From the Institut National de la Santé et de la
Recherche Médicale Unit 525, Faculté de médecine
Pitié-Salpétrière, 91 Boulevard de
l'Hôpital, 75013 Paris, France; the ¶ Unit 36-Collège
de France, 3 rue d'Ulm, 75005 Paris, France; the
Department
of Internal Medicine I, University of Rotterdam, dr Molewaterplein 40 3015 GD Rotterdam, The Netherlands; the ** Université Paris 6 and 7, CNRS UMR 7590, 4 place Jussieu, 75252 Paris Cedex 05, France; and the
Department of
Pharmacology and Toxicology and Department of Internal Medicine,
University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen,
The Netherlands
Received for publication, August 23, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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Angiotensin-converting enzyme (ACE), an
enzyme that plays a major role in vasoactive peptide metabolism, is a
type 1 ectoprotein, which is released from the plasma membrane by a
proteolytic cleavage occurring in the stalk sequence adjacent to the
membrane anchor. In this study, we have discovered the molecular
mechanism underlying the marked increase of plasma ACE levels observed
in three unrelated individuals. We have identified a
Pro1199 Angiotensin I-converting enzyme (DCP1, EC 3.4.15.1,
ACE)1 is a zinc
metallopeptidase that plays an important role in blood pressure
regulation by cleaving the inactive decapeptide angiotensin I to
angiotensin II, a potent vasopressor octapeptide. It also inactivates
bradykinin, a potent vasodilator peptide (1). There are two ACE
isoforms transcribed from a single ACE gene by two alternate promoters (2). Somatic ACE (170 kDa) is synthesized by
vascular endothelial cells as well as several types of epithelial cells, whereas testis ACE (110 kDa) is expressed exclusively by male
germinal cells (3, 4).
Somatic ACE consists of two homologous catalytic domains, a
juxtamembrane stalk region, a hydrophobic transmembrane domain of 17 amino acids, and a 30-residue C-terminal cytosolic domain (5). Thus ACE
is primarily an integral membrane protein anchored to the plasma
membrane by its C-terminal segment. But the membrane-bound enzyme can
be fully solubilized in vitro by detergents or limited proteolytic cleavage (6). In vivo, a soluble form of the
enzyme exists in plasma and other body fluids (7). Plasma ACE is known to be strongly modulated by a common polymorphism in linkage
disequilibrium with an insertion/deletion polymorphism (8). Although
this quantitative trait locus (QTL) was localized on the ACE
gene itself (9), the functional variant has not yet been identified.
Human plasma ACE is derived from endothelial cells by
post-translational cleavage. Several membrane-anchored proteins
are solubilized by limited proteolysis with release of their
extracellular domains. This common phenomenon, also referred to as
"shedding" displays typical characteristics such as induction by
phorbol esters, calcium ionophores, and unidentified serum factor;
inhibition of the shedding protease by hydroxamate-based
metalloprotease inhibitors; localization of the shedding event at the
cell surface; and structural requirements of the juxtamembrane stalk
that determine cleavage efficiency (10-12). Cleavage secretion of ACE
is topologically constrained to an accessible stalk region of at least
11 residues in length and requires a minimum distance of 3 residues
from the proximal extracellular domain and 8 residues from the membrane (10). Furthermore, mutational analysis of the stalk region has shown
that conservation of the amino acid sequence was not essential for
shedding, even though the secretase seems to have a weak preference for
cleavage after Arg or Lys residues (10, 13, 14). Thus, the critical
parameter, for cleavage efficiency seems to be the conformation of the
stalk more than the amino acid sequence. In addition, experiments using
the homologous stalk region of CD4 (which is not cleaved) to construct
a chimeric ACE revealed that the distal extracellular domain of ACE
also has a prominent role for determining the cleavage (14).
Here we report the identification of a Pro1199 Subjects and Biological Findings--
Three unrelated
individuals (HOL.2, HOL.10, and HOL.19) were identified through a
screening of plasma ACE levels in a population of patients referred to
the hospital for various causes. The study included the measurement of
several clinical, paraclinical, and biological parameters. Plasma ACE
enzymatic activity was measured by a commercial kit (ACEcolor, Fujizoki
Pharmaceutical Co. Ltd., Tokyo, Japan) using Gly-His-Leu as substrate.
The complete clinical report has been described in Kramers et
al.2
Reagents--
Phorbol 12-myristate 13-acetate (PMA), MCDB-131
medium, Dulbecco's modified Eagle's medium, CHAPS, and hydrocortisone
were from Sigma. Fetal calf serum was from Valbiotech (Paris, France). Phosphate-buffered saline, L-glutamine,
penicillin-streptomycin, and geneticin were from Life Technologies,
Inc. Human recombinant epidermal growth factor (hEGF) and
EXGEN 500 were from Euromedex (Souffelweyersheim, France). Compound 3 [N-[-D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl]-L-3-(tert-butyl-alanyl-L- alanine,
2- aminoethyl amide) was provided by Dr. Roy A. Black, Immunex Research
and Development Corp. (Seattle, WA).
