(Received for publication, June 9, 1995; and in revised form, August 24, 1995)
From the
Angiotensin-converting enzyme (ACE) belongs to the type I class
of ectoproteins and is solubilized by Chinese hamster ovary cells
transfected with the full-length human ACE cDNA. ACE release in Chinese
hamster ovary cells involves a proteolytic cleavage occurring in the
carboxyl-terminal region, between Arg-1137 and Leu-1138. The
subcellular localization of ACE proteolysis was established by
pulse-chase experiments, cell surface immunolabeling, and biotinylation
of radiolabeled mature proteins. The proteolysis of ACE takes place
primarily at the plasma membrane. The solubilization of ACE is less
than 2% within 1 h, is increased 2.4-fold by phorbol esters, but is not
influenced by ionophores. An ACE mutant lacking the transmembrane
domain and the cytosolic part (ACE), is secreted at
a faster rate without a carboxyl-terminal cleavage, and phorbol esters
or ionophores have no effect on its rate of production in the medium.
Therefore, the proteolysis of ACE is dependent on the presence of the
membrane anchor and suggests that the secretase(s) involved is also
membrane-associated. An ACE mutant lacking the amino-terminal domain
(ACE
) is secreted 10-fold faster compared with wild-type
ACE. The solubilization of ACE
occurs at the plasma
membrane and is stimulated 2.7-fold by phorbol esters, and the cleavage
site is localized between Arg-1227 and Val-1228. The amino-terminal
domain of ACE slows down the proteolysis and seems to act as a
``conformational inhibitor'' of the proteolytic process,
possibly via interactions with the ``stalk'' of ACE and the
secretase(s) itself.
Proteolytic release of the ectodomain of carboxyl-terminally
anchored proteins (type I class ectoproteins) leads to the production
of the protein in the extracellular space and, in some cases, in
biological fluids. It is a widespread phenomenon(1) ,
concerning several proteins with different biological functions such as
membrane-anchored growth factors like pro-transforming growth factor
(TGF-
)(
)(2) , the c-kit
ligand(3) , growth factor receptors like the tumor necrosis
factor receptor(4) , cell adhesion molecules(1) ,
ectoenzymes such as angiotensin I-converting enzyme (ACE) (1) or lactase-phlorizin hydrolase (5) and the amyloid
precursor protein (APP)(6, 7) . However, little is
known about the molecular basis of this secretion. A common mechanism
involving a processing enzyme named ``secretase'' has been
proposed(1, 2, 6) . The characteristics of
this putative enzyme, which could be itself membrane-associated are
broad sequence specificity (2, 6) and cleavage at a
certain distance from the membrane(8) .
ACE (EC 3.4.15.1) is a zinc-dipeptidyl carboxypeptidase (9) that also displays endopeptidase activity on some peptides(10) . ACE is an ectoenzyme found in most mammalian tissues bound to the external surface of the plasma membrane of endothelial, epithelial, neural, and neuroepithelial cells.
There are two ACE isoforms derived from a
single gene, by the transcription from two alternative
promoters(11, 12) , which are expressed in a
tissue-specific fashion. In somatic tissues, ACE is expressed as a
glycoprotein composed of a single polypeptide chain with an apparent
molecular mass of 170 kDa, containing two large homologous domains,
called the N and C domains, each domain bearing an active catalytic
site(13, 14) ; in male germinal cells, ACE is
synthesized as a lower molecular mass form of 110 kDa containing only
the active C domain of endothelial ACE(15) . Both isoforms
belong to the type I class of integral transmembrane ectoproteins (1, 16) with a large amino-terminal extracellular
domain, a 17-amino acid hydrophobic anchor located 30 amino acid
residues from the cytosolic carboxyl terminus (13) . The role
of this -helix in the membrane anchoring of ACE has been
demonstrated by studying a carboxyl-terminal truncated cDNA
(ACE
), which is secreted faster in the medium of
CHO cells(17) .
ACE is solubilized and circulates in many body fluids such as serum (18) . The source of circulating ACE in plasma is thought to be derived mainly from endothelial pulmonary cells. Wei et al.(17) showed that the full-length endothelial recombinant ACE cDNA, expressed in CHO cell lines, is secreted into the culture medium from the membrane form by a post-translational proteolytic cleavage occurring in the carboxyl-terminal region. Recently(19) , we have established by carboxyl-terminal microsequencing the same carboxyl terminus sequences AGQR for secreted recombinant ACE and for human plasma ACE, which corresponds to a cleavage site between Arg-1137 and Leu-1138 (see Fig. 1).
