From the
Endothelins (ET) are a family of potent vasoactive peptides that
are produced from biologically inactive intermediates, termed big
endothelins, via a proteolytic processing at
Trp
Endothelins are a family of 21-amino-acid peptides that possess
a wide variety of biological activities(1, 2) . The
first member of this family, endothelin-1 (ET-1),
Endothelins are
produced from
ECE-1
is abundantly expressed in vivo in endothelial cells and other
cell types known to produce mature ET-1. However, we failed to detect
ECE-1 expression in some cell types (e.g. neurons) that
produce endothelins(21) . This implied the existence of another
ECE in these cells. In addition, ECE-1 cleaves big ET-1 more
efficiently than big ET-2 or -3, suggesting the possibility that there
are additional ECE(s) that efficiently convert big ET-2 and/or big
ET-3. These considerations led us to search for structurally related
isoenzymes of ECE-1. In this study, we report cDNA cloning and
enzymological characterization of a novel ECE isoenzyme, ECE-2.
Transfection of ECE-2 cDNA conferred an ability to process and secrete
mature ET-1. Structurally, ECE-2 is similar to ECE-1. Both proteins
belong to the NEP-ECE-Kell family of type II membrane-bound
metalloproteases. ECE-2, like ECE-1, is inhibited by phosphoramidon and
the nonpeptidic ECE inhibitor FR901533 but not by thiorphan or
captopril. The isopeptide selectivity of ECE-2 is similar to ECE-1,
with preferential cleavage of big ET-1. The two enzymes differ in that
ECE-2 has an unusually acidic pH optima at around pH 5.5. This suggests
that ECE-2 is an intracellular processing enzyme that acts in acidified
compartments of the secretory pathway and not on the cell surface.
compares the
sensitivity of ECE-1 and ECE-2 with various protease inhibitors. Both
enzymes are inhibited by metal chelating agents, the metalloprotease
inhibitor phosphoramidon, and the specific ECE inhibitor FR901533. They
are not inhibited by the specific NEP inhibitor thiorphan, the
angiotensin-converting enzyme inhibitor captopril, or inhibitors of
other classes of proteases. The organic mercury thiol reagent pCMS
apparently augmented the activity of ECE-2 in this crude membrane-based
assay system. We feel that this is due to the inhibition of thiol
protease(s), which act to degrade the product ET-1 (and/or ECE-2
protein) under the acidic pH used in our ECE-2 assay.
Although ECE-1
and ECE-2 show a similar overall profile of inhibitor sensitivity,
dose-response analysis of the inhibition by phosphoramidon and FR901533
demonstrates a striking pharmacological difference between ECE-1 and
ECE-2 (Fig. 3C). The potency of phosphoramidon against
ECE-2 is
Fig. 3D shows the isopeptide substrate selectivity of
ECE-1 and ECE-2. Both enzymes have a strong substrate preference toward
big ET-1 at their respective optimal pH. As determined with
20-40-µg membrane proteins, ECE-1 cleaves big ET-2 and big
ET-3 only 5-7% and 1-3% as rapidly as it converts big ET-1,
respectively. Similarly, ECE-2 cleaves big ET-1, -2, and-3 at relative
rates of 100%, 7-10%, and 4-9%, respectively. To
confirm that this apparent selectivity toward big ET-1 is not due to a
difference in the stability of isopeptide substrates or products during
the enzyme reactions, we incubated each big and mature peptide (0.1
µM) with 20-µg crude membrane proteins under the
standard ECE-1 and ECE-2 assay conditions and measured the amount of
the remaining intact peptide after incubation. The reaction mixture did
not contain protease inhibitors, although 4-amidinophenylmethylsulfonyl
fluoride, pCMS, and pepstatin A were included in the initial
homogenization buffer for membrane preparation as described
previously(21) . Under the ECE-1 assay condition (pH 6.8), we
did not detect significant degradation of either of the big and mature
isopeptides after up to 4 h of incubation. In contrast, we observed
appreciable degradation of the substrates and products under the ECE-2
assay condition (pH 5.6); after incubation for 30 min (standard assay
period) the amounts of intact peptides decreased by approximately 15%,
and after 2 h of incubation, the remaining amounts of intact peptides
were 50-55%. Importantly, however, we did not observe an
appreciable difference in the rate of degradation among the different
isopeptides; big ET-1, -2, and -3 and mature ET-1, -2, and -3 were all
degraded at similar rates. Crude membranes from CHO/ECE-2 and
untransfected CHO cells exhibited similar rates of degradation,
indicating that the degradation is caused by endogenous acidic
protease(s) contained in our CHO membrane preparations. These findings
indicate that the >10-fold isopeptide selectivity we observed in the
specific cleavage of big peptides by ECE-1 and ECE-2 is not an artifact
due to a selective degradation of the isopeptide substrate or product.
