From the
Mammalian somatic angiotensin converting enzyme (EC 3.4.15.1,
ACE) consists of two highly homologous (N- and C-) domains encoded by a
duplicated gene. We have identified an apparent single-domain (67 kDa)
insect angiotensin converting enzyme (AnCE) in embryos of
Drosophila melanogaster which converts angiotensin I to
angiotensin II (K
Mammalian angiotensin converting enzyme (EC 3.4.15.1, peptidyl
dipeptidase A) (ACE)
Testicular ACE (tACE) is identical to the C-domain of sACE, apart
from a short N-terminal sequence (64-72 amino acid, depending on
species), and includes the transmembrane and the intracellular
C-terminal domains (14-17). Both sACE and tACE are transcribed
from a single gene with the testis-specific transcript under the
control of a tissue- and stage-specific intragenic
promoter
(18, 19, 20) . The physiological
function of tACE with its single catalytic domain is not known.
The
close similarity of the N-and C-domains of mammalian sACE and the fact
that exons 4-11 and 17-24 of the human ACE gene, which code
for the N- and C-domains, are very similar in size and have similar
codon phases at exon-intron boundaries
(20) strongly indicates
that the gene arose from duplication of an ancestral ACE gene coding
for a single-domain enzyme.
We have recently found a peptidyl
dipeptidase in heads of the housefly, Musca domestica, which
has very similar enzymatic properties to mammalian ACE
(21) . The
enzyme had an apparent M
A control transfection was
performed exactly as described above but in the absence of pCMV/AnCE
(``mock transfection''). COS-7 cells were also transfected
with the cDNA encoding wild-type human ACE (4.02 kb) with the
C-terminal membrane anchor
(5) , in the expression vector pECE
(pEACE). In addition, cells were transfected with a mutated human sACE
cDNA with a stop codon introduced before the membrane anchoring
sequence, also in pECE (pEACE
A 1000-fold purification of the soluble embryo peptidyl
dipeptidase was achieved using a lisinopril-Sepharose affinity column.
SDS-PAGE analysis of the affinity-purified enzyme revealed a single
protein band with an apparent molecular mass of 67 kDa (Fig. 1).
The purified enzyme converted AI to AII and removed Phe-Arg from the C
terminus of BK with K
A comparison of the deduced
amino acid sequence of AnCE with the sequences of the two domains of
mammalian sACEs reveals a number of regions that have common identity.
For example, the peptide sequence between residues 311 and 395 of AnCE
displays 72 and 76% identity with the same region in the N- and
C-domains of human sACE, respectively. This region includes the
consensus sequence HExxH in which the 2 histidines serve as zinc
ligands and the glutamate is the base donor in catalysis in human
ACE
(11) . In addition, Glu
Analysis of hydrophobicity confirms the presence of a potential
hydrophobic signal sequence at the N terminus of AnCE, but there is no
hydrophobic region toward the C terminus. This is in contrast to the
situation in mammals, where ACE is anchored to the plasma membrane by a
hydrophobic transmembrane domain close to the C terminus
(3) .
This suggested the possibility that the insect cDNA codes for a soluble
form of AnCE.
Southern blot analysis of genomic D. melanogaster DNA digested with BamHI, EcoRI,
HindIII, PstI, and XbaI gave single
hybridizing bands of 10.8, 19.1, 6.8, 3.7, and 7.7 kb when probed with
the complete AnCE cDNA (clone 53B11). Using the same cDNA probe, the
AnCE gene was localized to a single site at band 34A on
chromosome 2. Northern blot hybridization of total RNA extracted from
D. melanogaster embryos revealed a single transcript of around
2.1 kb (Fig. 5A).