Figure 1:
Wild-type ACE,
carboxyl-, and amino-terminally truncated mutants. A, diagram
of human ACEs encoded by the cDNA constructions. Human wild-type ACE,
encoded by the full-length cDNA, is composed of 1277 residues,
comprising the N and C homologous domains (egg-shaped
boxes) in the extracellular region and the hydrophobic segment of
17 amino acids near the carboxyl terminus (square boxes). The
carboxyl-terminally truncated ACE (ACE) comprises 1230 residues.
The 47 amino acids deleted from the carboxyl terminus correspond to the
transmembrane and the cytosolic regions. The amino-terminally truncated
ACE (ACE
) comprises residues 1-4
of the amino terminus followed by residues 572-1277 of mature ACE.
Antiserum Y1 directed against pure human kidney ACE is shown.
Sequences of synthetic peptides used for the production of antisera
28A, Clo, 5, and 3 are also shown in the diagram. B,
carboxyl-terminal sequence of ACE from Cys-1114 to Ile-1254. Cleavage
sites of human endothelial ACE (R1137), rabbit testicular ACE (R1203), and ACE
(R1227) are shown by an arrow. The hydrophobic domain is boxed.
Sen et al.(20) showed that the rabbit testicular ACE cDNA expressed in a mouse epithelial cell line is secreted in the culture medium by a proteolytic processing of its carboxyl-terminal region. The cleavage of the rabbit testicular ACE also occurs at a monobasic site between Arg-663 and Ser-664(21) . This cleavage site corresponds in endothelial human ACE to positions Arg-1203 and Ser-1204, downstream from the cleavage site we observed for the full somatic ACE (Fig. 1). The mutation of Arg-1137 to a glutamine residue did not prevent the secretion of ACE(19) . Altogether, these findings suggest several hypotheses since the enzyme(s) responsible for ACE cleavage is (are) not identified. 1) A single enzyme of broad specificity could be involved, which could accommodate an Arg to Gln substitution in the P1 site of the human ACE. 2) Several related enzymes could be implicated. 3) Species differences could account for the differences in the solubilization of human and rabbit ACE observed in the two cell lines.
The subcellular localization of the proteolytic cleavage of endothelial ACE in CHO cells is unknown and could occur intracellularly or at the plasma membrane. The molecular mechanisms of its regulation are also unknown.
The aim of the present study was to identify the
subcellular localization of the proteolytic cleavage of ACE and its
regulation by metabolic labeling in CHO cell lines permanently
transfected with the cDNA of endothelial ACE. This study shows that the
solubilization takes place at the plasma membrane and that ACE
solubilization under basal conditions is low and enhanced by phorbol
esters but not by ionophores, implying a calcium-independent protein
kinase C. The solubilization rate also depends on the presence of the N
domain; an amino-terminal truncated mutant (ACE) was
secreted 10-fold faster compared with the wild-type enzyme comprising
both domains, and its cleavage site was different.
After metabolic labeling and chase
periods, the culture medium was collected and centrifuged at 1,000
g for 5 min at 4 °C, and EDTA was added to the
resultant supernatant to a final concentration of 10 mM. Cell
extracts were prepared by washing labeled cells with ice-cold
phosphate-buffered saline (PBS) and were solubilized in 0.5 ml of lysis
buffer/dish, 1% Triton X-100 in 50 mM Tris-HCl buffer, pH 7.4,
containing 150 mM NaCl, 10 mM EDTA, and 0.15% SDS for
30 min at 4 °C. Cell lysates were collected, and insoluble material
was removed by centrifugation for 10 min at 10,000
g at 4 °C. Cell lysate supernatants and media were used
immediately or stored at -70 °C until further use.