We previously showed that CHO/ECE-1 cells
can also cleave exogenously added big ET-1, presumably because of the
location of some of the ECE-1 on the cell surface(21) . Since
ECE-2 has little activity at neutral pH, ECE-2 should be incapable of
cleaving extracellular big ET-1 under normal culture conditions. We
tested this by coculturing the stable transfectant CHO/prepro-ET-1
cells with either CHO/ECE-1 or CHO/ECE-2 cells and determining the
amount of mature ET-1 in the medium. Consistent with our previous
findings, CHO/ECE-1 cells produced significant amounts of mature ET-1
in the coculture assay (Fig. 5). The production of mature peptide
was readily inhibited by both phosphoramidon and FR901533 with
IC
We have described the cloning of ECE-2, a novel
metalloprotease that can convert big ET-1 into mature ET-1 both in
vitro and in transfected cells. ECE-2 qualifies as an
``endothelin-converting enzyme'' in that it can produce large
amounts of mature ET-1 from big ET-1 in test tubes and in live cells
(up to 90% of total endothelin peptides converted in the double
transfection assays). However, we have not yet formally tested whether
ECE-2 cleaves big endothelins at other site(s) than the
Trp
Although ECE-2 closely
resembles ECE-1, the two enzymes exhibited two significant differences. (i) The nanomolar sensitivity of ECE-2 to phosphoramidon in vitro resembles NEP rather than ECE-1(30) . (ii) The acidic pH optimum of ECE-2 with a narrow pH profile
is unusual for a metalloprotease. Although a pH optimum of
5.3-5.5 has been reported for a class of matrix metalloprotease
in cartilage, matrix metalloproteinase-3 (stromelysin-1), this enzyme
shows a broad pH profile spanning a pH range from 5 to
>8(31) . We are unaware of a metalloprotease that is inactive
at neutral pH. In contrast to these differences, we found that ECE-1
and ECE-2 have very similar isopeptide substrate selectivities; both
enzymes strongly preferred big ET-1 over big ET-2 and -3. This implies
that there may be yet other ECE(s) that cleave big ET-2 and/or big ET-3
more efficiently.
We previously reported that we did not detect
ECE-1 mRNA in neurons and glia in the bovine brain by in situ hybridization and speculated that these cells, which are known to
produce mature endothelins, may have another ECE(21) . The
Northern blots presented in this paper show that neural tissues
represent the site of the most abundant expression of ECE-2 mRNA.
Although we have not yet carried out in situ hybridization
histochemistry with ECE-2 probes, these findings suggest the
possibility that ECE-2 may be the major ECE in neurons, glia, and
certain neuroendocrine cells.
We have demonstrated that ECE-2 cannot
efficiently convert extracellular big ET-1 on the cell surface, as
expected from the acidic pH profile of the enzyme. However, double
transfection of CHO cells with ECE-2 and prepro-ET-1 led to a
significant production of mature ET-1, due to an intracellular cleavage
of endogenously synthesized big ET-1 in these cells. The deduced
structure of ECE-2 predicts that it is expressed as a type II integral
membrane protein, and its C-terminal catalytic domain faces the lumen
of secretory vesicles, where it encounters the substrate big ET-1. The
trans-Golgi network (and later compartments of the secretory pathway)
are known to provide a highly acidified intravesicular environment in
many cells(32) . The luminal pH of the trans-Golgi network has
been directly measured to be 5.5-5.7, which precisely matches the
optimal pH range of ECE-2. We feel that ECE-2 functions in these
acidified compartments of the secretory pathway. We do not know whether
the same ECE-2 molecules are located on the cell surface. However, we
speculate that cell surface ECE-2, if any, may not have functional
relevance except under pathological conditions where the interstitial
space is abnormally acidified. It is worth noting that small but
detectable amounts of mature ET-1 were produced in the
CHO/ECE-2+CHO/prepro-ET-1 coculture experiments (Fig. 5).