Recombinant AnCE from the medium of transfected cells, gave a
K
We have purified a peptidyl dipeptidase with an apparent
M
The purification of the Drosophila AnCE did not yield sufficient protein for sequence analysis which
would have been necessary for the design of oligonucleotide probes for
use in screening insect DNA libraries. An alternative strategy of using
the cDNA for human endothelial ACE as a heterologous probe led to the
isolation of cDNA clones with regions of homology to human ACE cDNA
from a 4-8-h Drosophila embryo cDNA library. The deduced
amino acid sequence of the AnCE protein predicted a number of
structural features of an insect protein with an M
The fact that
mammalian sACE consists of two highly homologous domains indicates that
the mammalian genes arose from duplication of an ancestral ACE gene.
The evidence obtained in this study indicates that the insect AnCE gene is much smaller than the corresponding ACE genes of mammalian
species and, therefore, might resemble the ancestral form of the ACE
gene prior to the duplication event. Comparison of the core regions of
the two mammalian sACE domains with the single AnCE core domain shows
three significant events in evolutionary terms: the divergence of the
higher insect and mammal lineages (usually put at 550 million years
ago), the radiation of the modern mammals (65 million years ago), and
the gene duplication event that led from a single-domain ACE-like
protein to the mammalian two domain sACE. As noted
previously
(6, 20) , the duplication event predates the
radiation of the mammals because the percent similarity between the
core domains is less than the percent similarity within the C- and
N-domain groupings (Fig. 7, 73% as against 91-93%). The
KITSCH algorithm used to produce the tree in Fig. 4attempts to
calculate time scales
(31) . Clearly the time spans it comes up
with are underestimates, presumably because the sequences involved are
under strong selection. It is possible to say that the duplication
probably occurred in the Deuterostome lineage, as seen by the greater
similarity between the N- and C-domains than between either N- or
C-domains and AnCE. Secondly, an estimate can be made of the latest
time at which the duplication must have occurred. Correcting for the
time compression introduced by selection gives a minimum estimate of
330-350 million years ago before present, at about the time of
the appearance and radiation of the amphibians, during the
mid-Paleozoic. Already there is biochemical evidence that Torpedo
marmorata has a double-domain ACE, based on size estimates of the
protein
(34) . This puts the date for the duplication much
earlier in history, at the time of the development of the Chordata, or
represents a different event, either another duplication or some other
alteration leading to a larger ACE protein.
Data are the mean values ± S.E. of three separate
transfections for each construct. Enzyme assays were performed as
described under ``Materials and Methods'' using
Bz-Gly-His-Leu (1 mM final concentration) as substrate.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
, 365 µM),
removes Phe-Arg from the C terminus of bradykinin
(K
, 22 µM), and is inhibited
by ACE inhibitors, captopril (IC
= 1.1
10
M) and trandolaprilat (IC
= 1.6
10
M). We also
report the cloning and expression of a Drosophila AnCE cDNA
which codes for a single-domain 615-amino acid protein with a predicted
17-amino acid signal peptide and regions with high levels of homology
to both the N- and C-domains of mammalian somatic ACE, especially
around the active site consensus sequence. Northern analysis identified
a single 2.1-kilobase mRNA in Drosophila embryos, and Southern
analysis of Drosophila genomic DNA indicates that the insect
gene is not duplicated. When expressed in COS-7 cells, the AnCE protein
is a secreted enzyme, which converts angiotensin I to angiotensin II
and is inhibited by captopril (IC
= 5.6
10
M) and trandolaprilat (IC
= 2
10
M). The
evolutionary significance of these results is discussed.
(
)
is a Zn
metallopeptidase, which plays an important role in blood pressure
homeostasis by cleaving the C-terminal His-Leu from angiotensin I (AI)
to generate the potent vasopressor, angiotensin II (AII)
(1) . A
number of other small peptides are also hydrolyzed by mammalian ACE,
including the vasodilatory peptide bradykinin
(2) . The bulk of
the protein, including the active sites, is extracellular, connected to
a short intracellular C-terminal tail by a hydrophobic sequence that
traverses the plasma membrane
(3, 4) . The enzyme exists
as two isoforms, a somatic form (M
140,000-180,000) which displays a wide tissue distribution
and a smaller form (M
90,000-110,000) found
exclusively in the testes
(2, 3) . Somatic ACE (sACE) is
composed of two highly homologous domains (N- and C-domains), both of
which contain the Zn
-binding motif HExxH found in
other Zn
metallopeptidases
(10) . It has
recently been shown that both the N- and C-domains bind Zn
and are catalytically active
(11, 12, 13) .
of approximately 80, 000,
thus resembling the single- domain enzyme found in mammalian testes.