Three different rabbit antisera were used for immunoprecipitation (see Fig. 1). Antiserum Y1 was obtained from rabbits immunized against pure human kidney ACE(13) . Antisera 28A and Clo were raised in rabbits against synthetic peptides corresponding to amino acids 1258-1277 at the carboxyl terminus and 1-18 at the amino terminus of the human endothelial ACE sequence, respectively(14, 19) . Cell lysates or culture media were incubated overnight at 4 °C with undiluted antisera and protein A-Sepharose (Pharmacia Biotech Inc.) (50 µl of a 50% suspension in lysis buffer). The immune complex protein A-Sepharose was collected by centrifugation and washed 4 times with 0.1% Triton X-100 in 50 mM Tris-HCl containing 1 mM EDTA, 0.15% SDS and once with 20 mM Tris-HCl, pH 6.8, and then dried. Samples were heated for 5 min at 100 °C in 25 µl of SDS-PAGE electrophoresis sample buffer. The Sepharose was removed by centrifugation, and proteins were resolved by 6 or 10% SDS-polyacrylamide gel electrophoresis (24) and revealed by autoradiography. Proteins were quantified from autoradiograms using a video densitometer (MC View Color, Agfa).
In some experiments a two-step immunoprecipitation method was used. Cells were pulse-labeled as described above and then chased in some cases in a serum-free medium for 4 or 16 h. Cell lysates (20 µl) were immunoprecipitated overnight with the antibody 28A, at the concentrations indicated in the figure legends. The immune complex protein A-Sepharose was removed, and the supernatant was heated at 100 °C for 5 min and immunoprecipitated for 4 h with antibody Y1 (3 µl).
Figure 2:
Analysis of cellular ACEs isoforms by
immunoprecipitation. A and B, ACE and
ACE cells were pulse-labeled 30 min without chase
to obtain the EHS forms of proteins, localized in the endoplasmic
reticulum. The nonglycosylated forms (NG) were obtained after
endo-H deglycosylation of these EHS precursors. Cells were chased 16 h
to determine the mature, N-glycosylated, and EHR forms of ACE
and ACE
, which are localized in the post-Golgi
compartment. C, ACE
cells were pulse-labeled 15
min without chase for EHS forms and chased 4 h to obtain EHR forms. The
nonglycosylated forms derived from endo-H deglycosylation of EHS forms.
Proteins were immunoprecipitated with antibodies Y1 (3 µl) or 28A
(3 µl), as indicated. Numbers on the left indicate positions of molecular
weights.
The two-step immunoprecipitation (tsIP) of EHS form of ACE showed that the protein was entirely immunoprecipitated with increasing concentrations of the 28A antibody (Fig. 3); all expressed EHS forms of ACE were immunoprecipitated with 5 µl of the 28A antibody since the subsequent immunoprecipitation step using the Y1 antibody (3 µl) did not detect ACE, indicating that all of the protein is in a membrane-anchored form.
Figure 3: Two-step immunoprecipitation of endo-H sensitive forms of ACE. Cells were pulse-labeled 30 min without chase to obtain endo-H sensitive forms. The same volume of cell lysates (20 µl) were first immunoprecipitated overnight without or with increased concentrations of 28A, from 0.01 to 5 µl. The supernatant of the first immunoprecipitation was heat-denaturated and then immunoprecipitated with Y1 (3 µl) for the tsIP procedure. The 28A and Y1 immune complexes were resolved by SDS-PAGE. Results are expressed as the percentage (% ±S.D., n = 3) of cell lysates immunoprecipitated with 28A. Inset, autoradiograms of two tsIP are shown. One tsIP experiment is represented with bracket; the first lane is the result of the first immunoprecipitation with the 28A antibody, the second lane is that of the supernatant immunoprecipitation with Y1 antibody (3 µl) and are indicated by 28A/Y1. Cell lysates were first immunoprecipitated without (0) or with 5 µl of 28A (5).
Brefeldin A (BFA) induces a resorption of the Golgi into the ER, blocks intracellular transport, and increases the protein pool from the ER into an intermediate compartment(25, 26) . BFA was used to confirm the absence of ER proteolytic cleavage. A 4-h pulse-chase with BFA (5 µg/ml) showed that ACE appeared exclusively as an intermediate glycosylated protein of 150 kDa (Fig. 4A). In cells, BFA-treated ACE cross-reacted with Y1 and 28A antibodies. In the medium, BFA treatment inhibited the secretion of ACE (data not shown). The 28A antibody (5 µl) immunoprecipitated all BFA-treated ACE cell lysates, as shown by the two-step immunoprecipitation (Fig. 4A).