PCR Amplification and Sequencing--
Genomic DNA of all
subjects was used as template for PCR amplification. Specific primers
were designed to amplify each of the 26 exons of the ACE
gene, according to our own sequence determination and to the published
data (GenBankTM/EBI accession numbers AC002345 and
AF118569). The primer sequences are available upon request. Exon 25 amplification was performed with oligonucleotides
5'-CATGTTGAGCTACTTCAAGC-3' (sense) and 5'-CCAGTGTTCCCATCCCAG-3'
(antisense), using the following cycling parameters: one denaturation
step at 95 °C, 4 min; 31 cycles (95 °C, 30 s; 60 °C,
30 s; 72 °C, 30 s); and one final elongation step at
72 °C, 10 min. Purified PCR products were submitted to direct
sequencing using fluorescent dideoxyterminator (ABI Prism BigDye
Terminator Cycle Sequencing Ready Reaction Kit, PE Biosystems) on an
ABI 377 sequencer.
Site-directed Mutagenesis--
A single Pro (CCG) to Leu (CTG)
mutation at position 1199 was introduced into pACE-WT (or peACE), an
expression vector containing the full-length somatic ACE cDNA
controlled by the SV40 early promoter (15). An oligonucleotide-directed
mutagenesis system (QuickChange site-directed mutagenesis kit,
Stratagene) was used according to the manufacturer's recommendation.
The following oligonucleotides containing the desired mutation were
used: 5'-GCAGTACAACTGGACGCTGAACTCCGCTCGC-3' (sense);
5'-GCGAGCGGAGTTCAGCGTCCAGTTGTACTGC-3' (antisense). Mutant cDNAs were screened by DNA sequencing. One mutant containing the desired mutation was selected and designed as
pACE(Leu1199). The entire cDNA fragment of this
construct was sequenced to ensure that no other mutations occurred. The
same protocol was used to introduce the Pro1199 Transient Expression of pACE-WT,
pACE(Leu1199), pACE-CF, and
pACE-CF(Leu1199)--
pACE-WT,
pACE(Leu1199), pACE-CF, and pACE-CF(Leu1199)
were expressed in COS-7 cells. Transient transfections were performed
by using polyethyleneimine suspension in a commercially available
solution (EXGEN 500). COS-7 cells (250 × 103/well)
were plated on a 6-well plate and incubated 24 h in Dulbecco's modified Eagle's medium supplemented by 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml
penicillin-streptomycin and then treated with a mix containing 2 µg
of plasmid, 4 µl of EXGEN 500, and NaCl (150 mM) in a
final volume of 100 µl. The same protocol was used to express pACE-WT
and pACE(Leu1199) in HMEC-1 (human microdermal endothelial
cells transfected and immortalized with a pBR-322-based plasmid
containing the coding region for the SV40 A gene product, large T
antigen, Ref. 17) except that they were grown in MCDB-131 medium
supplemented by 20% heat-nactivated fetal calf serum, 2 mM
L-glutamine, 100 units/ml penicillin-streptomycin, 10 ng/ml
hEGF, 1 µg/ml hydrocortisone, and 6 µl of EXGEN 500 were used for
transfection. Cells were incubated overnight with the transfection mix.
Expression of pACE(Leu1199) in CHO
Cells--
pACE(Leu1199) was introduced by cotransfection
with neomycin resistance plasmid pSV2neo into CHO cells. CHO cells
(1 × 106) were plated on a 10-cm2 Petri
dish, incubated 24 h, then treated with a mix containing 2.95 µg
of pACE(Leu1199), 500 ng of pSV2neo, 6 µl of EXGEN 500, and NaCl (150 mM) in a final volume of 100 µl. The
transfected cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml
penicillin-streptomycin, and 750 µg/ml geneticin. Single colonies of
primary G418-resistant transformants were assayed for the expression of
pACE(Leu1199) by Western blot analysis and enzymatic
assays (see below). Cell lines expressing pACE(Leu1199)
were selected and purified by subcloning using limiting dilution. Establishment of a CHO cell line expressing pACE-WT has been previously described (15).
Metabolic Labeling and Immunoprecipitation--
Wild-type and
mutant ACE CHO cell lines with similar cellular contents of ACE
activity were seeded in 60-mm dishes at a density of 2 × 106 cells/dish and grown to confluency. Metabolic labeling
and immunoprecipitation were performed as previously described (18),
except that immunoprecipitation was performed using antiserum HKE
obtained from sheep immunized against pure human kidney ACE and protein
G-Sepharose (Sigma). Proteins were resolved by 7.5% SDS-polyacrylamide
gel electrophoresis and revealed by autoradiography.
Preparation of Subcellular Fractions--
CHO cells expressing
wild-type ACE and ACE(Leu1199) were scraped and suspended
in buffer A containing 50 mM HEPES pH 7.5, 0.25 M sucrose, and 5 mM MgCl2. All
steps were performed at 4 °C. The cell suspension was homogenized
using a glass-Teflon homogenizer for 30 s and then centrifuged at
1,000 × g for 10 min. The pellet was resuspended in
buffer A and centrifuged at 10,000 × g for 10 min. The
pellet was solubilized in CHAPS (8 mM) and conserved as a
lysosomal fraction. The supernatant was centrifuged again at
105,000 × g for 1 h. The sedimented membranes
were washed with buffer A and solubilized in CHAPS (8 mM),
and the supernatant was conserved as a cytosolic fraction.