There was no evidence for a larger two-domain protein, implying that
the insect enzyme is the product of a non-duplicated gene. In the
present study we have purified a soluble 67-kDa ACE (AnCE) from embryos
of Drosophila melanogaster and have used a cDNA for human sACE
as a heterologous probe to clone a cDNA from a Drosophila embryo cDNA library, whose predicted translation product
has regions of homology to both the C- and N-domains of mammalian sACE.
The sequence data predicts that this cDNA encodes a secreted protein
with no recognizable C-terminal hydrophobic membrane-anchor sequence.
Functional expression of the cDNA in COS-7 cells confirms that we have
cloned the cDNA for an insect ACE.
Insect Culture
Standard culture methods were
employed to obtain large numbers of adult D. melanogaster (Oregon R strain) which were allowed to lay eggs on agar plates
impregnated with apple juice
(22) . Eggs were washed from the
agar with distilled water and were stored at -70 °C until
required.
Enzyme Preparation
Soluble enzyme was extracted
from eggs (4-16 h) by homogenization in 10 mM Tris-HCl
buffer, pH 8.4 (3 ml of buffer/g of eggs) using a glass homogenizer.
The homogenate was centrifuged at 200,000 g for 20 min
(Beckman 100.4 rotor for 20 min), and the pellet was resuspended in 10
mM Tris-HCl, pH 7.4, and subjected to centrifugation at
200,000
g for 20 min. The supernatant was kept and the
pellet washed in 10 mM Tris-HCl, 0.5 M NaCl, pH 7.4,
before being centrifuged for the last time to yield the final
supernatant and a pellet which was resuspended in 10 mM
Tris-HCl, pH 7.4. All procedures were performed at 4 °C. ACE
activity in the combined supernatants and the resuspended pellet were
assayed using Hip-His-Leu as the substrate and HPLC to quantify
hippurate (Hip) released
(21) .
Enzyme Purification
-Drosophila angiotensin converting enzyme (AnCE) was purified from eggs
(4-16 h) using a lisinopril-Sepharose affinity column
(23) and a procedure developed for the purification of AnCE from
the housefly, M. domestica.(
)
Briefly, eggs were homogenized in 10 mM Tris-HCl,
0.2 M NH
SO
, pH 8.4, and the
supernatant obtained after centrifugation (20,000
g for 20 min) was loaded onto the lisinopril-sepharose column (20
ml) equilibrated with 10 mM Tris-HCl, 0.2 M
NH
SO
, pH 8.5. The column was washed with the
loading buffer until all the unbound protein had been eluted, as
indicated by the UV absorbance (280 nm) of the eluate reaching a
base-line level. Bound AnCE was eluted by replacing the loading buffer
with 10 mM Tris-HCl, pH 8.5. Fractions were collected and
assayed for peptidyl dipeptidase activity as described above using
Hip-His-Leu as the substrate. Fractions containing enzyme activity were
concentrated using a CentriCell 20 filtration unit (Polysciences Inc.,
Park Scientific, Northampton, U.K.) with a 30-kDa molecular mass
cut-off. The purity of the enzyme was determined by SDS-PAGE using a
Pharmacia PhastSystem (Pharmacia Biotech, U.K.) and PhastGel
Homogeneous 7.5 polyacrylamide gels with SDS buffer strips and the
molecular mass was estimated by comparison to size-marker proteins
(Sigma, U.K.) which were analyzed in adjacent lanes on the same gel.