The inability of the two ECE inhibitors to inhibit this conversion at
low concentrations suggested that the conversion occurred
intracellularly. We speculate that the small amounts of conversion
observed in the CHO/ECE-2+CHO/prepro-ET-1 cocultures may be due to
an internalization of the extracellular big ET-1 by the CHO/ECE-2
cells, followed by cleavage within the acidified intracellular vesicles
and then resecretion of the mature peptide.
Further studies are
required to determine the physiological relevance of the intracellular versus cell surface conversion of big endothelins by the ECE
isoenzymes. Nevertheless, these observations indicate that the
development of ECE inhibitor requires a careful consideration on cell
permeability of inhibitor compounds. The live cell assay system
described in this and our previous study (21) should facilitate
the screening of therapeutically useful ECE inhibitors.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Sumio Kiyoto for a sample of FR901533;
Nobuhiro Suzuki and Hirokazu Matsumoto for the EIA antibodies; Damiane
deWit for technical assistance; and Mike Brown and Joe Goldstein for
reading this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Val/Ile
. We recently cloned and
characterized a membrane-bound metalloprotease that catalyzes this
proteolytic activation, endothelin-converting enzyme-1 (ECE-1) (Xu, D.,
Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and
Yanagisawa, M.(1994) Cell 78, 473-485). This enzyme was
shown to function in the secretory pathway as well as on the cell
surface. Here we report molecular cloning of another novel enzyme,
ECE-2, that produces mature ET-1 from big ET-1 both in vitro and in transfected cells. The cDNA sequence predicts that bovine
ECE-2 is a metalloprotease structurally related to ECE-1, neutral
endopeptidase 24.11, and human Kell blood group protein. The deduced
amino acid sequence of ECE-2 is most similar to ECE-1, with an overall
identity of 59%. ECE-2 resembles ECE-1 in that it is inhibited in
vitro by phosphoramidon and FR901533 but not by thiorphan or
captopril, and it converts big ET-1 more efficiently than big ET-2 or
big ET-3. However, ECE-2 also exhibits the following striking
differences from ECE-1. (i) The sensitivity of ECE-2 to
phosphoramidon is 250-fold higher as compared with ECE-1, while
FR901533 inhibits both enzymes at similar concentrations. (ii)
ECE-2 has an acidic pH optimum at pH 5.5, which is in sharp contrast to
the neutral pH optimum of ECE-1. ECE-2 has a narrow pH profile and is
virtually inactive at neutral pH. Chinese hamster ovary (CHO) cells,
which lack detectable levels of endogenous ECE activity, secrete mature
ET-1 into the medium when doubly transfected with ECE-2 and prepro-ET-1
cDNAs. However, ECE-2-transfected CHO cells do not efficiently produce
mature ET-1 when present with an exogenous source of big ET-1 through
coculture with prepro-ET-1-transfected CHO cells. These findings
suggest that ECE-2 acts as an intracellular enzyme responsible for the
conversion of endogenously synthesized big ET-1 at the trans-Golgi
network, where the vesicular fluid is acidified.
(
)was identified as an endothelium-derived
vasoconstrictor(3) . Three known members of the mammalian
endothelin family, ET-1, ET-2, and ET-3, are produced in various
tissues(4) . They act on two distinct subtypes of
G-protein-coupled receptors termed ET
and ET
,
which are expressed on a variety of target
cells(5, 6, 7) . Recent studies with specific
endothelin receptor antagonists have indicated that endothelins play
important roles in a number of animal models for vascular diseases and
possibly in certain pathological conditions in
humans(8, 9, 10, 11) . Mice carrying
targeted mutations in the ET-1, ET-3, and ET
receptor genes
exhibit developmental abnormalities that suggest a role for endothelins
in the development of neural crest-derived
tissues(12, 13, 14) .
200-residue prepropolypeptides, which are first
processed by the subtilisin family of prohormone processing enzyme(s) (15) into biologically inactive, 38-41-residue
intermediates called big ET-1, -2, and -3. The C-terminal halves of big
endothelins are then clipped off between Trp
and
Val/Ile
, yielding the N-terminal, 21-residue active
endothelins. This proteolytic conversion is catalyzed by specific
protease(s) called endothelin-converting enzyme(s) (ECE)(3) .