Figure 4: Subcellular localization of ACE proteolysis: endoplasmic reticulum and Golgi compartments. A, cells were labeled and chased for 4 h in the absence(-) and presence (+) of BFA (5 µg/ml), and then 20 µl of cell lysates were immunoprecipitated with Y1 (3 µl). Moreover, a tsIP with 20 µl of BFA-treated cell lysates was performed as described in the legend to Fig. 3using 28A (5 µl) and Y1 (3 µl). B, cells were labeled and chased for 4 h. Cell cultures were shifted from 37 to 20 °C at the beginning of the chase period (20 °C) for 4 h. Cells were then harvested and immunoprecipitated with 3 µl of Y1. A tsIP was performed as described in the legend to Fig. 3using 20 µl of 20 °C cell lysates. ¢, cell lysates.
These results show that the ACE precursor forms are not carboxyl-terminally truncated, which excludes ACE proteolysis in the ER compartment.
When the cells were cultured at 20 °C, the newly synthesized proteins were retained in the trans-Golgi network, reducing the access to the plasma membrane(27, 28) . When CHO cells were pulse-chased at 20 °C for 4 h, the rate of biosynthesis and glycosylation was reduced by approximately 20-30% compared with a pulse-chase at 37 °C but was not qualitatively modified. Cellular ACE was present as two forms of 160 and 170 kDa (Fig. 4B), the tsIP show that cellular ACE at 20 °C was entirely immunoprecipitated with the antibody 28A (5 µl).
These results show that the proteolytic cleavage of ACE does not occur in the Golgi compartment or between the post-Golgi compartment and the plasma membrane since neither intracellular EHS or EHR forms of ACE are carboxyl-terminally truncated. The soluble ACE found in the culture medium is the only species that lacks the cytoplasmic domain as shown by its absence of reactivity with the 28A antibody.
Figure 5:
Plasma membrane localization of the
proteolytic cleavage. A, cells were labeled and chased for 16
h and then immunolabeled with Y1 (10 µl), 1 h at 4 °C, as
described under ``Materials and Methods.'' Lane 1,
cell lysates (¢) (500 µl) were directly immunoadsorbed on
protein A-Sepharose; lane 2, the supernatant of
immunoadsorption was heat-denaturated and then immunoprecipitated with
Y1 (3 µl); lane 3, Y1-immunocomplexes after direct
immunoadsorption were heat-denaturated and then immunoprecipitated with
28A (3 µl). B, cells were pulse-chased for 16 h and
biotinylated with the NHS-LC-Biotin cross-linker (1 mg/ml) for 1 h at 4
°C (¢) and immunoprecipitated with Y1 (3 µl)
or 28A (3 µl) (lanes 1 and 2). Cells were
maintained at 37 °C for 16 h, and then the medium (m
) was removed and immunoprecipitated
with Y1 or 28A (lanes 3 and 4). Biotinylated proteins
were revealed with streptavidin after immunoprecipitations. ¢,
cell lysates; m, medium.
These results indicate that only mature cellular ACE proteins reached the plasma membrane and that the intracellular carboxyl terminus of the protein is not exposed to the extracellular space.
Using this method, the biotinylated ACE solubilization at 1 and 16 h was estimated at 1.9 ± 0.3% and 13.8 ± 3.1%, respectively. Furhermore, ACE solubilization quantified by the ratio of total soluble ACE in the medium after 1 and 16 h of chase over the total initially labeled cellular ACE at the chase time 0 was superimposable, estimated at 1.5 ± 0.2 and 18 ± 3%, respectively. Thus, the secretion of ACE in the medium closely corresponds to that of proteolytic cleavage occurring at the plasma membrane.
Figure 6:
Effects of PMA and A23187 on ACE
solubilization. Cells were stably transfected with ACE cells grown to
near confluency and then shifted in a serum-free medium for 1 h with or
without pharmacological reagents (10M).
Then, a time-course of ACE secretion, from 15 to 60 min was performed.
The solubilization was estimated by the ratio of enzymatic activity in
the medium over enzymatic activity in cellular homogenates. Results are
expressed in percentage (n = 6, ±S.D.) of
solubilization. C, basal solubilization without
pharmacological reagents.
To test whether PMA treatment altered the
proteolytic process of ACE, a pulse-chase of 16 h was used. After this
period, cells were washed and then incubated for 1 h in the absence or
presence of PMA (10M). The secretion of
ACE was increased with PMA in the same range, 2.2 ± 0.3 (n = 3), and soluble proteins did not cross-react with the 28A
antibody (data not shown). These results suggest that the proteolysis
of ACE in CHO cells is controlled by a calcium-independent protein
kinase C.
