to the membrane.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(TNF-
),1 involved in
inflammatory responses, several cytokine receptors,
-amyloid
precursor protein (
-APP) implicated in Alzheimer's disease, a
number of growth factors and their receptors, cell adhesion molecules,
and the vasoregulatory enzyme angiotensin-converting enzyme (ACE) (1,
2). Since a number of these proteins are involved in disease processes
such as inflammation, neurodegeneration, hypertension, apoptosis,
and oncogenesis, the secretases could also provide novel therapeutic targets.
, TNF-
,
L-selectin, interleukin-6 receptor
-subunit, and
-APP
(14). These observations led to the suggestion that a single secretase
or a small number of similar secretases rather than multiple
independent secretases are involved in the shedding process. The most
well characterized secretase is TNF-
-converting enzyme (TACE), the
one that cleaves proTNF-
. TACE has recently been purified and cloned
(15, 16), and mice lacking this protease were generated (17).
TACE-deficient cell populations derived from these mutant mice are
grossly deficient in shedding of several other ectoproteins in addition
to TNF-
. These include TGF-
, L-selectin, p75 TNF receptor, and
-APP (17-19). These results reinforce the suggestion that the
shedding process, which involves various structurally and functionally
diverse proteins, is probably mediated by the same or a similar type of proteases.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-[1-(S)-carboxy
3-phenylpropyl]-L-lysyl-L-proline) was
provided by Merck Sharp and Dohme Research Laboratories (Rahway,
NJ). Compound 3 (N-(dl-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl)-L-3-(tert-butyl)-alanyl-L-alanine,2-aminoethyl amide) was provided by Immunex Research and Development (Seattle, WA).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
following transfection of DNA encoding proTNF-
(19). ACE is not
naturally expressed in fibroblasts (data not shown); thus, to determine the ability of these mutant cells to secrete ACE, we transiently transfected the TACE-deficient and the wild-type fibroblasts with ACET cDNA. The synthesis and secretion of transfected
ACET were studied by Western blot analysis using an
anti-ACE antibody. The cell extracts (Fig.
1A, lane
1) and culture media (lane 2) of mutant (right panel) and wild-type
(left panel) cell populations exhibited
comparable amounts of ACET, indicating that the mutant cells are not deficient in expressing ACET and that both
cell types secreted ACET in a comparable fashion as well.
The secreted ACET exhibited a slightly greater
electrophoretic mobility than the corresponding cell-associated form
(compare lanes 1 and 2 in both panels). Our
previous results suggested that this was due to the cleavage removal of
the membrane-anchoring C-terminal segment of ACE during the secretion
process. We tested this directly by using an anti-peptide antibody that
recognizes an epitope near the C terminus of ACE. Although the anti-ACE
antibody recognized both the cell-bound and secreted ACET,
the anti-peptide antibody recognized the cell-bound form but not the
secreted form of ACET (lanes 3 and
4, respectively, in both panels), indicating that the
secreted ACE is missing the C-terminal fragment. These results demonstrate that mutant cells that cannot efficiently secrete TNF-
because of the absence of TACE are able to cleave and secrete ACET as efficiently as the wild-type cells.
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Fig. 1.
Cleavage and secretion of ACET
from wild-type and TACE-deficient cells. Wild-type (+/+) and
TACE-deficient ( /
) fibroblasts were transiently transfected with
ACET cDNA using the vaccinia virus T7 RNA polymerase
system. A, 4 h after transfection, cell extracts
(C) and culture media (M) were analyzed by
SDS-PAGE followed by Western blot analysis using anti-ACE
(lanes 1 and 2) or anti-C-terminal
peptide (lanes 3 and 4) antibodies.
B, following transfection, cells were pulse-labeled with
[35S]methionine for 30 min, and the label was chased for
4 h in the absence (lanes 3 and
4) or presence of Compound 3 (lanes 5 and 6) or PMA (lanes 7 and
8). Cell extracts (C) and culture media
(M) were immunoprecipitated with anti-ACE antibody and
analyzed by SDS-PAGE. Lanes 1 and 2,
without cDNA. The molecular mass of the mature ACET is
indicated in kilodaltons. Secretion was quantitated by PhosphorImager
analysis (see Table I).
Quantitation of ACET and ACET/CD4-5F secretion from
wild-type (+/+) and TACE-deficient (/
) fibroblasts
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Fig. 2.
A, cleavage and secretion of
ACET/CD4-5F from wild-type and TACE-deficient cells.
Wild-type (+/+) and TACE-deficient ( /
) fibroblasts were transfected
with ACET/CD4-5F cDNA using vaccinia virus T7 RNA
polymerase system. Pulse-chase analysis was performed in the absence
(lanes 3 and 4) or presence of
Compound 3 (lanes 5 and 6) or PMA
(lanes 7 and 8), followed by
immunoprecipitation, as described in the legend for Fig. 1B.
The molecular mass of the chimeric protein is indicated in kilodaltons.
B, cleavage and secretion of ACET from
TACE-deficient cells co-expressing ACET and TACE.