Several lines of evidence suggest that the physiologically relevant
ECE(s) are sensitive to the metalloprotease inhibitor
phosphoramidon(16) . Thus, in whole animal preparations and
isolated perfused tissues, the vasopressor actions of exogenously
administered big endothelins are inhibited by phosphoramidon. The
processing of endogenous big ET-1 in cultured endothelial cells is also
inhibited by phosphoramidon. Biochemical studies have shown that the
ECE from endothelial cells and other sources is a membrane-bound
metalloprotease(17, 18, 19) . One such
metalloprotease, ECE-1, was recently cloned(20, 21) .
ECE-1 was shown to be a type II membrane-bound metalloprotease that
processed endogenously produced big ET-1 intracellularly and
exogenously supplied big ET-1 on the cell surface(21) .
Reagents
Synthetic human big ET-1-(1-38),
big ET-2-(1-38), big ET-3-(1-41) amide, ET-1, ET-2, and
ET-3 were obtained from American Peptides. Phosphoramidon, thiorphan,
captopril, 1,10-phenanthroline, 4-amidinophenylmethylsulfonyl fluoride, p-chloromercuriphenylsulfonic acid (pCMS), N-ethylmaleimide, E-64, pepstatin A, and leupeptin were from
Sigma. FR901533 (WS79089B;
1,6,9,14-tetrahydroxy-3-(2-hydroxypropyl)-7-methoxy-8,13-dioxo-5,6,8,13-tetrahydrobenzo[a]naphthacene-2-carboxylateNa)
was a generous gift from Fujisawa Pharmaceutical Co., Ltd.
cDNA Cloning and Sequencing
A partial cDNA clone
encoding ECE-2 was obtained by RT-PCR (21) against bovine
adrenal cortex mRNA with highly degenerate primers based on a peptide
microsequence from purified bovine ECE-1. Unamplified gt10 bovine
adrenal cortex and bovine endothelial cell cDNA libraries (21) were screened with the
P-labeled RT-PCR
product as probe. Nine and five positive plaques were identified in the
adrenal cortex and endothelial libraries, respectively. The 5` end of
the cDNA was cloned by 5`-RACE (Life Technologies, Inc.) against bovine
adrenal cortex poly(A)
RNA. The first-strand cDNA was
synthesized with SuperScript reverse transcriptase (Life Technologies,
Inc.) by using a specific primer ACAGGGGCTCACTCCA (corresponding to
amino acids 134-139 of ECE-2). An oligo(dC) anchor was added to
the 3` end of the first-strand cDNA with terminal
deoxynucleotidyltransferase. The first round of PCR was performed as
recommended by the manufacturer with a specific 3` primer
CTCCAGGATTTTTCCAGCCACTCGA (amino acids 122-130) and a 5` anchor
primer. The product of this PCR reaction was subjected to the second
amplification by using a nested specific 3` primer
GGCCTCTGTGAGGCAAGTGCTATG (amino acids 113-120). The products from
three independent 5`-RACE reactions were separately subcloned into pCR
II plasmid (Invitrogen) and sequenced. For nucleotide sequencing,
overlapping restriction fragments of cDNA were subcloned in pBlueScript
plasmid vector (Stratagene), and double-stranded plasmid DNA was
PCR-sequenced by an Applied Biosystems model 373A DNA Sequenator. Both
strands of cDNA were covered at least twice. Some subclones were
manually sequenced by using the Sequenase kit (U. S. Biochemical
Corp.).
Northern Blotting
RNA was extracted from bovine
tissues by the LiCl/urea method(22) . Total RNA (10 µg) was
separated in a formaldehyde/1.1% agarose gel, transferred to a nylon
membrane, and prehybridized and hybridized in QuickHyb solution
(Stratagene) as recommended by the manufacturer. A 0.3-kb Eco47III-XhoI fragment of the bovine ECE-2 cDNA,
which encodes amino acids 198-283 and does not cross-hybridize
with the ECE-1 mRNA, was random primed P-labeled and used
as a probe. The membranes were washed finally in 0.1
SSC, 0.1%
SDS at 60 °C and exposed to an x-ray film for 24 h (ECE-2) and for
90 min (
-actin) at -80 °C with an intensifying screen.