Protein bands were visualized by staining with silver
nitrate
(24) , and the protein concentration of the purified
sample was estimated using a Bio-Rad Protein Assay Kit (Bio-Rad
Laboratories, U.K.) with bovine serum albumin as the protein standard.
K
The effect of AI concentration on the ACE activity
of the native Drosophila enzyme was determined by quantifying
the amount of AII formed by HPLC using a SuperPac Pep-S reversed phase
column (250 mm Determinations for the Hydrolysis
of AI and BK
4 mm; Pharmacia Biotech, U.K.), a 10 min
gradient of acetonitrile (16-38%) in 0.1% trifluoroacetic acid,
and a flow rate of 1 ml/min. The hydrolysis of BK generated
BK
, BK
, and
BK
, all of which were resolved by HPLC using the
SuperPac Pep-S reversed phase column (250
4 mm) and a gradient
of acetonitrile in 0.1% orthophosphoric acid (flow rate, 1 ml/min).
Initially the column was eluted with 5% acetonitrile for 3 min,
followed by a linear increase to 28% acetonitrile over 15 min.
Authentic peptides (Sigma, U.K.) were used as chromatography markers.
The rate of BK hydrolysis was determined by quantifying Phe-Arg. The
hydrolysis of AI and BK were performed in the presence of 0.1
M Tris-HCl, 0.3 M NaCl, 10 µM
ZnCl
, pH 8.3. Reactions were terminated by heating to 100
°C for 5 min, and samples were diluted with 0.1% trifluoroacetic
acid before HPLC analysis. Standard solutions of AII and Phe-Arg were
used for calibration, and a curve-fitting computer programme (P.fit,
Biosoft, U.K.) was used to obtain K
values.
Cloning of AnCE cDNA
A D.melanogaster 4-8 h embryo cDNA library
(25) was screened with a 3.33-kb fragment of the human sACE cDNA
(HEC1922 clone, Ref. 5). [
P]CTP
multiprime-labeled probe (Amersham International) was hybridized to the
D.melanogaster 4-8 h embryo cDNA library grid
in 4
SSPE, 2% SDS at 65 °C overnight, followed by
sequential washes in 0.5
SSC, 0.1% SDS (65 °C, 30 min) and
0.1
SSC, 0.1% SDS (65 °C, 30 min). Five hybridizing clones
(17C9, 37C5, 48A3, 52F10, and 53B11) were subcloned into
NotI/HindIII-digested pBluescript II SK
and SK
(Stratagene) and sequenced using a
Sequenase version 2.0 DNA sequencing kit (United States Biochemical
Corp.) with T3 and T7 primers and specific synthetic oligonucleotides
as internal primers.
Expression of Drosophila AnCE cDNA
A 2.03-kb
HindIII/NotI fragment of the Drosophila AnCE
cDNA was subcloned into the HindIII-NotI sites of
pRC/CMV expression vector (Invitrogen) under the transcriptional
control of the cytomegalovirus promoter for the immediate early gene.
COS-7 cells were cultured and transfected as described
previously
(26) . At 24-h post-transfection, the medium was
discarded and the cells were washed five times with phosphate-buffered
saline before a further 24-h incubation in fetal calf serum-free
Dulbecco's minimum essential medium, since the circulating form
of ACE is present in fetal calf serum. The culture medium was collected
and assayed for ACE-soluble activity. The cells were washed five times
with phosphate-buffered saline and were dislodged from the culture
flasks by scraping into 1 ml of 8 mM CHAPS detergent, 5
mM potassium phosphate pH 8.3, 10 µM ZnSO and were incubated at 4 °C for 1 h with agitation.
Solubilized proteins were recovered in the supernatant after
centrifugation at 12, 000
g for 15 min and were
assayed for membrane-bound ACE activity.
