Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified se cretase. Although testis and somatic ACE both share the same stalk and memb rane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beld ent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962 -28969]. These differences constitute a useful model system of the regulati on and substrate preferences of the ACE secretase, and hence we investigate d this further. In transfected Chinese hamster ovary cells, human somatic A CE (hsACE) was indeed shed less efficiently than human testis ACE, and shed ding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, an ti-peptide antibodies raised to specific sequences on either side of the re ported cleavage site (Arg(1137)/Leu(1138)) clearly recognized soluble porci ne somatic ACE, indicating that cleavage was C-terminal to Arg(1137). Secon d, a competitive ELISA gave superimposable curves for porcine plasma ACE, s ecretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, sugge sting similar C-terminal sequences. Third, mass-spectral analyses of digest s of released soluble hsACE or of eACE enabled precise assignments of the C -termini, in each case to Arg(1203). These data indicated that soluble huma n and porcine somatic ACE, whether generated in vivo or in vitro, have C-te rmini consistent with cleavage at a single site, the Arg(1203)/Ser(1204) bo nd, identical with the Arg(627)/Ser(628) site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an altern ative stalk site, but instead supports the hypothesis that the testis ACE e ctodomain contains a motif that activates shedding, which is occluded by th e additional domain found in somatic ACE.