Antibodies and Immunoblotting
Antibodies directed
against ECE-1 and ECE-2 were each produced by immunizing rabbits with
synthetic peptides, CPPGSPMNPHHKCEVW and CPVGSPMNSGQLCEVW,
corresponding to the C-terminal 16 amino acids of bovine ECE-1 and
ECE-2, respectively. Rabbits were immunized with keyhole limpet
hemocyanin-coupled peptides in complete adjuvant, and the antisera were
prepared. Immunoblot analysis was performed with horseradish
peroxidase-conjugated anti-rabbit IgG by using the ECL detection kit
(Amersham Corp.) as recommended by the manufacturer.
Cell Culture and Transfection
CHO-K1 cells were
cultured as described(21) . Since many batches of tissue
culture-grade trypsin preparations contained high levels of
metalloprotease contaminants with an ECE-like activity, a highly
purified crystallized preparation of trypsin (Sigma, catalog no. T7418)
was dissolved in phosphate-buffered saline at 0.013% (w/v) and used for
all trypsinization procedures. The coding region of bovine ECE-2 cDNA
was subcloned into pME18Sf- expression vector(23) . Stable
transfection of CHO cells and isolation of the transfectant clones, as
well as transient transfection of human prepro-ET-1 cDNA was performed
as described(21) . Twelve hours after the transient
transfection, cells were refed with fresh medium with or without ECE
inhibitors. The medium was conditioned for an additional 12 h and
directly subjected to enzyme immunoassay (EIA) for mature ET-1 (24).
ECE Assays
Solubilized crude membranes were
prepared in parallel from CHO/ECE-2 and CHO/ECE-1 cells as
described(21) . For protease inhibitor studies, membranes were
prepared without protease inhibitors. Standard reaction mixtures for
ECE-2 assay (50 µl) contained 0.1 M MES buffer (pH 5.5),
0.5 M NaCl, 0.1 µM human big ET-1 and enzyme
fraction. ECE-1 assay reactions contained 0.1 M phosphate
buffer (pH 6.8) instead of the MES buffer. For the pH profiling and
isopeptide selectivity studies, the buffer solution (0.1 M) or
the substrate (0.1 µM) was substituted as designated. For
the inhibitor studies, the reactions were preincubated at 37 °C
with protease inhibitor or vehicle for 15 min. The reaction was started
by the addition of substrate and incubated at 37 °C for 30 min in
siliconized 0.5-ml microcentrifuge tubes. Enzyme reactions were
terminated by adding 50 µl of 5 mM EDTA. The mixture was
then directly assayed for mature ET-1 as described(24) .
Duplicate assay wells were used for each enzyme reaction. For big ET-2
and big ET-3 conversion assays, mature ET-1 EIA (which fully
cross-reacts with mature ET-2) and mature ET-3 EIA were used with human
ET-2 and ET-3 as standards, respectively(25) . Protein
concentration was determined by the Bradford method (Bio-Rad) using IgG
as standard.
Cloning of ECE-2 cDNA
We previously reported the
purification and peptide microsequence analysis of bovine
ECE-1(21) . The N-terminal sequence of one of the Lys-C-digested
peptide fragments from the purified ECE-1 (residues 562-586 in Fig. 1) showed a significant similarity to amino acid residues
543-567 of human NEP (26). We designed a pair of highly
degenerate oligonucleotide primers based on this 25-residue sequence.
RT-PCR from bovine adrenal cortex RNA yielded cDNA products of the
predicted size. We subcloned these cDNA fragments into plasmid vectors
and determined the nucleotide sequence. Unexpectedly, the sequences
from several randomly picked plasmid clones revealed that the 75-base
pair cDNA product was a mixture of two distinct cDNA sequences; about
80% of the plasmid clones encoded the ECE-1 microsequence, whereas the
nucleotide sequences from the remaining clones predicted a closely
related polypeptide sequence in which 4 amino acid residues out of 25
differed from ECE-1 (see Fig. 1). Moreover, the third nucleotide
residues in the reading frame were frequently different between the two
cDNA sequences, suggesting that these cDNAs are derived from the
products of two different genes. Based on these findings, we named the
second putative protein ECE-2.
Figure 1:
Deduced amino acid sequence of
bovine ECE-2 aligned with bovine ECE-1. Dots represent amino
acid residues identical to those in the aligned ECE-1 sequence.
