Identification and characterisation of the angiotensin converting enzyme-3 (ACE3) gene: a novel mammalian homologue of ACE
© Rella et al; licensee BioMed Central Ltd. 2007
Received: 19 March 2007
Accepted: 27 June 2007
Published: 27 June 2007
Mammalian angiotensin converting enzyme (ACE) plays a key role in blood pressure regulation. Although multiple ACE-like proteins exist in non-mammalian organisms, to date only one other ACE homologue, ACE2, has been identified in mammals.
Here we report the identification and characterisation of the gene encoding a third homologue of ACE, termed ACE3, in several mammalian genomes. The ACE3 gene is located on the same chromosome downstream of the ACE gene. Multiple sequence alignment and molecular modelling have been employed to characterise the predicted ACE3 protein. In mouse, rat, cow and dog, the predicted protein has mutations in some of the critical residues involved in catalysis, including the catalytic Glu in the HEXXH zinc binding motif which is Gln, and ESTs or reverse-transcription PCR indicate that the gene is expressed. In humans, the predicted ACE3 protein has an intact HEXXH motif, but there are other deletions and insertions in the gene and no ESTs have been identified.
In the genomes of several mammalian species there is a gene that encodes a novel, single domain ACE-like protein, ACE3. In mouse, rat, cow and dog ACE3, the catalytic Glu is replaced by Gln in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, no evidence was found that the ACE3 gene is expressed and the presence of deletions and insertions in the sequence indicate that ACE3 is a pseudogene.
Angiotensin-converting enzyme (ACE; EC 126.96.36.199) is a well-characterised zinc metallopeptidase that plays a key role in the renin-angiotensin system . Through cleavage of a C-terminal dipeptide from angiotensin I to produce the potent vasoconstrictor angiotensin II, and through inactivation of the vasodilator bradykinin, ACE regulates blood pressure and cardiovascular homeostasis. Inhibitors of ACE, such as captopril and lisinopril, are front line therapeutics in a range of cardiovascular disorders, including hypertension, congestive heart failure, left ventricular hypertrophy and myocardial infarction .
ACE exists in two forms as a result of alternative use of promoters within the same gene on human chromosome 17 and mouse chromosome 11: the larger, two domain somatic ACE (1306 amino acids in humans) and the smaller, single domain germinal ACE (701 amino acids in humans) . The latter is identical to the C-terminal domain of somatic ACE, except for a unique region of 67 amino acids at its N-terminus. The two domains of somatic ACE are catalytically active and each contains the prototypical zinc binding motif HEXXH. In this motif, the two His residues are two of the zinc ligands, the third being a Glu on the C-terminal side of this motif . The Glu in the HEXXH motif is critically involved in catalysis, binding the activated water molecule which initiates a nucleophilic attack on the susceptible peptide bond in the substrate. Recently, the separate three-dimensional structures of the C-domain and of the N-domain of ACE in complex with the inhibitor lisinopril have been reported [5, 6]. Somatic and germinal ACE are synthesised with an N-terminal signal sequence that is cleaved off in the lumen of the endoplasmic reticulum and exist as type I integral membrane proteins anchored to the plasma membrane through a hydrophobic transmembrane domain at the C-terminus.
In 2000, we and another group independently reported the identification using genomics-based strategies of the first human homologue of ACE, termed angiotensin-converting enzyme-2 (ACE2) [7, 8]. Like ACE, ACE2 is a type 1 integral membrane protein, however, ACE2 contains only a single active site domain and consists of 805 amino acids. The gene encoding ACE2 is located on the X chromosome. ACE2 acts as a carboxypeptidase removing single amino acids from the C-terminus of its substrates, whereas ACE acts predominantly as a peptidyl dipeptidase removing C-terminal dipeptides . Studies from knockout mice indicate that ACE2 is an essential regulator of heart function  and is involved in the tissue response to injury . ACE2 is also the receptor for the severe acute respiratory syndrome (SARS) coronavirus . Homology modelling of the active site of ACE2 on the crystal structure of the C-domain of ACE  and the subsequent elucidation of the three-dimensional structure of the extracellular domain of ACE2  revealed that the catalytic mechanism of ACE2 closely resembles that of ACE. However, the substrate-binding pockets differ significantly explaining the differences in substrate specificity between the two enzymes and the failure of ACE inhibitors to bind to and inhibit ACE2 [7, 14].
