Metalloprotease-mediated cleavage secretion of pulmonary ACE by vascular endothelial and kidney epithelial cells

Abstract

The pulmonary isozyme of angiotensin-converting enzyme (ACEP) is present in the body both as a cell-associated protein in endothelial, epithelial, and monocytic cells and as a soluble protein in various body fluids including serum. The mechanism by which soluble ACEP is produced in vivo is unknown. Using in vitro transfected cell culture systems, we previously demonstrated that the rabbit testicular isozyme of ACE (ACET), which shares extensive homology with ACEP, is first synthesized as a plasma membrane-anchored ectoprotein and then secreted to the culture medium by cleavage removal of its COOH-terminal membrane-anchored tail. Here, using in vitro cultures of arterial endothelial cells and acutely isolated renal epithelial cells, we demonstrate that ACEP is also cleavage secreted from their natural producer cells. Biochemical and immunological characterization of the in vitro secreted ACEP protein revealed that it is missing the COOH-terminal membrane-anchored region of the cell-associated ACEP. Similar analysis of ACEP proteins present in rabbit serum, lung, and kidney established that ACEP secretion in vivo is also caused by the cleavage removal of the COOH-terminal region of the cell-associated protein. To characterize the proteolytic enzyme responsible for ACEP secretion, we employed rabbit renal proximal tubular epithelial cells and demonstrated significant inhibition of secretion by compound 3, a hydroxamic acid-based inhibitor of specific metalloproteases. In contrast, the inhibitors of chymotrypsin, trypsin, serine, aspartate, and cysteine proteases were ineffective. These results indicate that soluble ACEP production by vascular endothelial and renal epithelial cells, both in vitro and in vivo, is achieved by cleavage removal of its membrane-anchoring COOH-terminal tail by a metalloprotease.

Calmodulin Binds to the Cytoplasmic Domain of Angiotensin-converting Enzyme and Regulates Its Phosphorylation and Cleavage Secretion

“…If that were the case, the amino-terminal residues of the peptides would be different. However, when the amino-terminal sequences were determined by Edman degradation, the same sequences were obtained for peptides purified from untreated or PMA-treated cells: the sequence of 13 and 15 amino-terminal residues of the purified carboxyl-terminal peptides obtained from untreated and PMA-treated cells matched perfectly with that of ACE residues 664 -676 and 664 -678, respectively (22). Although, both purified peptide samples, as expected, contained both peptides of the doublet, no other major sequence was detected.…”

“…Rabbit Lung Extract-Extracts of rabbit lungs were obtained using previously described methods (22). 35 S Labeling of Cells, Immunoprecipitation, and Quantification of Cleavage Secretion-Confluent ACE89 or transiently transfected HeLa cells were first incubated in methionine/cysteine-free medium for 1 h, and then labeled with [ 35 S]methionine/cysteine (PerkinElmer Life Sciences) 0.5 h; the label was chased for the time periods as indicated in figures.…”

“…Both the isoforms of ACE are expressed on the cell surface as a type I ectoprotein, with a short cytoplasmic domain, a transmembrane domain, and a long extracellular domain containing the active site(s), and are cleaved on the membrane proximal region to release the enzymatically active extracellular domain in the body fluid, including serum (21)(22)(23). Our previous studies on rabbit gACE demonstrated that ACE shedding is a regulated event, which can be stimulated by phorbol esters (PMA) and inhibited by specific metalloprotease inhibitor (Compound 3/TAPI II) (22,24), the distal ectodomain of ACE plays a vital role in ACE shedding (25), and the ACE molecule is cleaved at 663 RS 664 on the membrane-proximal region to generate the soluble form (22). The protease responsible for ACE cleavage is the putative ACE secretase, a metalloprotease, which is different from TACE (23,26) and still remains unidentified.…”

A ligand-based approach to investigate the expression and function of angiotensin converting enzyme in intact human umbilical vein endothelial cells

“…Endothelial ACE may be regulated by the rate of its synthesis and the cleavage and/or shedding of surface ACE [5,17,27]. In human umbilical vein endothelial cells (HUVECs), the transcription of the ACE gene mRNA and ACE activity are known to be stimulated by protein kinase (PK) A and PKC [30,35,36] and slightly via NO-controlled PKG [28], and repressed by the inflammatory cytokines IL-- [25,29].…”

Altered cardiac bradykinin metabolism in experimental diabetes caused by the variations of angiotensin-converting enzyme and other peptidases

“…decrease the endothelial cells density where ACE resides. Secondly, ectopeptidases like ACE and NEP could theoretically be regulated by cleavage and shedding from the cell surface (Ramchandran et al, 1996), and there is evidence for an increased plasma ACE concentration in some diabetic patient subgroups and some animal diabetes model (Schernthaner et al, 1984;van Dijk et al, 2001).…”