TACE-deficient fibroblasts (
/
) were transiently co-transfected with
either ACET and an irrelevant cDNA (lanes
3 and 4) or co-transfected with ACET
and TACE cDNA (lanes 5 and 6).
Lanes 1 and 2, no cDNA. Unlike the
experiments described in Figs. 1 and 2A, a cytomegalovirus
promoter-driven system was used in these experiments (22). Cells were
pulse labeled with [35S]methionine and chased in the
presence of PMA. Cell extracts (C) and culture media
(M) were immunoprecipitated and analyzed, as described for
Fig. 1B. Quantitation of secretion by PhosphorImager
analysis is presented in Table III (left
panel).
ACEp activity in plasma of wild-type (+/+) and
TACE-deficient (/
) mutant mice
Co-transfection of ACET and TACE in TACE-deficient fibroblasts
) when both the substrate and TACE were in solution
(17). This is consistent with the evidence that shedding generally
requires the anchoring of the secretase and its target substrate (or
both) in the plasma membrane. Thus, we immobilized soluble ACE on
agarose beads by the lectin ConA or lisinopril, the competitive
inhibitor of ACE, by including ConA-agarose or lisinopril-Sepharose in
the incubation. Under both conditions, much of the uncleaved ACE
present in the detergent extract was converted to the cleaved form
(lanes 5 and 6). Again, the presence
of Compound 3 completely inhibited this conversion (lane
7, data for lisinopril-Sepharose and Compound 3 not
included). These results indicate that ACE-secretase, similar to TACE,
is unable to cleave its substrate in solution. However, if either the
substrate or the secretase (or both) was anchored to a solid surface,
such as agarose beads, efficient cleavage was observed. This cleavage
was blocked in the presence of Compound 3, confirming its authenticity.
Incidentally, much more cleaved ACE was generated when
lisinopril-Sepharose rather than ConA-Sepharose was used (compare
lanes 6 and 5). In both situations,
however, as determined in separate experiments, all of the ACE present in the detergent extract was bound to agarose beads (data not shown).
The reason for this difference is not known. In fact, different domains
of the ACE molecule are involved in binding to lisinopril or to ConA,
which in turn might alter the accessibility of ACE-secretase,
explaining the difference in efficiency of cleavage.
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Fig. 3.
Solubilization of ACE-secretase activity from
ACE89 cell membranes. A,
[35S]methionine-labeled ACE 89 cell membranes
(lanes 1-3) or Triton X-100 extract of the
labeled membranes (lanes 4-7) were incubated at
37 °C with added ConA-Sepharose (lanes 5 and
7), lisinopril-Sepharose (lane 6),
and/or Compound 3 (lanes 3 and 7, 50 µM). ACE-related proteins were immunoprecipitated,
deglycosylated, and analyzed on SDS-PAGE. The molecular mass of the
deglycosylated uncleaved ACET is shown on the
left. B, immunological characterization of the
product generated by solubilized ACE-secretase. Detergent extracts of
[35S]methionine-labeled ACE89 cell membranes were
incubated at 37 °C in the presence of lisinopril-Sepharose, followed
by immunoprecipitation, deglycosylation, SDS-PAGE, and transfer to
nitrocellulose membranes. Lane 1 shows a
autoradiogram of the blot. Lanes 2 and
3 show results of Western analysis performed by using an
anti-ACE antibody (lane 2) or an anti-C-terminal peptide
antibody (lane 3).
ACE-secretase activity in detergent extract of rabbit lung membranes
precursor and
-APP, the determinants are the
juxtamembrane domains. The structurally unrelated juxtamembrane domains
of the two proteins can mediate cleavage and secretion of an uncleaved
protein when substituted for the corresponding domain of that protein
(23). These important differences between ACE-secretase and the
secretase(s) that cleave TGF-
precursor or
-APP, on the other
hand, implicate involvement of multiple proteases. The current study
establishes in a definitive way that ACE-secretase is distinct from
TACE. Future isolation and cloning of ACE-secretase will reveal whether
the two enzymes bear a structural resemblance. Similarly, further
studies will be required to identify the secretases that catalyze the
cleavage and secretion of other membrane-bound proteins. One can
speculate that many related but distinct proteins belong to this family
of enzymes that carry out similar functions in cells.
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ACKNOWLEDGEMENT |
---|
We are grateful to Ganes C. Sen for providing constructive suggestions and critical reading of the manuscript. We thank JoAnne Holl for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL54297 and by a fellowship from the American Heart Association, Northeast Ohio Affiliate.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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of Molecular
Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation,
9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-9057; Fax:
216-444-9263; E-mail: seni{at}cesmtp.ccf.org.
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ABBREVIATIONS |
---|
The abbreviations used are:
ACE, angiotensin-converting enzyme;
ACET, testicular ACE;
ACEP, pulmonary ACE;
PAGE, polyacrylamide gel
electrophoresis;
TNF-, tumor necrosis factor-
;
TGF-
, transforming growth factor-
;
TACE, tumor necrosis
factor-
-converting enzyme;
-APP,
-amyloid precursor protein;
PMA, phorbol 12-myristate 13-acetate;
ConA, concanavalin A.
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REFERENCES |
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