Sequence gaps introduced for maximum match alignment are designated by dashes. Putative transmembrane domains are represented by doubleunderlines. The Cys residues conserved in all
known members of the NEP-ECE-Kell family are underscored.
Predicted N-glycosylation sites are designated by openboxes. The conserved zinc binding motifs are marked by a closedbox. A dottedunderscore shows the peptide microsequence of ECE-1 from which the degenerate
PCR primers were designed.
With the cloned ECE-2 RT-PCR product
as probe, we screened a cDNA library from bovine adrenal cortex. In an
initial screening, we detected 9 ECE-2 positive clones, as compared
with 15 ECE-1 clones detected from the same library. Partial sequencing
of these clones indicated that they contained overlapping cDNAs derived
from the same ECE-2 mRNA but lacked a 5` part of the coding region.
Since we could not clone the 5` part of the cDNA by rescreening the
library, we performed 5`-RACE by using a nested set of specific
internal primers. This yielded five overlapping extensions to the cDNA,
which covered all of the coding sequence. The full-length nucleotide
sequences of the ECE-2 cDNA revealed a 5` ATG triplet, which was
preceded by an in-frame stop codon and followed by a long open reading
frame. The predicted amino acid sequence of ECE-2 is shown in Fig. 1, aligned with the amino acid sequence of bovine
ECE-1(21) .
Structure of ECE-2
The ECE-2 cDNA sequence encodes
a novel 787-amino-acid polypeptide, which shares important structural
features with ECE-1. (i) The cDNA predicts a type II integral
membrane protein with a 82-residue N-terminal cytoplasmic tail, a
23-residue putative transmembrane helix (Fig. 1, doubleunderscore), and a large (682 residue) extracellular
C-terminal part. (ii) The extracellular portion of ECE-2
constitutes the putative catalytic domain and contains (residues
622-630) a highly conserved consensus sequence of a zinc-binding
motif, XHE
H
(where
and
represent an uncharged and hydrophobic amino acid,
respectively), that is shared by many Zn
metalloproteases(27) . (iii) ECE-2 has 10
predicted sites for N-glycosylation in the extracellular
domain, suggesting that ECE-2, like ECE-1(20) , is a highly
glycosylated protein. Immunoblot analysis with an anti-ECE-2 C-terminal
peptide antiserum shows that ECE-2 is expressed as a
130-kDa
protein in bovine adrenal medulla (see Fig. 3A). The
predicted molecular weight of the ECE-2 polypeptide is 88,952. We
assume that the difference between this value and the apparent
molecular weight of ECE-2 on immunoblots may be largely due to the
sugar side chains. (iv) There are 4 Cys residues in the
extracellular domain near the transmembrane helix that are conserved
among all proteins in the NEP-ECE-Kell family.
Figure 3:
In vitro characterization of
ECE-1 and ECE-2 from membrane fractions of stably transfected CHO
cells. A, immunoblot analysis of membrane fractions from
CHO/ECE-1 cells, CHO/ECE-2 cells, and bovine adrenal medulla. Membrane
proteins (75 µg/lane) were separated on a 6%
SDS-polyacrylamide gel under reduced conditions, blotted, and detected
by anti-C-terminal peptide antisera for ECE-1 and ECE-2. Preliminary
digestions with endoglycosidase H and F showed that the 110-kDa
species seen in the transfected cells are partially glycosylated
enzymes. B, pH profiles of ECE-1 and ECE-2. C,
concentration-dependent inhibition of ECE-1 and ECE-2 by phosphoramidon
and FR901533. D, isopeptide substrate selectivity of ECE-1 and
ECE-2.
A search of the
Entrez sequence data base detected a significant similarity of the
ECE-2 sequence to ECE-1, NEP, and the human Kell minor blood group
protein(28) . The sequence similarity is especially high within
the C-terminal one-third of the putative extracellular domain,
including the region around the zinc-binding motif. Within this region
(amino acids 582-787 of ECE-2), the identities of ECE-2 with
respect to ECE-1, NEP, and Kell are 71, 44, and 40%, respectively. ECE-1 and ECE-2 are 52% identical to each other in the
N-terminal portions (amino acids 1-581 in ECE-2), while they
resemble NEP and Kell only slightly in these regions. This indicates
that the ECE-1 and ECE-2 comprise a subfamily within this group of type
II membrane-bound metalloproteases.
