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A Human Homolog of Angiotensin-converting Enzyme

CLONING AND FUNCTIONAL EXPRESSION AS A CAPTOPRIL-INSENSITIVE CARBOXYPEPTIDASE*
Open AccessPublished:October 27, 2000DOI:https://doi.org/10.1074/jbc.M002615200
      A novel human zinc metalloprotease that has considerable homology to human angiotensin-converting enzyme (ACE) (40% identity and 61% similarity) has been identified. This metalloprotease (angiotensin-converting enzyme homolog (ACEH)) contains a single HEXXH zinc-binding domain and conserves other critical residues typical of the ACE family. The predicted protein sequence consists of 805 amino acids, including a potential 17-amino acid N-terminal signal peptide sequence and a putative C-terminal membrane anchor. Expression in Chinese hamster ovary cells of a soluble, truncated form of ACEH, lacking the transmembrane and cytosolic domains, produces a glycoprotein of 120 kDa, which is able to cleave angiotensin I and angiotensin II but not bradykinin or Hip-His-Leu. In the hydrolysis of the angiotensins, ACEH functions exclusively as a carboxypeptidase. ACEH activity is inhibited by EDTA but not by classical ACE inhibitors such as captopril, lisinopril, or enalaprilat. Identification of the genomic sequence of ACEH has shown that the ACEH gene contains 18 exons, of which several have considerable size similarity with the first 17 exons of human ACE. The gene maps to chromosomal location Xp22. Northern blotting analysis has shown that the ACEH mRNA transcript is ∼3.4 kilobase pairs and is most highly expressed in testis, kidney, and heart. This is the first report of a mammalian homolog of ACE and has implications for our understanding of cardiovascular and renal function.
      ACE
      angiotensin-converting enzyme
      ACEH
      angiotensin-converting enzyme homolog
      AnCE
      D. melanogaster angiotensin-converting enzyme
      ACEr
      D. melanogaster angiotensin-converting enzyme-related
      Hip
      hippuryl
      CHO
      Chinese hamster ovary
      HPLC
      high performance liquid chromatography
      RACE
      rapid amplification of cDNA ends
      PCR
      polymerase chain reaction
      PNGase F
      peptideN-glycosidase F
      PAGE
      polyacrylamide gel electrophoresis
      kb
      kilobase pair
      Angiotensin-converting enzyme (ACE,1 peptidyl-dipeptidase A, EC 3.4.15.1) is a well characterized zinc metalloprotease of the M2 family (
      • Corvol P.
      • Williams T.A.
      ). The predominant physiological function of ACE is in cardiovascular homeostasis through cleavage of the C-terminal dipeptide from angiotensin I to produce the potent vasoconstrictor, angiotensin II (
      • Skeggs L.T.
      • Kahn J.R.
      • Shumway N.P.
      ). ACE also inactivates the vasodilator, bradykinin, by the sequential cleavage of two C-terminal dipeptides (
      • Yang H.Y.T.
      • Erdös E.G.
      • Levine Y.
      ). ACE can also hydrolyze a wide range of other endogenous bioactive peptides (
      • Rieger K.-J.
      • Saez-Servent N.
      • Papet M.-P.
      • Wdzieczak-Bakala J.
      • Morgat J.-L.
      • Thierry J.
      • Voelter W.
      • Lenfant M.
      ).
      Two forms of mammalian ACE have been identified to date: the two-domain somatic ACE, containing two catalytic sites and a single domain germinal ACE (
