Cloning and expression of an evolutionary conserved single-domain angiotensin converting enzyme from Drosophila melanogaster.
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 (Km, 365 microM), removes Phe-Arg from the C terminus of bradykinin (Km, 22 microM), 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.
Cornell
M J
MJ
Department of Pure & Applied Biology, University of Leeds, United Kingdom.
Williams
T A
TA
Lamango
N S
NS
Coates
D
D
Corvol
P
P
Soubrier
F
F
Hoheisel
J
J
Lehrach
H
H
Isaac
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004331:Drosophila melanogaster / Q000201:enzymology*; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D007703:Peptidyl-Dipeptidase A