) which produces a
secreted enzyme
(4) . Due to differences in the promoter
activities of pECE and pRC/CMV, the culture medium of pEACE and
pEACE
transfected cells was changed to fetal calf
serum-free Dulbecco's minimum essential medium 48-h
post-transfection, and the culture medium and cells were collected
after a further 24 h of growth. ACE activity was determined as
described previously
(4) , using either Hip-His-Leu or AI as the
substrate.
Western Blotting
Samples of cell culture medium
from AnCE and mock transfected cells (800 µl, corresponding to 50
nmol of Hip produced/min from pCMV/AnCE transfected cell culture
medium) were concentrated using micron-10 filtration tubes (Amicon)
before SDS-PAGE and Western blotting, as described
previously
(4) . Antiserum to AnCE was obtained by three
subcutaneous injections of 50 µg of enzyme purified from the
housefly M. domestica into a New Zealand
White rabbit at 3-week intervals.
Genomic Southern Blot Analysis
Genomic DNA
isolated using standard procedures
(27) was digested with
BamHI, EcoRI, HindIII, PstI,
XbaI, and XhoI and run on a 0.8% agarose gel. The DNA
was transferred to a Hybond N membrane (Amersham International, U.K.)
and was hybridized with the full-length AnCE cDNA. Membranes were
washed in 0.1 SSC, 0.1% SDS at 65 °C and were exposed to
x-ray film.
Northern Blot Analysis
Total RNA (20 µg),
extracted from D. melanogaster embryos using the hot phenol
method
(27) , was subjected to electrophoresis on a 1% denaturing
agarose gel, transferred to a Hybond N membrane (Amersham
International, U.K.), oven baked, and hybridized to the full-length
AnCE cDNA (5 Denhardt's solution, 5
SSC, 1% SDS,
50% formamide at 42 °C overnight). The membrane was washed using
stringent conditions with a final wash in 0.1
SSC, 0.1% SDS at
65 °C. The membrane was exposed to a Fuji imaging plate (BASIIIs)
for 4 days, and the plate was then developed using a Fuji BAS 1000
PhosphoImager.
Chromosomal Localization
In situ hybridization of the full-length AnCE cDNA to polytene chromosomes
of salivary glands of third instar larvae was carried out using a
commercial kit (Life Technologies, Inc., U.K.) according to a standard
procedure
(22) .
Sequence Comparisons
The common core region
sequences
(20) of human sACE (exons 4-11 for the N-domain
and exons 17-24 for the C-domain, corresponding to amino acid
positions 162-569 and 770-1167) were used to identify the
equivalent domains in rat, mouse, rabbit, and bovine
(6) sACE
and the D. melanogaster AnCE. Sequences were compared using
the Wisconsin GCG package
(28) . Alignments produced using
ClustalV
(29) were analyzed using the PROTDIST (Dayhoff PAM 001
matrix)
(30) and KITSCH components of PHYLIP
(31) to
generate the dendrogram shown in Fig. 4(optimum tree from 25
randomized cycles of KITSCH).
Figure 4:
A,
cartoon showing the relative positions of the conserved features of
AnCE. CHO marks the conserved glycosylation site; the
hatched area represents the conserved core region found in all
ACE domains; the solid bars mark the
Zn-binding site, and the small upright bars the positions of the conserved cysteines. B, nucleic acid
sequence of AnCE with the predicted translation product. The putative
signal peptide is marked in italics, the three glycosylation
sites are underlined, and the conserved HExxH motif and the
conserved E
are double underlined. C,
alignments of the human, mouse, and rabbit tACE and AnCE. Con. is the consensus sequence; capitalized residues are
conserved in all sequences; lower case are conservative
substitutions. A dash represents a site where there is no
consensus, and a dot represents gaps inserted for optimal
alignment. Arrows mark the conserved cysteines;
underlining is as in B.
