Increased Shedding of Angiotensin-converting Enzyme by a Mutation Identified in the Stalk Region

Abstract

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 3 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 2Ser 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 3 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.Angiotensin I-converting enzyme (DCP1, EC 3.4.15.1, ACE) 1 is a zinc metallopeptidase that plays an important role in blood pressure regulation by cleaving the inactive decapeptide angiotensin I to angiotensin II, a potent vasopressor octapeptide. It also inactivates bradykinin, a potent vasodilator peptide (1). There are two ACE isoforms transcribed from a single ACE gene by two alternate promoters (2). Somatic ACE (170 kDa) is synthesized by vascular endothelial cells as well as several types of epithelial cells, whereas testis ACE (110 kDa) is expressed exclusively by male germinal cells (3, 4).Somatic ACE consists of two homologous catalytic domains, a juxtamembrane stalk region, a hydrophobic transmembrane domain of 17 amino acids, and a 30-residue C-terminal cytosolic domain (5). Thus ACE is primarily an integral membrane protein anchored to the plasma membrane by its C-terminal segment. But the membrane-bound enzyme can be fully solubilized in vitro by detergents or limited proteolytic cleavage (6). In vivo, a soluble form of the enzyme exists in plasma and other body fluids (7). Plasma ACE is known to be strongly modulated by a common polymorphism in linkage disequilibrium with an insertion/deletion polymorphism (8). Although this quantitative trait locus (QTL) was localized on the ACE gene itself (9), the functional variant has not yet been identified.Human plasma ACE is derived from endothelial cells by post-translational cleavage. Several membrane-anchored proteins are solubilized by limited proteolysis with release of their extracellular domains. This common phenomenon, also referred to as "shedding" displays typica...

Association between plasma activities of semicarbazide-sensitive amine oxidase and angiotensin-converting enzyme in patients with type 1 diabetes mellitus

“…The relation between soluble and membrane-bound human SSAO is not firmly established, but experiments with transgenic mice and rats strongly suggest that the major source of soluble SSAO is membrane-bound SSAO from endothelial cells and adipocytes [8,9]. Thus, it seems likely that the soluble form is formed from membrane-bound SSAO by shedding, as is the case for ACE [7,28]. The correlation between plasma SSAO and serum ACE activities might therefore be explained by a common sheddase or secretase.…”

High-resolution genetic mapping of the ACE-linked QTL influencing circulating ACE activity

“…Secondly, a more rare form of familial elevation of plasma ACE has been shown to be due to a mutation changing a proline to a leucine in the C terminal part of ACE. 25 This mutation is responsible for an increased shedding of ACE due to an increased rate of proteolytic cleavage of the membrane bound enzyme by the still unidentified ACE secretase.…”

Influenza vaccination in patients with pulmonary sarcoidosis: efficacy and safety

“…Greater immune activity at the inflammation sites (i.e., granulomatous lesions) and higher U Ca (as a result of higher cellular immune system activation at the inflammation sites) can be associated with greater antibody titer, as observed in our study. The negative association of ACE level with antibody response could be questioned because serum ACE level is under the influence of several factors 26 , 27 , 28 …”