Increased shedding of angiotensin-converting enzyme by a mutation identified in the stalk region

Angiotensin-converting enzyme (ACE), an enzyme that plays a major role in vasoactive peptide metabolism, is a type 1 ectoprotein, which is released from the plasma membrane by a proteolytic cleavage occurring in the stalk sequence adjacent to the membrane anchor. In this study, we have discovered the molecular mechanism underlying the marked increase of plasma ACE levels observed in three unrelated individuals. We have identified a Pro(1199) -> Leu mutation in the juxtamembrane stalk region. In vitro analysis revealed that the shedding of [Leu(1199)]ACE was enhanced compared with wild-type ACE. The solubilization process of [Leu(1199)]ACE was stimulated by phorbol esters and inhibited by compound 3, an inhibitor of ACE-secretase. The results of Western blot analysis were consistent with a cleavage at the major described site (Arg(1203) down arrow Ser(1204)). Two-dimensional structural analysis of ACE showed that the mutated residue was critical for the positioning of a specific loop containing the cleavage site. We therefore propose that a local conformational modification caused by the Pro(1199) -> Leu mutation leads to more accessibility at the stalk region for ACE secretase and is responsible for the enhancement of the cleavage-secretion process. Our results show that different molecular mechanisms are responsible for the common genetic variation of plasma ACE and for its more rare familial elevation.