Enzymatic Characterization--
Cells were scraped and washed
with phosphate-buffered saline and then centrifuged at 1,200 × g for 10 min. The pellet was dissolved in CHAPS (8 mM). After stirring for 12 h to solubilize the
membrane-bound ACE, the suspension was centrifuged at 12,000 × g for 10 min, and the supernatant was used for enzymatic assays.
Secreted ACE was obtained from the culture medium of transfected cells
grown in serum free medium.
All enzymatic studies were performed using
p-benzoyl-L-glycyl-L-histidyl-L-leucine
(Hip-His-Leu, Bachem, Switzerland) as substrate. The detection and
quantification of hippuric acid released from the Hip-His-Leu were
performed by HPLC as previously described (15). Kinetic parameters for
the hydrolysis of Hip-His-Leu were determined from Michaelis-Menten
plots using ENZFITTER software. All enzymatic studies were performed
under initial rate conditions.
Western Blot Analysis--
Two different rabbit antisera were
used to characterize expressed forms of ACE: antisera 3 and 4 (18)
which were raised against synthetic peptides corresponding to sequences
of the C-terminal ACE (see Fig. 1). Membrane-bound and secreted ACE
were collected as described above and analyzed by SDS-PAGE on a 7.5%
polyacrylamide gel, transferred to a nitrocellulose membrane (Pall
Gelman Sciences) in a buffer containing 25 mM Tris-HCl, pH
8.3, and 150 mM glycine using a semi-dry transfer system
(Bio-Rad). Western blotting was performed in TBST buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween
20) with antiserum 3 (1:500 dilution) or antiserum 4 (1:500 dilution).
The peroxidase-conjugated anti-rabbit IgG (Jackson Immunoresearch)
(1:5000 dilution) was used as secondary antibody. The detection step
was carried out with ECL Western blotting detection reagents (Amersham
Pharmacia Biotech) as recommended by the manufacturer.
Computer-assisted Structural Analysis--
Multiple alignment of
domain 1 (N-terminal domain) and domain 2 (C-terminal domain) of
several ACE orthologs was performed using ClustalW program (19). The
sequence data used are available in the Swiss-Pro database under
accession numbers P12821, P09470, and Q10751.
Hydrophobic cluster analysis (HCA), a two-dimensional structure
prediction method based on the detection and comparison of the
structural segments constituting the hydrophobic core of globular protein has been described elsewhere (20, 21). ACE sequences were
submitted to HCA analysis, and domain 1 and domain 2 of ACE orthologs
were aligned according to hydrophobic clusters determined by HCA plots.
Optimal alignment was performed using combined data from ClustalW and
HCA analysis.
Biochemical Parameters--
Three unrelated subjects with marked
increases in plasma ACE levels were identified through a screening of
patients referred to a general hospital (55.8, 83.8, and 86.6 units/liter). Using the colorimetric assay ACEcolor, normal values of
plasma ACE fluctuate between 8.3 and 21.4 units/liter. Plasma ACE
levels are 2.5-4-fold higher than highest levels found in the normal
population. All other biological and paraclinical parameters measured
were normal.
Sequencing of the ACE Gene Exons--
A heterozygote point
mutation was identified in exon 25 of the ACE gene. This
point mutation is a C-T transition, introducing a leucine in position
1199 instead of a proline found normally at this position (Fig.
1). No other mutation was present in
ACE gene exons from these subjects. Cotransmission of high
plasma ACE levels and the Pro1199
To elucidate the mechanism by which this mutation was responsible for
the high plasma ACE levels observed in these individuals, an in
vitro study using site-directed mutagenesis was undertaken.
Expression of Wild-type ACE and [Leu1199]ACE in
COS-7 and HMEC-1 Cells--
The expression plasmid pACE-WT (or peACE)
was used to direct the synthesis of wild-type ACE (16). The expression
plasmid pACE(Leu1199) is identical to pACE-WT except for a
single Pro (CCG) to Leu (CTG) mutation at position 1199 of the coding
sequence of somatic ACE and was used to direct the synthesis of
[Leu1199]ACE.
The kinetic parameters for Hip-His-Leu hydrolysis of the wild-type ACE
and of the [Leu1199]ACE, calculated from a
Michaelis-Menten plot, were not significantly different (Fig.
2). The Km and the
Vmax values of the [Leu1199]ACE
were similar to that of the wild-type ACE (3.33 ± 0.11 mM versus 2.51 ± 0.12 mM and
0.35 ± 0.007 nmol/ml/min
Both plasmids were transiently expressed in COS-7 cells for comparing
the ability of cells to secrete [Leu1199]ACE with that of
the wild-type ACE (Fig. 3, A,
C, and E). Transfected cells were grown in
serum-free medium for indicated times before the ACE enzymatic activity
was measured with a Hip-His-Leu substrate by HPLC. No significant
difference was observed in levels of cell-associated ACE between the
wild-type form and [Leu1199]ACE during the time-course
study (Fig. 3A). In contrast, levels of soluble
[Leu1199]ACE activity were higher than wild-type ACE
suggesting that the rate of release was enhanced (Fig. 3C).