The nonglycosylated form of
ACE migrated as a 135-kDa protein after endo-H
treatment (Fig. 2B). Intracellular traffic of
ACE
is similar to that of the wild-type ACE. The
EHS form of ACE
(155 kDa) is entirely converted in
the EHR form (165 kDa) after 16 h of chase (Fig. 2B). 8
h were required for conversion of half of the 30-min pulse-labeled ACE
and ACE
molecules to the EHR forms.
The secreted
form of ACE appeared in the medium after a 4-h chase, whereas the
secreted form of ACE appeared after a 1-h chase, in
the same experimental conditions (Fig. 7, A and B). Quantification of secretion using the ratio of total
soluble ACEs after a 16-h chase, over initially labeled cellular ACEs
at chase time 0, revealed that 97 ± 17% of the labeled
glycosylated ACE
was secreted compared with 18.5
± 1.8% of ACE after a chase of 16 h when 90% of cellular
proteins are mature.
Figure 7:
Pulse-chase analysis of the wild-type ACE,
ACE and ACE
. A and B, CHO-ACE and CHO-ACE
cells were
pulse-labeled with [
S]methionine-cysteine for 30
min and then chased with serum-free medium from 0 to 16 h. C,
CHO-ACE
cells were pulse-labeled for 15 min and then
chased from 0 to 4 h. An aliquot of cell lysates (50 µl; one-tenth)
and media (1 ml; one-half) were immunoprecipitated with Y1 (3 µl).
Solubilization was quantified by the ratio of total soluble forms in
the medium from 1 to 4 and 16 h of chase over the total initially
labeled cellular forms at the chase time 0.
ACE is not found at the
plasma membrane by cell surface immunolabeling and biotinylation (data
not shown). Western blot analysis showed that both antisera 5 and 3
cross-react with soluble forms of ACE
(Fig. 8), suggesting that ACE
is
secreted into the medium without modification of its carboxyl-terminal
end.
Figure 8:
Western blot analysis of
ACE and ACE
. Western blot analysis of
secreted ACE
and ACE
was performed
using antisera Y1, 5, 3, and 28A. Y1 and antiserum 3 cross-reacted with
soluble ACE
, thus indicating the absence of a
cleavage between Arg-1137 and Leu-1138. Antibody 3 cross-reacted with
soluble ACE
, consistent with the mutant being cleaved at
the carboxyl terminus between Arg-1227 and
Val-1228.
In addition, the percentage of ACE
secretion after 1 h, in absence of PMA, determined by enzymatic
activity, was 20.6 ± 1.01% (n = 6). No
differences were observed under PMA (10
M)
or A23187 (10
M) reagents: the secretion
was estimated, respectively, to 23.2 ± 1.4% and 19.2 ±
0.78%.
Thus, the proteolysis of wild-type ACE depends on the presence of its membrane-anchor and possibly on a plasma membrane-associated secretase.
Since, the rate of biosynthesis of ACE was faster than that of ACE, the conditions of the metabolic
labeling were adapted. Cells were labeled for 15 min with
[
S]methionine-cysteine and chased in serum-free
medium for 0.5, 1, 2, and 4 h (Fig. 7C). After 4 h of
chase, 90% of the initially labeled ACE
was converted to
the EHR form of the protein. Under these conditions, the biosynthesis
and secretion of ACE
could be compared with that of a 16-h
chase of ACE, where 90% of the initially labeled ACE was converted to
the EHR form of ACE.
The nonglycosylated form of the ACE migrated as a 75-kDa protein after endo-H treatment (Fig. 2C). ACE
was initially synthesized
as an EHS protein (95 kDa), which was entirely converted to an EHR form
(105 kDa) after 4 h of chase (Fig. 2C). 2 h were
required for conversion of half of the 15-min pulse-labeled initial
molecules to the EHR form.
The secreted ACE appeared in
the medium (Fig. 7C), after a 30-min chase. After 4 h
of chase, when 90% of initially labeled proteins were mature, 35% of
ACE
was secreted as a carboxyl-terminal truncated protein
with a decrease of 5 kDa in molecular mass (100 versus 105
kDa). Western blot analysis (Fig. 8) showed that both antisera 5
and 3 cross-react with the soluble form of ACE
, suggesting
that the cleavage site of ACE
is localized between
Arg-1227 and Val-1228 (see Fig. 1). This cleavage did not occur
intracellularly, as shown by the two-step immunoprecipitation (Fig. 9A).