ACE-like proteins also occur in non-mammalian species [15, 16]. Drosophila melanogaster has two functionally active, single domain, soluble ACE-like proteins (termed Ance and Acer) that share 36% amino acid sequence identity with human ACE . There are four other ACE-like genes in D. melanogaster that encode proteins which are predicted to be catalytically inactive as they lack critical residues involved in zinc binding or catalysis . In the mosquito Anopheles gambiae there are 9 genes which code for proteins with similarity to mammalian ACE , while in Caenorhabditis elegans there is a single ACE-like gene which also encodes a protein that is predicted to be catalytically inactive . Orthologues of ACE2 have been described recently in a range of non-mammalian vertebrates .
In this study we have identified and characterised a gene in the genomes of several mammalian species that encodes a novel, single domain ACE-like protein, that we have termed ACE3. In mouse, rat, cow and dog ACE3, the catalytic Glu is replaced by Gln (HQXXH) in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, we could find no evidence that the ACE3 gene is expressed and the presence of deletions and insertions in the sequence indicate that in humans ACE3 is a pseudogene.
Predicted ACE3 sequences in mouse, rat, dog and cow genomes
Blast searches using human somatic ACE against the mouse genome and gene prediction programmes on the NCBI and Ensembl databases identified a potential homologous region on chromosome 11 (region E1) downstream of the ACE gene.
Percentage identity of the murine ACE family.
Genomic sequence analysis of the murine ACE3 gene
Exon/intron boundary prediction for mouse ACE3.
Exon size (bp)
5' Splice donor
Intron size (bp)
3' Splice acceptor
Expression of ACE3 in murine tissues
Modelling of the substrate binding site of murine ACE3
Genomic sequence analysis of the human ACE3 gene
The restored human ACE3 protein sequence (adjusted for several base deletions/insertions causing frame shifts and premature stop codons, see Fig. 1) shows strong conservation with ACE and other ACE3 sequences. It contains the typical HEMGH zinc binding motif present in other members of the ACE family and is 54% identical to the C-domain of human ACE and 71% identical to the mouse ACE3 catalytic domain, respectively. The ancestral ACE3 gene is predicted to contain at least 14 exons, interspersed with 13 introns and spans 13 kb (Fig. 6). All of the predicted intron-exon junction sequences follow the GT/GA rule (data not shown). Hydropathy analysis and transmembrane prediction revealed two hydrophobic regions at the C-terminus, in agreement with ACE3 from other species, and an N-terminal signal peptide.
The human ACE3 gene sequence, however, has several frame shifts and premature stop codons (see Fig. 1). For example, there is a one base deletion directly after the sequence encoding the HEMGH zinc binding motif that causes a frameshift and a premature stop codon. To confirm this deletion, genomic DNA was isolated from 3 different human cell lines (Eahy926, Hek293, SH-SY5Y) and PCR performed using primers flanking this region. In all three cell lines sequencing of the PCR product confirmed that there was a one base deletion directly after the codon for the second His of the zinc binding motif (data not shown).
RT-PCR was used in an attempt to identify potential mRNA transcripts of human ACE3. However, RT-PCR using various primer sets on an RNA panel that included total RNA from human brain, heart, kidney, liver, lung, testis, colon, small intestine, placenta and skeletal muscle failed to identify a transcript. The specificity of the primers was checked by using them to PCR genomic DNA extracted from Hek293 cells from which they amplified a band of the predicted size that corresponded to the sequence of ACE3 (data not shown). To date, blast searches of the EST databases has not identified any ESTs for human ACE3. Thus, point mutations that give rise to premature stop codons and the failure to identify RNA transcripts or ESTs all suggest that human ACE3 is likely a pseudogene.
Comparison of the ACE3 sequences.
Length (amino acids)
Within another zinc metalloprotease family, the ADAM (a d isintegrin a nd m etalloprotease) family, several members have subtle or more complete mutation of the HEXXH motif . For example, in ADAM4 the Glu is mutated to Ala, while in ADAM29 it is His. Interestingly many of these catalytically inactive members of the ADAM family are present exclusively in the testis, where some have critical roles in fertilisation . It remains to be determined whether ACE3 has a role to play in fertilisation, although the recent report of its presence in membrane vesicles released by the acrosome reaction  is suggestive of such a role. There is a further parallel between the ADAM family members and ACE3, in that a large number of the ADAMs present in the mouse genome are pseudogenes in humans . In the present study we could find no evidence for human ACE3 to be expressed and the presence in the gene of multiple base insertions and deletions would, if expressed, generate a severely truncated protein lacking residues that are critical for zinc binding and/or catalysis in ACE and ACE2 [5, 12, 13]. This, and the localisation of ACE3 within the ACE gene, point to ACE3 being a pseudogene in humans.