The percentage of solubilization, which was defined as the ratio of the
enzymatic activity in the medium to the total enzymatic activity
(medium + cells) was in average 1.5-fold higher for
[Leu1199]ACE, as compared with wild-type ACE in COS-7
cells with a maximum difference of 2.5-fold at 24 h (Fig.
3E).
We also investigated the rate of secretion of
[Leu1199]ACE in HMEC-1, an endothelial cell line, which
does not express ACE, because endothelium is a major site of expression
of ACE in vivo and the source for plasma ACE (Fig. 3;
B, D, and F). Higher levels of soluble
[Leu1199]ACE activity were observed, as compared with
wild-type ACE, and lower cell associated ACE activity was observed. The
rate of solubilization was in average 2.5-fold higher for
[Leu1199]ACE compared with the wild-type form.
These data showed that a leucine in position 1199 instead of a proline
in ACE protein leads to an enhancement of cleavage and secretion of
ACE. Thus, both in an endothelial cell type and a nonendothelial cell
type, differences between wild-type ACE and [Leu1199]ACE
release observed in vitro are consistent with the plasma ACE
increase observed in subjects bearing this mutation.
Biosynthesis and Secretion of Metabolically Labeled Wild-type ACE
and [Leu1199]ACE--
CHO cells stably transfected with
the expression plasmid pACE-WT or pACE(Leu1199) were used
to investigate the role of the Pro1199
The metabolic labeling results were consistent with the kinetic
experiments based on ACE activity, indicating that
[Leu1199]ACE was released more efficiently from the cell
surface than wild-type ACE.
Subcellular Localization of [Leu1199]ACE--
To
test whether the Pro1199
ACE activity measured in the cytosol for wild-type ACE and
[Leu1199]ACE was very low, indicating that these proteins
are rapidly addressed to the membrane. A higher proportion of total ACE
(38.6 ± 8.5% for wild-type ACE and 24.9 ± 9.7% for
[Leu1199]ACE at 3 h) was detected in lysosomal
fraction. At 3 h (Fig. 5A) only 6% of total
[Leu1199]ACE was detected in membranous compartment,
whereas high levels of [Leu1199]ACE (34% of total ACE)
was observed in the medium. At the same time, wild-type ACE levels were
similar in membranous compartment and medium (17 and 16% of total ACE
respectively). This difference between [Leu1199]ACE and
wild-type ACE was more pronounced at 24 h (Fig.
5B).
Immunological Characterization of Membrane-bound and Soluble
[Leu1199]ACE--
To map the cleavage site of
[Leu1199]ACE, Western blot analyses using specific
antibodies were performed (Fig. 6).
Antiserum 3 was raised against sequences from the C-terminal side of
the Arg1203
CHO cells stably transfected with either wild-type ACE and
[Leu1199]ACE were grown to subconfluence and then
incubated in serum-free medium for 48 h. Medium and
CHAPS-solubilized cell lysates were analyzed on SDS-PAGE gel followed
by Western blotting. Antiserum 3 failed to recognize wild-type or
[Leu1199]ACE secreted in the medium, indicating that
cleavage occurred before amino acid 1214. In contrast antiserum 4 was
able to recognize the soluble form of wild-type and
[Leu1199]ACE, indicating that cleavage occurred after
amino acid 1162. Both antisera recognized wild-type and
[Leu1199]membrane-bound ACE.
Effects of PMA and Compound 3 on [Leu1199]ACE
Secretion--
To document the involvement of the already
characterized ACE secretase in increased shedding of
[Leu1199]ACE, we investigated the effects of compound 3 on the [Leu1199]ACE solubilization process. Transfected
cells were grown in a serum-free medium containing (or not) compound 3 (50 µM), and the time course of secretion was
investigated over a 48 h period (Fig.
7A). After 3 h, 31% of
[Leu1199]ACE was secreted from the untreated cells,
whereas in compound 3-treated cells, only 4% of
[Leu1199]ACE was secreted at the same time. This effect
was still observed after 48 h of treatment. So compound 3 inhibited [Leu1199]ACE to the same extent that it
inhibited wild-type ACE secretion.
COS-7 cells were transfected with wild-type ACE and
[Leu1199]ACE and were thereafter grown in serum-free
medium containing (or not) PMA (500 nM). The time course of
secretion was investigated over a 3-h period (Fig. 7B). PMA
enhanced the rate of [Leu1199]ACE cleavage secretion
considerably. For example, after 3 h, 55% of
[Leu1199]ACE was secreted from the PMA-treated cells,
whereas, in untreated cells, only 20% of [Leu1199]ACE
was secreted at the same time. We observed a significant difference in
solubilization of PMA-treated cells compared with untreated cells as
early as 15 min after treatment. No similar effect was observed with
wild-type ACE secretion.
Role of the N-terminal Domain: Expression of
[Leu1199]ACE-CF in COS-7 Cells and Effect of PMA on
Secretion--
The possible effect of the N-terminal domain on
[Leu1199]ACE solubilization was investigated using
expression plasmid pACE-CF containing full-length somatic ACE cDNA
except the coding sequence for the N-terminal domain (16). pACE-CF and
pACE-CF(Leu1199) were transiently expressed in COS-7 cells
(Fig. 8A). At 6 h, nearly
50% of ACE-CF was solubilized in medium whereas at the same time only
20% of wild-type somatic ACE was secreted (compare Figs. 3E
and 8A), confirming the lower solubilization of the two domain-containing enzyme. We observed a significantly higher rate of
release for [Leu1199]ACE-CF as compared with ACE-CF after
1 h (31 versus 7%, respectively). The percentage of
solubilization was in average 1.8-fold higher for
[Leu1199]ACE-CF as compared with ACE-CF in COS-7
cells.
