Mouse angiotensin-converting enzyme is a protein composed of two homologous domains

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that converts angiotensin I into the potent vasoconstrictor angiotensin II. We have used cDNA and genomic sequences to assemble a composite cDNA, ACE.315, encoding the entire amino acid sequence of mouse converting enzyme. ACE.315 contains 4838 base pairs and encodes a protein of 1278 amino acids (147.4 kDa) after removal of a 34-amino acid signal peptide. Within the protein, there are two large areas of homologous sequence, each containing a potential Zn-binding region and catalytic site. These homologous regions are approximately half the size of the whole ACE protein and suggest that the modern ACE gene is the duplicated product of a precursor gene. Mouse ACE is 83% homologous to human ACE in both nucleic acid and amino acid sequence, and like human ACE, contains a hydrophobic region in the carboxyl terminus that probably anchors the enzyme to the cell membrane (Soubrier, F., Alhenc-Gelas, F., Hubert, C., Allegrini, J., John, M., Tregear, G., and Corvol, P. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9386-9390). Northern analysis of mouse kidney, lung, and testis RNA demonstrates that the testicular isozyme of ACE is encoded by a single, smaller RNA (2500 bases) than the two message sizes found in kidney or lung (4900 and 4150 bases), and that this testicular RNA hybridizes to the 3' portion of ACE.315.     

The isolation of angiotensin-converting enzyme cDNA

Angiotensin-converting enzyme (ACE) is an Zn(II)-containing dipeptidyl carboxypeptidase that converts angiotensin I to the potent vasoconstrictor, angiotensin II. Using oligonucleotide probes based on the amino acid sequence of mouse kidney ACE, cDNA encoding this protein has been isolated. One cDNA, ACE.31, encodes the N-terminal 332 amino acids of mouse ACE, a portion of the protein containing a putative 34-amino acid leader sequence and the N terminus of the mature protein. Northern analyses with cloned ACE cDNA revealed that both mouse kidney and lung express two ACE mRNAs, one of 4900 and another of 4150 bases. Southern analysis suggests that cDNA ACE.31 is the product of a single gene, and thus these data add evidence to the hypothesis that the converting enzymes produced by epithelial and endothelial cells are identical.     

The unique N-terminal sequence of testis angiotensin-converting enzyme is heavily O-glycosylated and unessential for activity or stability.

The testis-specific isozyme of angiotensin-converting enzyme (ACE) is identical, from residue 68 to the C terminus, to the second half or C-terminal domain of somatic ACE. However, the first 67 residues, comprising the signal peptide and a Ser-/Thr-rich 36-residue sequence that constitutes the N terminus of mature testis ACE, are unique. We have expressed a mutant human testis ACE lacking this 36-residue N-terminal sequence and find that compared to the wild-type protein the mutant is 15 kDa smaller due to the loss of greater than 90% of all O-linked sugars, but that it retains full enzymatic activity and is stable in culture. Heavy O-glycosylation is a property of testis ACE that is not shared by the somatic enzyme and is attributable to this unique sequence.     

Angiotensin-converting enzyme: zinc- and inhibitor-binding stoichiometries of the somatic and testis isozymes.

The blood pressure regulating somatic isozyme of angiotensin-converting enzyme (ACE) consists of two homologous, tandem domains each containing a putative metal-binding motif (HEXXH), while the testis isozyme consists of just a single domain that is identical with the C-terminal half of somatic ACE. Previous metal analyses of somatic ACE have indicated a zinc stoichiometry of 1 mol of Zn2+/mol of ACE and inhibitor-binding studies have found 1 mol of inhibitor bound/mol of enzyme. These and other data have indicated that only one of the two domains of somatic ACE is catalytically active. We have repeated the metal and inhibitor-binding analyses of ACE from various sources and have determined protein concentration by quantitative amino acid analysis on the basis of accurate polypeptide molecular weights that are now available. We find that the somatic isozyme in fact contains 2 mol of Zn2+ and binds 2 mol of lisinopril (an ACE inhibitor) per mol of enzyme, whereas the testis isozyme contains 1 mol of Zn2+ and binds 1 mol of lisinopril. In the case of somatic ACE, the second equivalent of inhibitor binds to a second zinc-containing site as evidenced by the ability of a moderate excess of inhibitor to protect both zinc ions against dissociation. However, active site titration with lisinopril assayed by hydrolysis of furanacryloyl-Phe-Gly-Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either isozyme, indicating that the principal angiotensin-converting site likely resides in the C-terminal (testicular) domain of somatic ACE and that binding of inhibitor to this site is stronger than to the second site.(ABSTRACT TRUNCATED AT 250 WORDS)     

ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors

A human zinc metalloprotease (termed ACEH or ACE2) with considerable homology to angiotensin-converting enzyme (ACE) (EC 3.4.15.1) has been identified and subsequently cloned and functionally expressed. The translated protein contains an N-terminal signal sequence, a single catalytic domain with zinc-binding motif (HEMGH), a transmembrane region, and a small C-terminal cytosolic domain. Unlike somatic ACE, ACEH functions as a carboxypeptidase when acting on angiotensin I and angiotensin II or other peptide substrates. ACEH may function in conjunction with ACE and neprilysin in novel pathways of angiotensin metabolism of physiological significance. In contrast with ACE, ACEH does not hydrolyse bradykinin and is not inhibited by typical ACE inhibitors. ACEH is unique among mammalian carboxypeptidases in containing an HEXXH zinc motif but, in this respect, resembles a bacterial enzyme, Thermus aquaticus (Taq) carboxypeptidase (EC 3.4.17.19). Collectrin, a developmentally regulated renal protein, is homologous with the C-terminal region of ACEH but has no similarity with ACE and no catalytic domain. Thus, the ACEH protein may have evolved as a chimera of a single ACE-like domain and a collectrin domain. The collectrin domain may regulate tissue response to injury whereas the catalytic domain is involved in peptide processing events.     

Nucleotide sequence of a cDNA clone encoding mouse protamine 1

The nucleotide sequence of a 404-base cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine has been determined. This insert, isolated from a mouse testis cDNA library, encodes a polypeptide of 50 amino acids of which 28 are arginine, 9 are cysteine, and 3 are tyrosine. The insert contains the complete 3' noncoding region of 151 bases and most of the 5' noncoding region. The predicted amino acid sequence of mouse protamine 1 is about 80% homologous to boar protamine and 67% homologous to bull protamine and contains the central, highly basic domain of four arginine clusters found in the trout protamines. The identification of a cDNA clone for a mouse protamine will facilitate studies of the evolution, regulation, and protein-DNA interaction of this nuclear protein unique to haploid spermatogenic cells.