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

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Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn 631 to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE NQ ) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE NQ was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15°C revealed that ACE NQ was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn 635 -Ser 636 bond, three residues N-terminal to the normal secretase cleavage site at Arg 638 -Ser 639 . These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.

Section: Ace Nq Is Cleaved At a Different Bond In The Juxtamembranesupporting

confidence: 65%

Section: Ace Nq Is Secreted From the Imr-32mentioning

confidence: 95%

mentioning

confidence: 99%

See 1 more Smart Citation

A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase

Alfalah

Parkin

Jacob

et al. 2001

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“…Two antibodies, raised against peptides flanking the cleavage site determined for both human testicular and somatic ACE (26), were used to determine whether [Leu 1199 ]ACE was cleaved at the same location as the wild-type enzyme. Indeed, [Leu 1199 ]ACE is cleaved between amino acid 1162 and 1214, a result consistent with the cleavage site determined for the in vitro expressed wild-type somatic and testicular enzymes, and for the seminal plasma ACE (26).…”

Section: Discussionmentioning

confidence: 99%

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

Eyries

Michaud

Deinum

et al. 2001

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...

“…2A). ACE is known to be secreted both in vivo and in vitro from the cell surface at low levels in an ␣-secretase-dependent manner (38). The concentration of conditioned medium from HEK293 and CHO cells transiently transfected with ACE cDNA was found to contain low levels of ACE protein, whereas control cells transfected with the empty vector had no corresponding signal (Fig.…”

Section: Expression and Characterization Of Transfected Human Ace-tomentioning

confidence: 99%

Amyloid β-Protein Is Degraded by Cellular Angiotensin-converting Enzyme (ACE) and Elevated by an ACE Inhibitor

Hemming

Selkoe

2005

295

211

Human genetic data have associated angiotensin-converting enzyme (ACE) with Alzheimer disease (AD), and purified ACE has been reported to cleave synthetic amyloid ␤-protein (A␤) in vitro. Whether deficiency in ACE activity, arising from genetic alteration or pharmacological inhibition, can decrease A␤ degradation and allow A␤ accumulation in intact cells is unknown. We cloned ACE from human neuroblastoma cells and showed that it had posttranslational processing and enzymatic activity typical of the endogenous protease. Cellular expression of ACE promoted degradation of naturally secreted A␤40 and A␤42, leading to significant clearance of both species. Using site-directed mutagenesis, we determined that both active sites within ACE contribute to A␤ clearance, and an ACE construct bearing mutations in each catalytic domain had no effect on A␤ levels. Pharmacological inhibition of ACE with a widely prescribed drug, captopril, promoted the accumulation of cell-derived A␤ in the media of ␤-amyloid precursor-protein expressing cells. Together, these results show that ACE can lower the levels of secreted A␤ in living cells and that this effect is blocked by inhibiting the protease's activity with an ACE inhibitor. This work, combined with the genetic studies, supports the hypothesis that ACE may modulate the susceptibility to and progression of AD via degradation of A␤. Our data encourage further analyses of the ACE gene for disease association and raise the question of whether currently prescribed ACE inhibitors could elevate cerebral A␤ levels in humans. An early and pathogenically important feature of Alzheimer disease (AD)2 is the progressive accumulation and deposition of the amyloid ␤-protein (A␤) in brain regions serving memory and cognition. Biochemical, cell biological, animal modeling, genetic, and emerging clinical data all suggest that A␤ is an upstream initiator of the disease process and its associated neuropathology (1-4). Although no proven diseasemodifying treatments are currently available, recent efforts to treat AD have focused on both decreasing the production of A␤ and enhancing its clearance from the brain. One little studied approach to A␤ clearance is augmenting the degradation of the peptide by various proteases expressed in the brain. Thus far, the metalloproteases neprilysin (NEP) (5), insulin-degrading enzyme (IDE) (6), and the endothelin-converting enzymes 1 and 2 (7) have each been implicated as A␤-degrading proteases in the mammalian brain. The serine protease plasmin has been implicated in A␤ degradation in vitro (8), although genetic plasmin deficiency did not promote accumulation of murine A␤ in vivo (9). Supporting a role for therapeutic regulation of A␤-degrading proteases, the overexpression of IDE or NEP in a murine model of AD decreased cerebral A␤ levels and produced significant attenuation of A␤-associated neuropathology (10).Somatic angiotensin-converting enzyme (ACE) is a zinc metalloprotease containing two homologous regions, termed the N-and C-domains, each of which is prot...