We also investigated the effect of PMA on ACE-CF and
[Leu1199]ACE-CF solubilization. (Fig. 8B). PMA
enhanced the rate of solubilization of ACE-CF and
[Leu1199]ACE-CF. After 15 min, 14% of ACE-CF and 62% of
[Leu1199]ACE-CF were secreted from PMA-treated cells,
whereas in untreated cells only 5% of ACE-CF and 20% of
[Leu1199]ACE-CF were solubilized at the same time. A PMA
effect was more pronounced on [Leu1199]ACE-CF
solubilization in the early times of a kinetic analysis, but the
difference was less evident between ACE-CF and
[Leu1199]ACE-CF after 2 h.
Structural Analysis--
The HCA plots of domain 1 (N-terminal
domain) and domain 2 (C-terminal domain) of several ACE orthologs were
compared to produce an optimal alignment. Fig.
9 shows this alignment for the region of
interest, i.e. close to proline 1199. HCA plots revealed
that the shape of the hydrophobic cluster immediately upstream of this proline is typical of a We report a mutation of the ACE gene, which was found
in three apparently healthy unrelated individuals from the same ethnic origin and selected for having very high plasma ACE levels.
Pathological causes of high plasma levels could be eliminated in these
individuals. Moreover, the genetic origin of the very high plasma ACE
levels was supported by the Mendelian transmission of the trait in the families of these subjects (Kramers et al.).2
Familial elevations of plasma ACE levels were already described in
Japan and Italy, but the molecular basis has not been elucidated (23,
24).
In view of its large effect, the genetic increase of plasma ACE levels,
which was observed in these subjects could not be attributed to the
common polymorphism of the ACE gene, which explains 30% of
plasma ACE variance in the Caucasian population and which is in linkage
disequilibrium with an insertion/deletion polymorphism located within
intron 16 of the ACE gene (8, 25).
The complete sequencing of the exons of the ACE gene was
performed to detect a mutation specific to the three individuals. DNA
sequencing revealed a mutation in exon 25 leading to a
Pro1199 Because the Pro1199 Two antibodies, raised against peptides flanking the cleavage site
determined for both human testicular and somatic ACE (26), were used to
determine whether [Leu1199]ACE was cleaved at the same
location as the wild-type enzyme. Indeed, [Leu1199]ACE is
cleaved between amino acid 1162 and 1214, a result consistent with the
cleavage site determined for the in vitro expressed
wild-type somatic and testicular enzymes, and for the seminal plasma
ACE (26). Thus, our results do not favor the hypothesis of another cleavage site created by the mutation but suggest a more active cleavage rate at the major site. However we cannot exclude that a small
fraction of secreted ACE, not detected with antiserum 4, is cleaved at
a secondary cleavage site, such as the one described between
Arg1137 and Leu1138 for human plasma ACE
(13).
The cleavage secretion of ACE is an enzymatic process which can be
modulated by pharmacological agents. A hydroxamic acid-based inhibitor
of metalloproteases, compound 3, was shown to block very effectively
the cleavage-processing activity of testicular ACE in a mouse
epithelial cell line (ACE 89) stably transfected with the rabbit
testicular ACE (27). Our data show that compound 3 completely abolished
[Leu1199]ACE secretion as well as wild-type ACE over a
prolonged period of time (12 h), after which a progressive degradation
of the inhibitor likely enabled secretion to rise again. This clearly
indicates that the increased cleavage-secretion process observed for
[Leu1199]ACE results from the similar enzymatic
process to the wild-type and not from cleavage because of another type
of enzyme or from a nonspecific leakage from the membrane.
Phorbol esters, which activate protein kinase C, were shown to enhance
the rate of ACE cleavage-secretion, with a marked difference between
the somatic and testicular isoforms, the latter being more strongly
solubilized than the first, even after a short period of incubation
(26). The phorbol ester PMA markedly and rapidly increased
[Leu1199]ACE solubilization (4-5-fold), in contrast to
wild-type ACE whose secretion was not significantly increased by
PMA.