Identification of a Soluble 67-kDa AnCE in Drosophila
Embryos
Peptidyl dipeptidase activity in the homogenate of
D. melanogaster embryos (4-18 h) measured using
Hip-His-Leu as the substrate was found predominantly (77.8 ±
1.9%; n = 4, ± S.E.) in the supernatant after
centrifugation at 200,000 g for 20 min. Washing the
200,000
g pellet with 10 mM Tris-HCl buffer,
pH, 7.4, and subsequently with 10 mM Tris-HCl, 0.5 M
NaCl, pH 7.4, left only 11.4 ± 1.1% (n = 4,
± S.E.) of the total peptidyl dipeptidase activity associated
with the membrane fraction. The hydrolysis of Hip-His-Leu by the
soluble enzyme was completely abolished in the presence of either 10
µM captopril or 10 µM trandolaprilat (data
not shown).
values of 365 and
22 µM for AI and BK, respectively (Fig. 2, A and B). The K
for the
hydrolysis of Hip-His-Leu was 2.0 mM (Fig. 2C),
and IC
values of 1.1
10
M and 1.6
10
M were
obtained for captopril and trandolaprilat, respectively (Fig. 3).
Figure 1:
SDS-polyacrylamide gel electrophoresis
of AnCE purified from Drosophila embryos. Affinity-purified
AnCE (0.1 µg) was loaded on to a Pharmacia PhastGel
Homogeneous 7.5 polyacrylamide gel which was subjected to
electrophoresis (Pharmacia PhastSystem) with SDS buffer strips.
Proteins were visualized by staining with silver nitrate and the
molecular mass of AnCE was determined by reference to marker proteins
indicated by arrows.
Figure 2:
Michaelis-Menten plots for the hydrolysis
of AI (panel A), BK (panel B), and Hip-His-Leu
(panel C) by Drosophila AnCE. The activity of
affinity-purified AnCE was measured with a range of substrate
concentrations under conditions which resulted in less than 20%
degradation of the substrate. The hydrolysis of AI was measured by
quantifying the amount of AII formed using HPLC. Peptidyl dipeptidase
activity toward BK was determined by quantifying Arg-Phe by HPLC. HPLC
methods and the assay of the hydrolysis of Hip-His-Leu are described
under ``Materials and Methods.'' Data are the mean ±
S.E. of triplicate assays. The Michaelis-Menten plots and the K values derived from them were generated by a curve-fitting
computer program (P.fit, Biosoft).
Figure 3:
Inhibition of Drosophila AnCE
activity by captopril () and trandolaprilat (
). The
activity of affinity-purified AnCE from Drosophila embryos was
assayed in the presence of different concentrations of inhibitor with
Hip-His-Leu as the substrate, as described under ``Materials and
Methods.'' The enzyme was preincubated with inhibitor for 15 min
at 37 °C before the addition of substrate. Data are expressed as a
percentage of the uninhibited activity and are the mean of triplicate
determinations ± S.E. The inhibition plot and the IC
values were generated by a curve-fitting computer program (P.fit,
Biosoft).
Cloning and Characterization of AnCE cDNA
A cDNA
comprising the nucleotide sequence between position 690 and 4024 of the
human sACE cDNA sequence
(5) was used to screen a 4-8-h
D. melanogaster embryo cDNA library. Five strongly hybridizing
clones (17C9, 37C5, 48A3, 52F10, and 53B11) were identified and
partially sequenced. Clone 53B11 had a long open reading frame of 1845
bp which was flanked at the 5` and 3` ends by 90 and 66 bp of
untranslated sequence. Partial sequencing of clones 48A3, 37C5, and
52F10 indicated that they contain partial sequences. The sequence of
the full-length cDNA was compiled from the direct sequencing of 17C9
and the partial sequencing of 37C5, 48A3, and 52F10
(Fig. 3B). The open reading frame encodes a protein of
615 amino acids with a hydrophobic N terminus, which is probably the
signal peptide for the secreted protein (Fig. 4, A and
B). There is a good consensus sequence for the hydrolysis of
the peptide bond between residues 17 and 18 by a signal
peptidase
(32) . The calculated M for the
protein including the predicted signal sequence is 70,746. The deduced
primary structure of the AnCE gene product has a high degree
of homology to both the N- and C-domains of mammalian sACE and to
mammalian tACE (see Fig. 4C for the comparison of AnCE
with tACEs). This strong sequence similarity to mammalian ACE suggests
that the clone 17C9 is a cDNA for an insect ACE which, like mammalian
tACE, is a single-domain protein. The acronym AnCE, rather than ACE,
has been given to this insect gene and its protein product, since ACE
has already been accepted as the abbreviation for the D.