Tissue Distribution of ECE-2 mRNA
Northern blot
analysis of bovine tissues revealed relatively large amounts of the
3.3-kb ECE-2 mRNA in the neural tissues, i.e. cerebral cortex,
cerebellum, and adrenal medulla (Fig. 2). Small amounts of the
3.3-kb mRNA were detected also in myometrium and testis. Low amounts of
a longer mRNA (4.7 kb) were detected in ovary and cultured
endothelial cells, as well as in the aforementioned neural tissues. A
long (96-h) exposure of the blots showed that the 4.7-kb mRNA is
expressed at very low levels in many other tissues. Screening of an
endothelial cell cDNA library confirmed that the 4.7-kb species is an
authentic ECE-2 mRNA with an extended 3`-noncoding region. The coding
sequences of the 3.3- and 4.7-kb mRNA were identical. In all tissues
examined, the absolute amounts of ECE-2 mRNAs were much smaller than
the ECE-1 mRNA (data not shown). The intensity of the ECE-1 mRNA
signals in similar Northern blots was severalfold higher than that for
ECE-2 mRNA even in the brain, where ECE-1 mRNA expression is
comparatively low(21) . In cultured endothelial cells, we
estimated the amount of ECE-2 mRNA is only 1-2% of that of ECE-1
mRNA.
Figure 2:
Northern blot analysis of ECE-2 mRNA in
bovine tissues and cultured coronary artery endothelial cells.
Rehybridization with -actin probe is shown as an internal standard
for the amounts of RNA loaded.
In Vitro Characterization of ECE-2
We previously
showed that CHO cells do not possess detectable levels of endogenous
ECE activity, as assayed both in vitro and in live
cells(21) . By transfecting a ECE-2 expression construct driven
by the SR viral promoter(23) , we generated a stable
transfectant cell line, CHO/ECE-2. Immunoblot analysis shows that
membrane fractions from CHO/ECE-2 and CHO/ECE-1 (21) cells
contain high levels of ECE-2 and ECE-1 proteins, respectively (Fig. 3A). To compare the enzymological properties of
ECE-1 and ECE-2, we assayed the membrane-associated ECE activities from
these cell lines in parallel. Initial experiments under our standard
ECE-1 assay conditions detected little ECE activity in multiple ECE-2
transfectant clones, which had been confirmed to express high levels of
ECE-2 mRNA by Northern and immunoblot analyses. We subsequently found
that this was because ECE-2 is virtually inactive at the neutral pH
(6.8) used in our ECE-1 assay. Fig. 3B compares the pH
profiles of ECE-1 and ECE-2. An optimal activity of ECE-1 and ECE-2 was
obtained at pH 6.8 and 5.5, respectively. Both enzymes have a sharp pH
dependence; one enzyme is virtually inactive at the optimal pH for the
other. Based on these findings, we performed ECE-1 and ECE-2 assays at
pH 6.8 and 5.5, respectively, in all subsequent experiments. Crude
membranes from untransfected CHO cells did not have a detectable ECE
activity as assayed either at pH 5.5 or 6.8. A majority of the ECE-2
activity in the CHO/ECE-2 cell homogenates was found in the membrane
fraction. We did not detect significant ECE-2 activity in the culture
supernatants from CHO/ECE-2 cells.
250-fold higher than against ECE-1 (IC
values, 1 µM and 4 nM for ECE-1 and ECE-2,
respectively). In contrast, FR901533 inhibits both enzymes with similar
potencies (IC
values, 2 and 3 µM for ECE-1
and ECE-2, respectively). Phosphoramidon is a competitive
inhibitor for this family of metalloproteases(16) . In order to
confirm that the observed difference in the apparent IC
values for phosphoramidon is not due to a large
difference in substrate affinity between ECE-1 and ECE-2, we determined K
values for the substrate by
measuring the initial rate of cleavage under increasing concentrations
of big ET-1 (0.01-10 µM). The apparent K
values of ECE-1 and ECE-2 were
both within 1-2 µM ranges as determined with 20
µg of crude membrane proteins per reaction (30 min of incubation).