The cleavage secretion of somatic ACE is lower compared with testicular
ACE, and it has been proposed that the N-terminal domain could have an
inhibitory effect on its secretion. Because both the basal and phorbol
ester-induced cleavage secretion of [Leu1199]ACE
resembled what is observed for the testicular isoform of ACE, we
investigated the hypothesis that the mutation could suppress an effect
of the N-terminal domain on solubilization. This was achieved by
introducing the mutation in an ACE expression vector lacking the
N-domain of ACE (pACE-CF) (16). At the basal level, secretion of
[Leu1199]ACE-CF was increased 4-fold as compared with the
wild-type ACE-CF in COS-7 cells. After treatment with PMA, an early
4-fold increase of [Leu1199]ACE-CF secretion rate was
observed as compared with the wild-type ACE-CF. Two kinds of arguments
drawn from our results indicate that the mutation is not acting through
the suppression of an inhibitory effect of the N-terminal domain on the
secretion rate. First, cleavage-secretion of somatic
[Leu1199]ACE is lower compared with C-terminal domain
[Leu1199]ACE. Second, the effect of the
Pro1199 It was recently shown that, in the ACE89 cell line, testicular ACE can
make a complex with protein kinase C subunits, and this complex
dissociates in the presence of phorbol ester (28). A subsequent
enhanced effect of ACE secretase was proposed to explain the phorbol
ester-induced cleavage-secretion. In the cases of both somatic and
C-terminal domain ACE (ACE-CF) with the Pro1199 Because no data are available on the ACE three-dimensional structure,
we performed multiple alignment deduced from HCA plots of the
N-terminal and the C-terminal domains of several ACE orthologs to
predict secondary structures around proline 1199. It is remarkable that
proline 1199 is highly conserved among ACE family sequences. Furthermore, this amino acid is the last common residue of the two
domains preceding a loop specific for the C-terminal domain and which
contains the cleavage site. The insertion of a fairly long loop between
two secondary structures might be an indication of a better
accessibility for proline 1199 by the C-terminal domain compared with
the homologous proline 601 in the N-terminal domain. Proline is known
to induce conformational constraints by creating twists and turns in
peptidic sequences. We proposed that the presence of a leucine in
position 1199 instead of a proline confers more flexibility in this
region leading to better accessibility and action of ACE secretase.
Thus, even though it was shown that the cleavage-secretion process is
not constrained by a specific amino acid sequence but is much
more dependent on topological parameters (10), we show here that an
amino acid sequence important for the conformational state of the
juxtamembrane stalk region is critical for ACE release. More
investigation in the structural conformation of the stalk region would
provide interesting data for the study of the regulated shedding of ACE
and other shedded membrane-associated proteins.
The elucidation of the molecular basis of a QTL affecting the plasma
concentration of ACE has implications that go beyond the ACE
gene itself. First, it illustrates that a QTL is not necessarily acting
by modifying the expression level of the gene affected by the QTL, but
conformational changes can modify its secretion rate and, hence, its
concentration in a particular biological fluid where it can be measured
in clinical practice. Second, it shows that the plasma concentration is
a biological phenotype, which can be of limited value, because it does
not necessarily reflect the level of expression at the cellular level.
In the case described here, the membranous concentration of ACE is even slightly decreased, and the high plasma concentration is not associated with any detectable pathological effect. In contrast, it has been shown
that the above mentioned common polymorphism of ACE also affects the
cellular level of the enzyme, where it can cause potential deleterious
effects (29, 30). Finally, because the mutation described here cannot
explain interindividual variations of plasma ACE associated with an
insertion/deletion polymorphism of the ACE gene, our results
clearly demonstrate that different molecular mechanisms account for
strong genetic influences on plasma ACE.
Leu mutation in the juxtamembrane
stalk region. In vitro analysis revealed that the
shedding of [Leu1199]ACE was enhanced compared
with wild-type ACE. The solubilization process of
[Leu1199]ACE was stimulated by phorbol esters and
inhibited by compound 3, an inhibitor of ACE-secretase. The
results of Western blot analysis were consistent with a cleavage
at the major described site
(Arg1203
Ser1204). Two-dimensional structural
analysis of ACE showed that the mutated residue was critical for the
positioning of a specific loop containing the cleavage site. We
therefore propose that a local conformational modification caused by
the Pro1199
Leu mutation leads to more accessibility at
the stalk region for ACE secretase and is responsible for the
enhancement of the cleavage-secretion process. Our results show
that different molecular mechanisms are responsible for the common
genetic variation of plasma ACE and for its more rare familial elevation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu
mutation found associated with high plasma ACE levels in three
unrelated Dutch individuals. In vitro study of this mutation
was performed, and effects of this mutation on the solubilization
process of ACE were investigated. We present experimental results
showing that this single amino acid change in the stalk region is
responsible for an enhancement of the cleavage and secretion of ACE in
an ACE-secretase activity-dependent manner. A hypothesis
implicating a modification of the ACE structure caused by the point
mutation that leads to better accessibility for the ACE secretase is proposed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu
mutation into plasmid pACE-CF containing the coding sequence for the
signal peptide, the C-terminal domain, and the transmembrane domain of
human endothelial ACE (16). The mutant containing the desired mutation
was designed as pACE-CF(Leu1199).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu mutation was
observed in the family, although this mutation was not found in a large
sample of Dutch subjects (Kramers et al.).2
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Fig. 1.
C-terminal sequence of wild-type ACE from
Gly1135 to Glu1230. The proline mutated
into leucine in the three individuals is underlined. The cleavage site
previously described is shown by an arrow. Sequences of
synthetic peptides used for the production of antisera 4 and 3 are
indicated.
1 versus
0.34 ± 0.008 nmol/ml/min
1).
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Fig. 2.
Lineweaver-Burk plots for hydrolysis of
Hip-His-Leu by wild-type ACE and [Leu1199]ACE.
Wild-type ACE ( ) was compared with [Leu1199]ACE (
).
Assay was performed in a mix containing 100 mM potassium
phosphate, pH 8.3, 300 mM NaCl, and 10 mM
ZnSO4 at 37 °C.