melanogaster acetylcholinesterase.
is conserved, which
provides the third amino acid zinc ligand in mammalian ACE
(33) .
The AnCE sequence consistently shows a slightly more favorable
alignment with tACEs and with the C-domains of sACEs as opposed to the
N-domains. The alignment of the AnCE amino acid sequence with
human
(14, 16) , rabbit
(15) , and mouse
(17) tACEs (Fig. 4C) identifies 5 conserved
cysteines. There are three consensus sequences for
N-glycosylation sites in AnCE, the first of which has been
conserved and corresponds to the first potential
N-glycosylation site of both the N- and C-domains of sACE.
Figure 5:
Northern (A) and Western
(B) blot analysis of AnCE expression. A, Northern
hybridization of 17C9 to 20 µg of total D.melanogaster RNA from embryos. A single band can be seen
at 2.1 kilobase pairs. B, Western blot analysis of recombinant
AnCE. Culture medium (800 µl) from AnCE and mock transfected COS-7
cells were concentrated and analyzed by SDS-PAGE followed by Western
blotting using antiserum raised against housefly AnCE. The analysis
identified a protein band with an M of 74,000
which was absent from the mock transfected cell medium. The migration
of molecular weight markers is indicated on the
left.
Expression of AnCE in COS-7 Cells
Expression of
AnCE in COS-7 cells resulted in 93% of the total ACE activity in the
serum-free medium, which was inhibited by trandolaprilat (10
µM), with little enzymatic activity in the cells
(), whereas medium and cells from mock transfected cell
cultures contained negligible levels of peptidase activity. COS-7 cells
transfected with pEACE, which codes for human sACE
minus the membrane spanning and cytoplasmic domains, also produced a
soluble form of ACE. In contrast, cells transfected with pEACE, which
codes for the entire sequence of human sACE including the C-terminal
membrane anchor, synthesized membrane-bound ACE ().
However, 43% of this activity was found in the medium, which we assume
resulted from post-translational cleavage of the C-terminal anchor
peptide of the membrane-bound enzyme, as observed previously when pEACE
was expressed in Chinese hamster ovary cells
(4) . Immunoblot
analysis of serum-free medium taken from pCMV/AnCE transfected cells
with a polyclonal antibody raised to housefly AnCE revealed a band with
an M
of 74,000 on SDS-PAGE, which was not present
in medium from mock transfected COS-7 cells (Fig. 5B).
of 1.64 mM for the hydrolysis
of Hip-His-Leu, and this activity was inhibited by captopril (IC
value, 5.6
10
M) and
trandolaprilat (IC
value, 2
10
M), both of which are potent and selective inhibitors of
mammalian ACE (Fig. 6). Recombinant AnCE also catalyzed the
conversion of AI to AII, and this activity was inhibited by both
captopril and trandolaprilat.