Cleavage of Big ET-1 by Live ECE-2-transfected
Cells
To examine whether ECE-2 can convert big ET-1 in
physiological context in transfected cells, we first used a double
transfection assay described previously(21) . CHO/ECE-2 cells
and untransfected CHO cells were transiently transfected in parallel
with a prepro-ET-1 construct, and mature ET-1 secreted from these cells
into the medium was determined by EIA. As shown in Fig. 4,
parental CHO cells transfected with prepro-ET-1 cDNA did not secrete a
significant amount of mature ET-1, consistent with the finding that CHO
cells do not have detectable ECE activity. In contrast, CHO/ECE-2 cells
transfected with the prepro-ET-1 construct produced large amounts of
mature peptide, indicating that ECE-2 cDNA conferred these cells the
ability to convert endogenously supplied big ET-1.
Figure 4:
Production of mature ET-1 by CHO/ECE-2
cells transiently transfected with prepro-ET-1 cDNA; cleavage of
endogenously produced big ET-1. Doubly transfected cells were cultured
for 12 h in the absence or presence of the designated concentrations of
ECE inhibitors, and mature ET-1 in the conditioned medium was
determined. Negative control experiments with parental CHO cells are
shown by dashedlines.
Phosphoramidon
has previously been shown to inhibit the secretion of mature ET-1 from
cultured endothelial cells, with a concomitant increase of big ET-1
secretion(29) . We also showed previously that phosphoramidon at
high concentrations is capable of inhibiting the conversion of big ET-1
in cells doubly transfected with ECE-1 and prepro-ET-1
cDNAs(21) . Fig. 4shows that phosphoramidon inhibits the
production of mature ET-1 by the ECE-2-prepro-ET-1 double transfected
cells in a concentration-dependent manner. The apparent IC value in this live cell assay was about 20 µM, which
is much higher than the IC
for the inhibitor determined in
the test tube (4 nM, see Fig. 3C). Furthermore,
FR901533, which efficiently inhibits the conversion of big ET-1 by
ECE-2 in vitro, did not appreciably inhibit the conversion in
the transfected cells when added to the medium at up to 1 mM.
We previously showed that FR901533 is quite stable under culture
conditions, and therefore the inability of the compound to inhibit the
conversion is not due to degradation of FR901533(21) . These
observations indicate that ECE-2 is processing big ET-1 inside the
cells, where these inhibitors have limited access. The concentration of
phosphoramidon required to inhibit big ET-1 conversion in the live
CHO/ECE-2 cells was much lower than those in CHO/ECE-1 cells (IC
> 200 µM in a parallel assay; data not shown).
This presumably reflects the higher in vitro sensitivity of
ECE-2 to phosphoramidon.
values (about 1 µM) similar to those seen
in the in vitro assay, indicating that the conversion takes
place on the cell surface. In contrast, only very small amounts of
mature peptide were produced in the coculture of CHO/ECE-2 and
CHO/prepro-ET-1 cells. Moreover, mature peptide production was not
inhibited by FR901533 at up to 100 µM or by phosphoramidon
at up to 3 µM (Fig. 5). This is compatible with the
notion either that ECE-2 is not expressed on the cell surface or that
ECE-2 cannot convert exogenously supplied big ET-1 on the cell surface
under normal tissue culture conditions, even if there is surface
expression.
Figure 5:
Production of mature ET-1 by
CHO/ECE-1CHO/prepro-ET-1 and CHO/ECE-2CHO/prepro-ET-1 cocultures;
cleavage of exogenously supplied big ET-1. Cells were cocultured for 24
h in the absence or presence of the designated concentrations of
phosphoramidon (Phos) or FR901533 (FR), and mature
ET-1 in the medium was determined. Negative control experiments were
performed by coculturing parental CHO cells with CHO/prepro-ET-1
cells.
-Val/Ile
cleavage site. We found that
crude membrane fractions from CHO cells contain large amounts of mature
ET-1-degrading protease activities at pH 5.5. The absence of such
ET-1-degrading activity at neutral pH previously enabled us to perform
a more detailed in vitro analysis of cloned ECE-1 in crude
membranes(21) . To characterize ECE-2 further, we need a
purified preparation of recombinant ECE-2.
Table: Protease inhibitor profile of ECE from
solubilized CHO/ECE-1 and CHO/ECE-2 membranes
/EMBL Data Bank with accession number(s) U27341.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.