Cloning and expression of an evolutionary conserved single-domain angiotensin converting enzyme from Drosophila melanogaster

Abstract

Mammalian somatic angiotensin converting enzyme (EC 3.4.15.1, ACE) consists of two highly homologous (N- and C-) domains encoded by a duplicated gene. We have identified an apparent single-domain (67 kDa) insect angiotensin converting enzyme (AnCE) in embryos of Drosophila melanogaster which converts angiotensin I to angiotensin II (K(m), 365 μM), removes Phe- Arg from the C terminus of bradykinin (K(m), 22 μM), and is inhibited by ACE inhibitors, captopril (IC50 = 1.1 x 10-9 M) and trandolaprilat (IC50 = 1.6 x 10-8 M). We also report the cloning and expression of a Drosophila AnCE cDNA which codes for a single-domain 615-amino acid protein with a predicted 17-amino acid signal peptide and regions with high levels of homology to both the N- and C-domains of mammalian somatic ACE, especially around the active site consensus sequence. Northern analysis identified a single 2.1-kilobase mRNA in Drosophila embryos, and Southern analysis of Drosophila genomic DNA indicates that the insect gene is not duplicated. When expressed in COS-7 cells, the AnCE protein is a secreted enzyme, which converts angiotensin I to angiotensin II and is inhibited by captopril (IC50 = 5.6 x 10-9 M) and trandolaprilat (IC50 = 2 x 10-8 M). The evolutionary significance of these results is discussed.