Figure 6:
Inhibition of recombinant AnCE activity by
captopril () and trandolaprilat (
). The activity of
recombinant AnCE from transfected COS-7 cells was assayed in the
presence of different concentrations of inhibitor with Hip-His-Leu as
the substrate (4). The enzyme was preincubated with inhibitor for 2 h
at 37 °C before the addition of substrate. Data are expressed as a
percentage of the uninhibited activity and are the mean of triplicate
determinations ± S.E. The inhibition plot and the IC
values were generated by a curve-fitting computer program (P.fit,
Biosoft).
of 67,000 from embryos of D. melanogaster using a lisinopril-Sepharose column which is routinely used to
affinity purify ACE from various mammalian sources (5, 23, 34, 35). The
enzyme is very similar to mammalian ACE in that it converts AI to AII
(K
, 365 µM), removes the
C-terminal dipeptide from BK (K
, 22
µM), and is inhibited by the ACE inhibitors, captopril and
trandolaprilat. Some differences between the insect and mammalian
proteins can be inferred from the lower affinity of the Drosophila enzyme for both AI and BK (K
for
hydrolysis of AI and BK by mammalian ACEs, 16-70 and
0.18-1.00 µM, respectively) (36-38). The
insect enzyme is much smaller than mammalian sACE (M
140,000-180,000) and tACE (M
90,000-110,000), both of which are heavily glycosylated,
but is similar in size to the non-glycosylated form of the
single-domain tACE (76-84 kDa, depending on species)
(3, 39) and the N-domain ACE (68 kDa), recently found in human
ileal fluid
(35) .
of 68,921 after cleavage of the putative signal peptide. The
strong sequence homology around the active site region and the
conservation of the amino acid residues known to contribute to
catalysis and coordination with the active site Zn
of
human ACE provides strong evidence for the identification of a cDNA
coding for a Drosophila AnCE. The functional expression of the
AnCE cDNA in COS-7 cells confirmed that the translated protein
product is an ACE which is inhibited by the selective ACE inhibitors,
captopril and trandolaprilat. The potencies of these inhibitors against
the recombinant (captopril, IC
= 5.6
10
M; trandolaprilat, IC
= 2
10
M) and native
enzyme (captopril, IC
= 1.1
10
M; trandolaprilat, IC
= 1.6
10
M) are very similar. The molecular mass
of the recombinant AnCE expressed in COS-7 cells appears to be
5
kDa larger than the affinity-purified enzyme and is probably the
consequence of differences between insect and mammalian cells in
post-translational modification of the protein. The absence of a
hydrophobic region toward the C-terminal part of the protein identifies
an important difference between insect and mammalian ACEs. Most of the
mammalian ACE occurs as a membrane-bound form, although a soluble
two-domain form of enzyme is found in plasma, cerebrospinal fluid,
seminal fluid, and lymph
(3, 40) . It has been clearly
shown in mammalian cells that the soluble two-domain ACE can be derived
from membrane sACE by a proteolytic cleavage, which releases the
extracellular domain of this enzyme
(4, 41, 42) .
Recently, a soluble N-domain of human sACE has been found in human
ileal fluid, and it is believed that this enzyme is derived from
proteolysis of the intact two-domain sACE of the intestine
(35) .
There is no evidence for the direct biosynthesis in mammalian tissues
of a soluble ACE from an alternatively spliced mRNA. In contrast, the
AnCE cDNA from Drosophila embryos codes for a soluble
enzyme which is secreted from transfected COS-7 cells. A hydrophobic
signal sequence is predicted, which in some ectopeptidases is not
cleaved and serves as a membrane anchor
(43) . We cannot at
present be certain that this does not occur to some extent with the
product of the AnCE gene in vivo, but we have shown
that only around 11% of the AnCE activity of Drosophila embryos is associated with cellular membranes.
Figure 7:
Dendrogram showing the relationships
between the core sequences of the mammalian N- and C-domains, and
D. melanogaster AnCE. The line below gives the estimated time
points for the original divergence (450 million years before present),
the duplication event (270 million yearsr before present), and the
radiation of the mammals (50 million years before present) as
calculated by the KITSCH algorithm based on these sequences. The table
at right summarizes the average relative percent similarities within
and between domains and between the N- and C-domains of the mammalian
enzyme (as defined under ``Materials and Methods'') and AnCE,
as calculated using the GCG Distances
programme.
Table:
Expression of recombinant enzymes in COS-7 cells
/EMBL Data Bank with accession number(s) U25344.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.