      • Soubrier F.
      • Alhenc-Gelas F.
      • Hubert C.
      • Allegrini J.
      • John M.
      • Tregear G.
      • Corvol P.
      ,
      • Lattion A.-L.
      • Soubrier F.
      • Allegrini J.
      • Hubert C.
      • Corvol P.
      • Alhenc-Gelas F.
      ,
      • Ehlers M.R.W.
      • Fox E.A.
      • Strydom D.J.
      • Riordan J.F.
      ). Both enzymes are derived from the same gene through the use of alternative promoters, and it has been suggested that the ACE gene arose from the duplication of an ancestral gene coding for a single domain enzyme (
      • Hubert C.
      • Houot A.-M.
      • Corvol P.
      • Soubrier F.
      ). Somatic ACE exists as a type I integral membrane protein anchored to the plasma membrane through a transmembrane domain near the C terminus (
      • Hooper N.M.
      • Keen J.
      • Pappin D.J.C.
      • Turner A.J.
      ). However, it can also be found in plasma and other body fluids as a soluble enzyme lacking the transmembrane and cytosolic domains (
      • Das M.
      • Hartley J.L.
      • Soffers R.L.
      ,
      • El-Dorry H.A.
      • MacGregor J.S.
      • Soffer R.L.
      ,
      • Yasui T.
      • Alhenc-Gelas F.
      • Corvol P.
      • Menard J.
      ). This form is thought to arise predominantly by post-translational proteolytic cleavage at the cell surface through the action of ACE secretase (
      • Hooper N.M.
      • Keen J.
      • Pappin D.J.C.
      • Turner A.J.
      ,
      • Oppong S.Y.
      • Hooper N.M.
      ,
      • Woodman Z.L.
      • Oppong S.Y.
      • Cook S.
      • Hooper N.M.
      • Schwager S.L.H.
      • Brandt W.F.
      • Ehlers M.R.W.
      • Sturrock E.D.
      ).
      ACE-like enzymes have also been found in other non-mammalian species. In particular Musca domestica and Drosophila melanogaster have both been shown to contain single domain, ACE-like proteins (AnCE) (
      • Lamango N.
      • Isaac R.E.
      ,
      • Cornell M.J.
      • Williams T.A.
      • Lamango N.S.
      • Coates D.
      • Corvol P.
      • Soubrier F.
      • Hoheisel J.
      • Lehrach H.
      • Isaac R.E.
      ), and more recently, a second ACE-like protein, termed ACEr, has been identified in D. melanogaster(
      • Taylor C.A.M.
      • Coates D.
      • Shirras A.D.
      ). ACEr and AnCE appear to be alternatively expressed duringD. melanogaster pupal development (
      • Houard X.
      • Williams T.A.
      • Michaud A.
      • Dani P.
      • Isaac R.E.
      • Shirras A.D.
      • Coates D.
      • Corvol P.
      ), suggesting different roles for the two enzymes.
      In this study we have identified a novel, single domain, human zinc metalloprotease cDNA (ACEH) whose predicted amino acid sequence has significant similarity with mammalian ACE. Furthermore, the genomic structure of ACEH indicates a remarkable exon size similarity with the first 17 exons of ACE. Expression of a soluble, truncated form of ACEH, lacking the transmembrane and cytosolic domains, produced a 120-kDa glycosylated protein that hydrolyzed the C-terminal residue from angiotensins I and II. The transcript for this cDNA is highly expressed in heart, kidney, and testis, implying that the translated protein may play a role in the regulation of cardiovascular and renal function, as well as fertility.

