Cleavage Processing of Angiotensin-Converting Enzyme by a Membrane-Associated Metalloprotease

Abstract

Angiotensin-converting enzyme (ACE) is synthesized as a type 1 ectoprotein. It is released from the cell surface by a proteolytic cleavage-secretion process which is enhanced by treatment of the cells with phorbol esters. Here, we report the development of an in vitro cell-free assay system for the cleavage-secretion, its characterization, and the identification of a potent inhibitor of this process. Membranes prepared from ACE89 cells secreted the testicular isozyme of ACE (ACET) in a temperature- and time-dependent fashion. As expected, the in vitro secreted ACET lacked the membrane-anchoring carboxy-terminal tail of the cell-associated ACET. The in vitro secretase activity was resistant to high salt extraction and to inhibitors of serine, chymotrypsin, trypsin, cysteine, aspartate, and elastase type proteases. However, the activity was sensitive to metal ion chelators and to a synthetic hydroxamic acid derivative, compound 3, a known inhibitor of certain metalloproteases. Compound 3 very efficiently blocked both basal and phorbol ester-stimulated ACET secretion by ACE89 cells. The inhibition was rapid, dose-dependent, and reversible, and ACET synthesis, glycosylation, and transport were not affected. Cleavage-secretion of ACET in transiently transfected HeLa cells was also inhibited by compound 3. Finally, in vitro cleavage-secretion of the other isozyme of ACE, ACEP, by membranes isolated from rabbit lungs was strongly inhibited by compound 3. These results indicate that the cleavage-secretion of both isozymes of ACE is carried out by an integral membrane metalloprotease which is specifically inhibited by compound 3.

Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer

“…values in the low micromolar range. The TAPI-2 result is consistent with the recent finding that this agent can inhibit the release of the testicular isoform of rabbit ACE from a transfected mouse epithelial cell line [14]. The reduced activity shown by compound 1 in particular is especially interesting, because it differs from batimastat only in the absence of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety.…”

“…ACE secretase was shown to be a metalloprotease that co-localized with ACE in a number of tissues including pig kidney, lung and testis, and human lung and placenta, although the activity was absent from human kidney and human and pig small-intestinal brush-border membranes [11]. Within pig kidney the secretase was enriched in the highly purified brush-border membranes to a similar extent as ACE and alkaline phosphatase, and was shown to be an integral plasma membrane protein [11] as confirmed subsequently by others [12][13][14].…”

Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

“…The only unglycosylated site in tACE was identified at Asn 620 . It has been shown that ACE is released from the plasma membrane of the CHO cells into the medium by a cleavage between Arg 627 and Ser 628 (8,20). The exact nature of the protease(s) involved in the cleavage reaction is not clear, but it is possible that if the nearby Asn 620 was glycosylated, this proteolytic modification might be sterically hindered.…”

Further evidence for a common mechanism for shedding of cell surface proteins

“…A recent study demonstrated that TAPI blocks the release of L-selectin and TGFcc from transfected cells [14] and hydroxamates have also been shown to inhibit L-selectin shedding from primary cells [15][16][17]. Other proteins whose release is blocked by TAPI or related compounds include the thyrotropin receptor [18], Fas ligand [19], heparin-binding EGF-like growth factor [20] and angiotensin-converting enzyme [21]. No inhibitors other than hydroxamates have been found to prevent these shedding events under physiological conditions.…”