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Fig. 3.
Time course of wild-type and
[Leu1199]ACE secretion. Enzymatic activity
of wild-type ACE ( ) and [Leu1199]ACE (
) transiently
expressed in COS-7 cells (A, C, E) or
in HMEC-1 (B, D, F) was measured for
48 h in serum-free medium. Medium samples (C,
D) and detergent lysates (A, B) were
collected at indicated time points and assayed with ACE substrate
Hip-His-Leu. E and F are representations of the
time course of ACE secretion, respectively, in COS-7 and HMEC-1 cells.
The results are mean ± S.E. of duplicate determinations of two
independent experiments. Where error bars are not evident,
S.E. is less than the figure resolution.
Leu mutation on
ACE secretion. Cells were labeled for 30 min with
[35S]methionine/cysteine and chased in serum-free medium
for the indicated periods of time (Fig.
4). Affinity precipitates of cell lysates
of transfected cells revealed two bands of immunoreactive ACE around
170 kDa. The lower band, which disappeared after 24 h of chase,
corresponds to the underglycosylated form of ACE, and the upper band,
which appeared at 1 h of chase, corresponds to the glycosylated
form of ACE. The secreted [Leu1199]ACE appeared earlier
than soluble wild-type ACE at 4 h of chase (Fig. 4B),
whereas only traces of soluble wild-type ACE were visible after 8 h of chase (Fig. 4A). Furthermore, soluble
[Leu1199]ACE increased rapidly in contrast with wild-type
ACE, which was difficult to detect in the medium even after 16 h
of chase.
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Fig. 4.
Pulse-chase analysis of the wild-type ACE and
[Leu1199]ACE. CHO cells expressing
wild-type ACE (A) or [Leu1199]ACE
(B) were pulse-labeled with
[35S]methionine/cysteine for 30 min and then chased with
serum-free medium from 0 to 16 h. An aliquot of cell lysates (200 µl) and medium (1 ml) were immunoprecipitated with HKE antiserum (3 µl).
Leu mutation influences the
intracellular trafficking of ACE, distribution of ACE was determined in
various compartments of CHO cells stably transfected with either wild-type ACE or [Leu1199]ACE grown for 3 or 24 h in
serum-free medium. Medium was then collected, and lysosomal, cytosolic,
and membranous compartments were separated by successive
centrifugations. ACE activity was measured in each fraction and
expressed as percentage of total ACE activity (Fig.
5). We determined that total ACE activity
was equivalent for wild-type ACE and [Leu1199]ACE.
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Fig. 5.
Subcellular localization of wild-type and
[Leu1199]ACE. Enzymatic activity of wild-type ACE
(open bar) or [Leu1199]ACE (filled
bar) was measured in subcellular compartments of CHO cells. The
results are mean ± S.E. of three independent experiments.
A, subcellular localization of wild-type and
[Leu1199]ACE in CHO cells grown 3 h in serum-free
medium. B, subcellular localization of wild-type and
[Leu1199]ACE in CHO cells grown 24 h in serum-free
medium.
Ser1204 cleavage site (22) and
antiserum 4 was directed against a synthetic peptide located at the
N-terminal side of this cleavage site (Fig. 1).
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Fig. 6.
Western blot analysis of wild-type and
[Leu1199]ACE. Wild-type ACE (WT) or
[Leu1199]ACE (MUT) expressed in CHO cells were
analyzed on SDS-PAGE gel. Medium (M) and cell lysates
(L) were collected immediately (To)
or 48 h (T48) after a switch to serum-free
medium. Western blot analysis was performed using antisera 3 and
4.
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Fig. 7.
Effects of PMA and compound 3 on wild-type
and [Leu1199]ACE solubilization. COS-7 cells
transiently expressing wild-type ACE (squares) or
[Leu1199]ACE (circles) were grown in a
serum-free medium with (black symbols) or without
(white symbols) pharmacological reagents for indicated
times. The solubilization was estimated by the ratio of enzymatic
activity in the medium over total enzymatic activity (medium + cells).
Results, expressed in percentage, are mean ± S.E. of duplicate
determinations of two independent experiments. A, effect of
compound 3 (50 µM) on ACE solubilization. Time course was
performed from 3 to 48 h. B, effect of PMA (500 nM) on ACE solubilization. Time course was performed from
15 to 180 min.
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Fig. 8.
Time course of ACE-CF and
ACE-CF(Leu1199) secretion and effect of PMA. A,
rate of solubilization of ACE-CF ( ) and ACE-CF(Leu1199)
(
) transiently expressed in COS-7 cells. Medium samples and
detergent lysates were collected at indicated time points and assayed
with ACE substrate Hip-His-Leu. The results are mean ± S.E. of
duplicate determinations of two independent experiments. B,
effect of PMA (500 nM) on ACE-CF and
ACE-CF(Leu1199) solubilization. COS-7 cells transiently
expressing ACE-CF (squares) or ACE-CF(Leu1199)
(circles) were grown in a serum-free medium with
(black symbols) or without (white symbols)
pharmacological reagents for indicated times. Results, expressed in
percentage, are mean ± S.E. of duplicate determinations of two
independent experiments.
