©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endothelin-converting Enzyme-2 Is a Membrane-bound, Phosphoramidon-sensitive Metalloprotease with Acidic pH Optimum (*)

Noriaki Emoto (§) , Masashi Yanagisawa (¶)

From the (1)Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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),()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) .

Endothelins are produced from 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) .

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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, XHEH (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.

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 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 ICvalues for phosphoramidon is not due to a large difference in substrate affinity between ECE-1 and ECE-2, we determined Kvalues for the substrate by measuring the initial rate of cleavage under increasing concentrations of big ET-1 (0.01-10 µM). The apparent Kvalues 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).

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.

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.

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 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.




DISCUSSION

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-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.

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.

  
Table: Protease inhibitor profile of ECE from solubilized CHO/ECE-1 and CHO/ECE-2 membranes



FOOTNOTES

*
This study is supported in part by research grants from the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U27341.

§
Associate of the Howard Hughes Medical Institute.

Associate Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9050. Tel.: 214-648-5082; Fax: 214-648-5068.

The abbreviations used are: ET, endothelin; ECE, endothelin-converting enzyme; pCMS, p-chloromercuriphenylsulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; EIA, enzyme immunoassay; RT-PCR, reverse transcription-polymerase chain reactions; RACE, rapid amplification of cDNA ends; kb, kilobase(s); CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Yanagisawa, M.(1994) Circulation89, 1320-1322 [Medline] [Order article via Infotrieve]
  2. Rubanyi, G. M., and Polokoff, M. A.(1994) Pharmacol. Rev.46, 325-415 [Medline] [Order article via Infotrieve]
  3. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T.(1988) Nature332, 411-415 [CrossRef][Medline] [Order article via Infotrieve]
  4. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K., and Masaki, T.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 2863-2867 [Abstract]
  5. Arai, H., Hori, S., Aramori, I., Ohkubo, H., and Nakanishi, S.(1990) Nature348, 730-732 [CrossRef][Medline] [Order article via Infotrieve]
  6. Sakurai, T., Yanagisawa, M., Takuwa, Y., Miyazaki, H., Kimura, S., Goto, K., and Masaki, T.(1990) Nature348, 732-735 [CrossRef][Medline] [Order article via Infotrieve]
  7. Bax, W. A., and Saxena, P. R.(1994) Trends Pharmacol. Sci.15, 379-386 [CrossRef][Medline] [Order article via Infotrieve]
  8. Ohlstein, E. H., Nambi, P., Douglas, S. A., Edwards, R. M., Gellai, M., Lago, A., Leber, J. D., Cousins, R. D., Gao, A., Frazee, J. S., Peishoff, C. E., Bean, J. W., Eggleston, D. S., Elshourbagy, N. A., Kumar, C., Lee, J. A., Brooks, D. P., Weinstock, J., Feuerstein, G., Poste, G., et al.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 8052-8056 [Abstract]
  9. Clozel, M., Breu, V., Burri, K., Cassal, J.-M., Fischli, W., Gray, G. A., Hirth, G., Loffler, B.-M., Muller, M., Neldhart, W., and Ramuz, H. (1993) Nature365, 759-761 [CrossRef][Medline] [Order article via Infotrieve]