Figure 9:
Subcellular localization of the
proteolytic cleavage of ACE. A, part 1,
CHO-ACE
cells were pulse-labeled for 15 min without chase
to obtain EHS forms localized in the endoplasmic reticulum; part
2, cells were pulse-labeled and chased for 4 h in presence of BFA; part 3, cells were pulse-labeled and chased for 4 h at 37 or
20 °C. In each case, a tsIP was performed as described in the
legend to Fig. 3with 28A (5 µl) and Y1 (3 µl). B, CHO-ACE
cells were labeled and chased for 4 h
and then immunolabeled with Y1 (10 µl) for 1 h at 4 °C. Cell
lysates were directly immunoadsorbed on protein A-Sepharose (lane
1). CHO-ACE
cells were pulse-chased for 4 h and
biotinylated as indicated in the legend to Fig. 5(¢
, lane 2). Biotinylated cells
were maintained at 37 °C for 1 h, in the absence(-) or
presence (+) of PMA (10
M). Then media (m
) were removed, and biotinylated
proteins were revealed, after immunoprecipitation with Y1 (3 µl) or
28A (3 µl) (lanes 3-6) by
streptavidin.
Like ACE, ACE is expressed at the
cell plasma membrane (Fig. 9B, lane 1) and was
studied by biotinylation (Fig. 9B, lanes
2-6). After 4 h of chase and 1 h of incubation at 37 °C
in a serum-free medium, 17.7 ± 9.2% of initially
ACE
biotinylated proteins appeared in the medium (Fig. 9B, lanes 3 and 4). This
protein did not cross-react with the 28A antibody.
Solubilization of
biotinylated ACE in the medium 1 and 4 h after the
biotinylation was 17.7 ± 9.2 and 35.1 ± 2%, respectively.
The solubilization, quantified by the ratio of soluble ACE
in the medium after 1 and 4 h of chase over the initially labeled
cellular ACE
at the chase time 0, was similar, estimated
at 19.4 ± 7% and 33.03 ± 2.7%, respectively. Thus, as for
wild-type ACE, the proteolytic cleavage of ACE
takes place
at the plasma membrane and occurs at the carboxyl-terminal end.
ACE solubilization is also stimulated by phorbol esters
since PMA (10
M) increases the
solubilization 2.7 ± 0.8-fold, which is inhibited by
staurosporin (10
M). The ionophore A23187
has no effect on ACE
solubilization (data not shown). In
order to verify that PMA stimulates the proteolysis of membrane-bound
ACE
, cells were pulse-labeled, chased 4 h and biotinylated
as described above. After 1 h in the presence of PMA (10
M), the solubilization of biotinylated ACE
was increased (Fig. 9B, lane 5) compared
with control (Fig. 9, lane 3). Moreover, soluble
biotinylated ACE
, after PMA treatment, did not cross-react
with the 28A antibody (Fig. 9, lane 6).
These data
show that basal ACE solubilization is clearly impaired by the presence
of the amino-terminal domain since deletion of this domain increases
the rate of solubilization of ACE without altering the
PMA-induced solubilization process.
The general phenomenon of the solubilization of
carboxyl-terminal membrane-anchored proteins involves a
carboxyl-terminal proteolytic cleavage that takes place near or at the
plasma membrane. This is the case for the secretion of the
carboxyl-terminally truncated secreted APP(8, 29) , or
TGF-(2) . The mechanism of ACE secretion in CHO cells
involves a proteolytic cleavage of the carboxyl-terminal part of the
protein between Arg-1137 and Leu-1138(19) . The present study
shows that the processing of membrane-anchored ACE operates primarily
at the cell surface. Indeed, all intracellular ACE retained in the ER
or in Golgi compartment before the membrane, cross-reacts with the
carboxyl-terminal antibody, and the two-step immunoprecipitation method
failed to detect any soluble intracellular forms of ACE even when
intracellular blockers (brefeldin A or lowering temperature to 20
°C treatments) were used. After 16 h of chase, 90% of initially
radiolabeled ACE was present as endo-H resistant form, N-glycosylated, and present at the plasma membrane.