-strand, which would presumably end at that
particular proline. An insertion of seven residues (NSARSEG in human
ACE) is readily detectable before the next downstream pattern of
hydrophobic residues. This insertion contains the cleavage site after
Arg1203 of domain 2 except in the case of chicken domain 2 whose amino acid sequence differs at that site (Fig. 9). No hydrophobic
residue was found within this insertion, thus suggesting that it
corresponds to a loop. Interestingly, proline 1199 is located at the
N-terminal side of this putative loop. Following the insertion, the
shape of the hydrophobic cluster, although it presents an ambivalent character, might indicate the presence of an
-helix in domain 2 compatible with the constraint of linking the first extracellular domain to the membrane anchor.
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Fig. 9.
HCA plots of the N-terminal
(D1) and C-terminal (D2) domains of
somatic ACE. This figure is limited to the C-terminal ends of both
domains, i.e. when considering the human sequence between
1135 and 1227 for D2 and 537 and 622 for D1. On these plots, the
sequence, which is duplicated to keep neighbors at the boundaries of
the plot, is written following the parallel and nearly
vertical lines. Four residues are represented by symbols as
indicated at the bottom of the figure. Hydrophobic clusters
are drawn when two hydrophobic residues (FILMVWY) are neighbors in this
picture, and a cluster is broken by the presence of one proline inside
it. It has been statistically demonstrated (31) that these clusters fit
with secondary structure elements. Gray shaded areas
correspond to conserved parts of the clusters among sequences. The last
cluster before Pro1199 is clearly a -strand element. The
cluster following this proline does not have a conserved shape that
allows elucidation of the nature of the corresponding secondary
structure. The last two secondary structures before Pro1199
are indicated by solid bar and zigzag symbols
below the figure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu mutation of the somatic form of ACE. This
mutation was not found in a large series of Caucasian subjects, and no
other mutation was found in the coding sequence of the three selected subjects.
Leu mutation was located in the
stalk region, which links the transmembrane domain to the first
extracellular domain, near the identified cleavage site (22), it was
considered as a good candidate, and its functional effect was further
investigated. We introduced the Pro1199
Leu mutation
into a somatic ACE expression vector, and we observed, as expected, no
difference in enzymatic properties between the [Leu1199]ACE and the wild-type ACE. The cleavage
secretion process was therefore the most likely to be affected by the
mutation. We investigated the release rate in two different cell lines,
the COS-7 cells already used in previous studies, and the HMEC-1, an
endothelial dermal microvessel cell line, which does not synthesize
ACE. In both cell lines, an increased secretion rate of
[Leu1199]ACE was observed, as compared with wild-type
ACE. The effect was however more pronounced in endothelial cells with a
2.5-fold increased secretion observed in average during the time-course study. Metabolic labeling experiments confirmed that
[Leu1199]ACE is secreted more efficiently than wild-type
ACE. Subcellular localization of [Leu1199]ACE in CHO
cells revealed that this effect is associated with a slight but
selective decrease of membranous ACE together with an increase in ACE
concentration of the medium, indicating that a global increase of ACE
production cannot explain the phenomenon observed. It is remarkable
that an important part of total ACE is retrieved in the lysosomal
fraction, indicating that a high proportion of both mutated and
wild-type ACE is degraded before it is addressed to the membrane or
that it is recycled from the membrane.
Leu mutation on secretion increase is also
observed with the recombinant ACE molecule lacking the N-terminal domain.
Leu
mutation, the preservation of a higher solubilization rate under PMA
indicates that the increase in speed of shedding induced by the
putative PMA-induced complex dissociation can still be enhanced by a
change in conformation.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Marie-Thérèse Chauvet for her invaluable help and for several reagents, and we thank Prof. Pierre Corvol for his continuous support. We thank Dr. Roy A. Black for providing compound 3. We thank the Center National de Séquencage and the Center National de Génotypage (Evry, France) for their help in the sequencing of the ACE gene.
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FOOTNOTES |
---|
* This work was supported by INSERM, by a grant from the French Ministry of Research, and by an unrestricted grant from Bristol-Myers-Squibb.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.
§ Recipient of a Ph.D. grant from the Société Francaise d'Athérosclérose and the Fondation de France.
§§ To whom correspondence should be addressed: INSERM U525, Faculté de médecine Pitié-Salpétrière, 91, boulevard de l'hôpital, 75013 Paris, France. Tel.: 33-1 40 77 97 25; Fax: 33-1 40 77 97 28; E-mail: florent.soubrier@chups.jussieu.fr.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M007706200
2 C. Kramers, S. M. Danilov, J. Deinum, I. V. Balyasnikova, N. Scharenberg, M. Looman, F. Boomsma, M. H. De Keijzer, C. Van Duijn, S. Martin, F. Soubrier, and G. J. Adema, submitted manuscript.
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ABBREVIATIONS |
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The abbreviations used are: ACE, angiotensin-converting enzyme; PMA, phorbol 12-myristate 13-acetate; CHO, Chinese hamster ovary; HMEC, human microdermal endothelial cell; hEGF, human epidermal growth factor; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; SV40 simian virus 40, QTL, quantitative trait loci; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; HCA, hydrophobic cluster analysis; WT, wild-type; PCR, polymerase chain reaction.
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