      DISCUSSION

      Following the identification of a novel zinc metalloprotease from an EST data base, we have determined its cDNA sequence, expressed it as a soluble protein, and determined its activity toward potentially important physiological substrates. As a single domain enzyme, ACEH is similar to AnCE and ACEr, the insect members of the ACE family. When compared with the human ACE isoforms, ACEH shares considerable homology which is particularly marked around the HEXXH zinc-binding domain. This sequence (HEMGH) is identical in ACE and ACEH. A conserved glutamic acid residue, 24 amino acids downstream of the HEXXH motif in ACEH, aligns with the critical glutamate necessary for the catalytic activity of ACE (
      • Williams T.A.
      • Corvol P.
      • Soubrier F.
      ). This glutamate serves as the third zinc coordinating ligand. ACEH also contains 8 cysteine residues 6 of which are conserved in the N- and C-terminal domains of endothelial ACE and of testicular ACE. ACEH contains 7 potentialN-linked glycosylation sites (compared with 10 and 7 in the N- and C-terminal domains of endothelial ACE, respectively) and is therefore likely to be glycosylated. This is further reinforced by the molecular mass of truncated, expressed ACEH that migrates at ∼120 kDa compared with the deglycosylated polypeptide that migrates at 85 kDa.
      There is a putative transmembrane domain of 22 amino acids near the C terminus followed by a cluster of charged residues that are likely to constitute a stop-transfer sequence. In contrast, the transmembrane sequence of ACE is predicted to be only 17 amino acids, which is a minimal requirement for a membrane-spanning region. Together with the 17-amino acid signal sequence at the N terminus of ACEH, the enzyme has all the features of a type I integral membrane protein, like ACE. We have shown that a soluble form of ACEH, lacking the transmembrane and cytosolic domains, is secreted from CHO cells and that this form is catalytically active. Surprisingly, ACEH appears to be acting specifically as a carboxypeptidase, rather than as a peptidyl dipeptidase, as it is able to cleave exclusively the C-terminal residues from both angiotensin I and angiotensin II. Bradykinin, which has a C-terminal arginyl residue, is not hydrolyzed, suggesting a carboxypeptidase A-like specificity for ACEH. However, ACEH does not have a typical carboxypeptidase A-like zinc-binding motif (
      • Vendrall J.
      • Querol E.
      • Avilés F.X.
      ). Several ACE inhibitors (lisinopril, captopril, and enalaprilat) were not able to inhibit the cleavage of angiotensin I by ACEH, although the metal-chelating agent, EDTA, was an effective inhibitor, showing complete inhibition at 10 mm. This reinforces the proposition that ACEH is a metalloprotease, but with a distinct substrate and inhibitor specificity from ACE. It is perhaps not unexpected that the typical ACE inhibitors do not inhibit ACEH as they have been designed to compete with peptides that are hydrolyzed to release C-terminal dipeptides. Hence, positioning of the inhibitors in the active site should not be in the correct conformation to affect the cleavage of a single amino acid from the C terminus of the substrate.
      The high expression of ACEH mRNA in heart and kidney is of interest as these organs are important contributors to blood pressure homeostasis. The highest expression of ACEH mRNA, however, is in testis. Testicular ACE is known to play a key role in fertility (
      • Krege J.H.
      • John S.W.M.
      • Langenbach L.L.
      • Hodgin J.B.
      • Hagaman J.R.
      • Bachman E.S.
      • Jennette J.C.
      • O'Brien D.A.
      • Smithies O.
      ,
      • Esther Jr., C.R.
      • Howard T.E.
      • Marino E.M.
      • Goddard J.M.
      • Capecchi M.R.
      • Bernstein K.E.
      ), and ACEH may also therefore have reproductive functions. The tissue distribution of ACE mRNA is more widespread than ACEH, with both 3.5- and 4.2-kb species present in most of the tissues examined.
      The genomic sequence of ACEH holds many similarities to the structure of the ACE gene. The sizes of many of the exons are identical. There is, however, a discrepancy at exons 5 and 6 of ACEH, which together appear to correspond with exon 5 of ACE, suggesting a fusion of two exons. The HEXXH motif is therefore located in exon 9 of ACEH but exon 8 of the ACE gene.
      The ACEH gene is located on the X chromosome (Xp22) which is similar to the location (Xp22.1) of another membrane metalloproteinase, the product of the PEX gene associated with X-linked hypophosphatemic rickets (
      • Francis F.
      • Hennig S.
      • Korn B.
      • Reinhardt R.
      • de Jong P.
      • Poustka A.
      • Lehrach H.
      • Rowe P.S.N.
      • Goulding J.N.
      • Summerfield T.
      • Mountford R.
      • Read A.P.
      • Popowska E.
      • Pronicka E.
      • Davies K.E.
      • O'Riordan J.L.H.
      • Econs M.J.
      • Nesbitt T.
      • Drezner M.K.
      • Oudet C.
      • Pannetier S.
      • Hanauer A.
      • Strom T.M.
      • Meindl A.
      • Lorenz B.
      • Cagnoli M.
      • Mohnike K.L.
      • Murken J.
      • Meitinger T.
      ). The Pex protein is a member of the neprilysin (NEP) family (
      • Turner A.J.
      • Tanzawa K.
      ).
      Taken together, these findings indicate that ACEH is a metalloprotease that may have a significant role not only in cardiovascular homeostasis but also in fertility. However, before the physiological roles of ACEH can be elucidated, further enzyme characterization is needed to identify selective inhibitors and the key residues that distinguish its activity from that of ACE itself.

      Acknowledgements

      We thank Dr David Coates (School of Biology, University of Leeds) for helpful discussion and criticism of the manuscript and Dr. J. Keen (University of Leeds) for matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis.

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