Traditional, non-processed or duplicated pseudogenes characteristically contain introns as they are duplications of the genomic DNA, whereas processed or retrotransposed pseudogenes are duplicated from RNA and lack introns . In addition, non-processed pseudogenes are usually adjacent to their original functional copies. As human ACE3 contains introns and is localised to the same region of the genome as ACE, it appears to be a non-processed pseudogene. Such non-processed pseudogenes are often silenced by point mutations, insertions or deletions, as seen in human ACE3. This 'pseudogenization' of genes in humans, as seen here with ACE3, results in the human degradome containing only 561 functional genes, while the mouse degradome is larger with 641 genes . Interestingly, many of these additional mouse genes are involved in fertilisation and immunity.
While the ACE3 gene being located on the same chromosome just downstream of the ACE gene in all five genomes analysed may just reflect an evolutionary relationship, it is possible that this location alongside the ACE gene may reflect a potential functional role at the genomic level, as pseudogenes can regulate the expression of a related gene. For example, the makorin1 protein is evolutionarily conserved from nematodes to mammals and encodes an RNA binding protein . Normally makorin1 is expressed throughout the animal but the disruption of the makorin1 pseudogene markedly reduced the expression of the makorin1 gene. This implies that for normal expression of makorin1, the presence of the RNA for the makorin1 pseudogene is required. However, it should be noted that a subsequent report failed to replicate this finding  and the technical problems associated with such studies are discussed more fully in . Thus it is possible, but remains to be determined, that the ACE3 gene may regulate the expression of the ACE gene.
Multiple ACE-like genes have been identified in a range of non-mammalian species and here we report the identification and characterisation of a third ACE-like gene in the genomes of several mammalian species. This gene encodes a novel, single domain ACE-like protein, ACE3. In the mouse, rat, cow and dog, where there is evidence that ACE3 is expressed, the catalytic Glu is replaced by Gln in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, although the predicted ACE3 protein would contain an intact HEXXH zinc binding motif, no evidence was found for the expression of the gene. This lack of expression, along with the presence of deletions and insertions in the sequence, indicate that in humans ACE3 is a pseudogene.
Blast searches for potential ACE3 sequences using human somatic ACE were performed against the mouse genome and gene prediction programmes on the NCBI  and Ensembl  databases. According to NCBI annotations, the alternate Celera genome assembly is composed of DNA from five different mouse strains: A/J, DBA/2J, 129X1/SvJ, 129S1/SvImJ and C57BL/6J, whereas the reference assembly is based on C57BL/6J. Sequences used for bioinformatics sequence analysis and homology modelling were obtained from the NCBI RefSeq database. Sequence analysis was performed using the Emboss software suite , Clustal W  and 3DCoffee . Hydropathy analysis was performed as described . Transmembrane and signal peptide regions were predicted using TMpred  and SignalP , respectively. Homology modelling of mouse ACE3 was undertaken with the SWISS-MODEL software  based on its alignment to the human testicular ACE template (PDB code 1o86).
Human and murine (from Swiss Webster mice) total RNA was from Ambion (Europe) Ltd. (Huntingdon, UK). Genomic DNA was isolated from Eahy926, Hek293 and SH-SY5Y cells using TRIzol (GE Biosciences). Reverse transcription-PCR (RT-PCR) was performed with a one step Titanium RT-PCR kit (BD Biosciences, Oxford, UK) with 1 μg of RNA and the appropriate forward and reverse primers at 50 pmol/μl. For mouse ACE3 the primers were: forward 5'-GGGCGGGAAGTGGAGTGCCACA-3'; reverse 5'-GTTGACCTCCTCCTCTGAATCCTGG-3'. For human ACE3 the primers were: forward 5'-TCTGCCTGGAACTTCCCAGGACG-3'; reverse 5'-CCCATTTCGTGGAAAGATGGAGAGCG-3'. For mouse actin the primers were: forward 5'-GTGGGCCGCTCTAGGCACCAA-3'; reverse 5'-CTCTTTGATGTCACGCACGATTTC-3'. After incubation at 50°C for 1 h and initial denaturation for 5 min at 94°C, 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 68°C for 1 min, followed by a final extension at 68°C for 2 min was performed.
angiotensin converting enzyme
a disintegrin and metalloprotease
expressed sequence tag
We thank the Medical Research Council of Great Britain and Pfizer (Sandwich, UK) for financial support of this work. The help of Stephen Ballard (Pfizer, UK) in the initial stages of this project is gratefully acknowledged. M. R. thanks the University of Leeds for a Research Scholarship.
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