  10. Giaid, A., Yanagisawa, M., Langleben, D., Michel, R. P., Levy, R., Shennib, H., Kimura, S., Masaki, T., Duguid, W. P., and Stewart, D. J. (1993) N. Engl. J. Med.328, 1732-1740 [Abstract/Free Full Text]
  11. Douglas, S. A., Meek, T. D., and Ohlstein, E. H.(1994) Trends Pharmacol. Sci.15, 313-316 [CrossRef][Medline] [Order article via Infotrieve]
  12. Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K., Nagai, R., Oda, H., Kuwaki, T., Cao, W., Kamada, N., Jishage, K., Ouchi, Y., Azuma, S., Toyoda, Y., Ishikawa, T., Kumada, M., and Yazaki, Y.(1994) Nature368, 703-710 [CrossRef][Medline] [Order article via Infotrieve]
  13. Baynash, A. G., Hosoda, K., Giaid, A., Richardson, J. A., Emoto, N., Hammer, R. E., and Yanagisawa, M.(1994) Cell79, 1277-1285 [Medline] [Order article via Infotrieve]
  14. Hosoda, K., Hammer, R. E., Richardson, J. A., Baynash, A. G., Cheung, J. C., Giaid, A., and Yanagisawa, M.(1994) Cell79, 1267-1276 [Medline] [Order article via Infotrieve]
  15. Seidah, N. G., Day, R., Marcinkiewicz, M., and Chretien, M.(1993) Ann. N. Y. Acad. Sci.680, 135-146 [Medline] [Order article via Infotrieve]
  16. Opgenorth, T. J., Wu-Wong, J. R., and Shiosaki, K.(1992) FASEB J.6, 2653-2659 [Abstract/Free Full Text]
  17. Ahn, K., Beningo, K., Olds, G., and Hupe, D.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 8606-8610 [Abstract]
  18. Okada, K., Arai, Y., Hata, M., Matsuyama, K., and Yano, M.(1993) Eur. J. Biochem.218, 493-498 [Abstract]
  19. Takahashi, M., Matsushita, Y., Iijima, Y., and Tanzawa, K.(1993) J. Biol. Chem.268, 21394-21398 [Abstract/Free Full Text]
  20. Shimada, K., Takahashi, M., and Tanzawa, K.(1994) J. Biol. Chem.269, 18275-18278 [Abstract/Free Full Text]
  21. Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and Yanagisawa, M.(1994) Cell78, 473-485 [Medline] [Order article via Infotrieve]
  22. Inoue, A., Yanagisawa, M., Takuwa, Y., Mitsui, Y., Kobayashi, M., and Masaki, T.(1989) J. Biol. Chem.264, 14954-14959 [Abstract/Free Full Text]
  23. Sakamoto, A., Yanagisawa, M., Sawamura, T., Enoki, T., Ohtani, T., Sakurai, T., Nakao, K., Toyo-oka, T., and Masaki, T.(1993) J. Biol. Chem.268, 8547-8553 [Abstract/Free Full Text]
  24. Suzuki, N., Matsumoto, H., Kitada, C., Masaki, T., and Fujino, M. (1989) J. Immunol. Methods118, 245-250 [CrossRef][Medline] [Order article via Infotrieve]
  25. Matsumoto, H., Suzuki, N., Onda, H., and Fujino, M.(1989) Biochem. Biophys. Res. Commun.164, 74-80 [Medline] [Order article via Infotrieve]
  26. Malfroy, B., Kuang, W. J., Seedburg, P. H., Mason, A. J., and Schofield, P. R.(1988) FEBS Lett.229, 206-210 [CrossRef][Medline] [Order article via Infotrieve]
  27. Rawlings, N. D., and Barrett, A. J.(1993) Biochem. J.290, 205-218 [Medline] [Order article via Infotrieve]
  28. Lee, S., Zambas, E. D., Marsh, W. L., and Redman, C. M.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 6353-6357 [Abstract]
  29. Sawamura, T., Kasuya, Y., Matsushita, Y., Suzuki, N., Shinmi, O., Kishi, N., Sugita, Y., Yanagisawa, M., Goto, K., Masaki, T., and Kimura, S.(1991) Biochem. Biophys. Res. Commun.174, 779-784 [Medline] [Order article via Infotrieve]
  30. Roques, B. P., Noble, F., Dauge, V., Fournie-Zaluski, M.-C., and Beaumont, A.(1993) Pharmacol. Rev.45, 87-146 [Medline] [Order article via Infotrieve]
  31. Wilhelm, S. M., Shao, Z.-H., Housley, T. J., Seperack, P. K., Baumann, A. P., Gunja-Smith, Z., and Woessner, J. F., Jr.(1993) J. Biol. Chem.268, 21906-21913 [Abstract/Free Full Text]
  32. Anderson, R. G. W., and Orci, L.(1988) J. Cell Biol.106, 539-543 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.