Immunolabeling of mature radiolabeled ACE shows that the amino-terminal
part of ACE is exposed in the extracellular space, whereas the
carboxyl-terminal end remained intracellular, excluding the possibility
that the carboxyl terminus forms a hairpin loop and is exposed in the
extracellular space. Biotinylation of mature radiolabeled ACE present
at the cell surface clearly shows that the proteolytic cleavage of ACE
takes place at the plasma membrane since the biotinylated ACE secreted
in the medium is carboxyl-terminally truncated. The solubilization rate
estimated for biotinylated proteins was the same as that determined for
total cellular ACE. Therefore, the cleavage at the plasma membrane
appears to be the predominant pathway for the generation of soluble
ACE. However, we cannot exclude the possibility that some cleavage
takes place in a specialized vesicular compartment near the plasma
membrane via a degradation/recycling pathway, as described for
APP(30, 31) . If this processing occurs, it would
involve another sequence than the consensus endocytic sequence
SXYQRL, which is not present in the cytosolic part of ACE.
Experiments using an ACE mutant with its transmembrane -helix
and cytosolic parts deleted (ACE
) show a faster
rate of secretion than ACE, without a difference in glycosylation or
pattern of cellular traffic. This mutant is not expressed at the plasma
membrane and is not cleaved intracellularly between Arg-1137 and
Leu-1138, showing that membrane-anchoring of the protein and its
cytosolic region are necessary for the proteolytic cleavage to take
place at the plasma membrane, as for other proteins of this class.
Very little is known about the molecular mechanisms that may
influence the secretion rate of C domain anchored proteins. One general
finding, however, seems to be the increase in the solubilization rate
induced by phorbol esters. The solubilization of the membrane-bound
form of APP, pro-TGF-, and ACE in CHO cells, transfected with the
cDNA of APP, pro-TGF-
(32) , or ACE (present study) is low,
being 5, 1, and 2%, respectively, after 1 h of chase. The proteolytic
cleavage of pro-TGF-
(33, 34, 35) and
APP (36, 37, 38) is enhanced in both cases by
phorbol esters or ionophores. A series of experiments were designed to
evaluate this observation with respect to the ACE mutants. Proteolytic
cleavage of recombinant human ACE in CHO cells is stimulated 2.4-fold
by phorbol esters but is not influenced by ionophores. These results
are in agreement with those published by Sen and co-workers (21) on the phorbol ester-induced stimulation of rabbit
testicular ACE secretion. In the present study, the degree of this
stimulation does not depend on the amino-terminal part of ACE, since
phorbol ester stimulation is quantitatively similar to that observed
for ACE
. In addition, the secretion of ACE
is not regulated by phorbol esters and ionophores, suggesting
that the PMA effect depends on the membrane-anchoring of the protein.
Experiments using biotinylated mature radiolabeled ACE
show that phorbol esters stimulate the proteolysis of the
membrane-bound protein, suggesting that the implicated secretase(s) is
probably also membrane-associated, a hypothesis consistent with the
membrane-associated secretase(s) proposed for ACE(39) ,
APP(8, 40) , or pro-TGF-
(41) .
The
increased solubilization of ACE (endothelial or testicular) by phorbol
esters (2-fold over basal conditions) is 2.5 times less than the
solubilization observed for pro-TGF- or APP in CHO
cells(32) . Another difference is that the secretion of
pro-TGF-
and APP in CHO cells is stimulated by ionophores
involving a calcium-dependent protein kinase C, whereas a
calcium-independent protein kinase C appears to be implicated in the
proteolytic processing of rabbit testicular ACE (21) and
endothelial ACE (present study).
Each class I ectoprotein could
possess its own specific determinants to control the plasma membrane
proteolysis(2, 6) . One mechanism involved is the
primary structure of the cytoplasmic tail, since a specific role for
the carboxyl-terminal valine has been clearly demonstrated for
pro-TGF- (42) but is not involved in the solubilization of
the tumor necrosis factor receptor(43) . The role of the
cytoplasmic tail of ACE has not yet been described. A second mechanism
involved is the extracellular ectodomain of the protein. Deletion of
285 amino acids from the amino-terminal part of APP increases the
solubilization of secreted APP(8) . Construction of a chimeric
protein of APP, where amino acids 1-647 of APP ectodomain were
replaced by a human secreted alkaline phosphatase derivative, (44) shows that the amino-terminal part of APP could inhibit
the solubilization of secreted APP.
The role of the amino-terminal
part of APP (8) in its solubilization as well as the increased
rate of solubilization of testis ACE (21) compared with
endothelial ACE (present study) prompted us to study the specific
effect of the ACE amino-terminal domain in the solubilizing process.
The proteolytic release of ACE is increased 10-fold by the deletion of
the amino-terminal domain of ACE, reaching 35% (Fig. 10) after 4
h of chase. Several hypotheses could be envisaged to explain this
increase of solubilization. 1) The intracellular traffic of ACE could be faster than that of ACE, resulting in an artificial
increase in solubilization rate expressed as the ratio of soluble
ACE
in the medium over cellular ACE
. However,
ACE
is expressed at the plasma membrane, and biotinylated
mature ACE
is secreted in the medium after 1 h of chase
10-fold faster than ACE, under the same experimental conditions, i.e. when 90% of all mature proteins have reached the plasma
membrane. Moreover, ACE
, like ACE, is primarily cleaved at
the cell surface and not intracellularly. Thus, the increased
solubilization observed with ACE
is not a consequence of
accelerated biosynthesis or another cellular proteolytic process. 2)
Through structural interactions with ACE itself or the secretase(s),
the amino-terminal domain of ACE could act as a ``conformational
inhibitor'' of the proteolytic process of membrane-anchored ACE.
The amino-terminal domain could act on extracellular ACE conformation,
near the anchoring domain; when the amino-terminal domain is present,
the region near the membrane anchor may have a conformation distinct
from that when the N domain is absent. Indeed, the amino-terminal amino
acids of the native protein cannot be reached by immunolabeling with
the Clo antiserum, directed against the first 18 amino acids of the
human endothelial sequence (data not shown). Accordingly, the
amino-terminal part of the protein is more or less masked by the rest
of the protein and/or in close-contact with the membrane. It is
possible that a part of the amino-terminal domain interacts with the
``stalk'' of ACE, the region situated between the end of the
globular part of the C domain and the hydrophobic membrane-spanning
domain. Another possibility is that the amino-terminal domain of ACE
affects the conformation of the membrane-bound secretase(s) itself, via
protein-protein interactions.
Figure 10:
Subcellular localization of ACE and
ACE. Schematic role of the amino-terminal domain of ACE in
the solubilization. All intracellular (IC) species
cross-reacts with 28A (28A
). 90% of initially labeled
proteins reach the membrane (pm) after 16 h of chase for ACE
and 4 h of chase for ACE
. Under these comparable
conditions, solubilization of ACE from the plasma membrane is 10-fold
lower than ACE
. In the medium (extracellular; EC)
ACE and ACE
do not cross-react with 28A (28A
). The inset shows the proposed
``conformational inhibitor'' role of the ACE amino-terminal
domain. On the left, the amino-terminal domain interacts with
ACE stalk and the secretase(s) leading to a cleavage between Arg-1137
and Leu-1138. On the right, deletion of the amino-terminal
domain modifies interactions of the secretase(s) with the ACE stalk,
which cleaves between Arg-1227 and
Val-1228.
Two distinct cleavage sites are
involved in the proteolysis of ACE and ACE. ACE is cleaved
between Arg-1137 and Leu-1138, whereas ACE
is probably
cleaved between Arg-1227 and Val-1228. Therefore, a model can be
proposed where ACE and ACE
are cleaved by one or two
secretase(s) (Fig. 10), Arg-1137 and Arg-1227 being alternative
cleavage sites. Interaction of the amino-terminal domain with the
``stalk'' and secretase(s) could induce a preferential
cleavage in Arg-1137 with a low turnover. Alternatively, the deletion
of the amino-terminal domain would involve a conformational change of
ACE stalk and/or of the secretase(s), which cleaves then after Arg-1227
with a more rapid turnover.
It is possible to envisage a general model for the processing of ACE. ACE could be cleaved at the plasma membrane by a secretase(s), localized like ACE to the plasma membrane. The amino-terminal domain would act as a conformational inhibitor of the proteolytic solubilization via structural interactions between the ACE stalk and the secretase(s) itself. Under these conditions, endothelial ACE would be slowly solubilized in plasma leading to the presence of most ACE activity at the endothelial cell level. Since protein-protein interactions vary in the plasma membrane and in the extracellular space, it is possible to conceive a ``conformational'' change in the cleavage site of ACE, leading to an increased ACE solubilization, under certain biological circumstances.