Abstract
Mammalian angiotensin-converting enzyme (ACE) is one of several biologically important ectoproteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic cleavage. It has been suggested that a common proteolytic system is responsible for the cleavage of a diverse group of membrane ectoproteins, and tumor necrosis factor-alpha-converting enzyme (TACE), a recently purified disintegrin-metalloprotease, has been implicated in the proteolytic cleavage of several cell surface proteins. Mice devoid of TACE have been developed by gene targeting. Such mice could provide a useful system to determine if TACE is responsible for the cleavage of other ectoproteins. Cultured fibroblasts without TACE activity, when transfected with cDNA encoding for the testicular isozyme of ACE (ACET), synthesized and secreted ACET normally after a proteolytic cleavage near the C terminus. In addition, similar quantities of the soluble, C-terminally truncated somatic isozyme of ACE (ACEP) were present in the serum of wild-type and TACE-deficient mice. These results demonstrate that TACE is not essential in the generation of soluble ACE under physiological conditions. Finally, we also report solubilization of ACE-secretase, the enzyme that cleaves ACE, from mouse ACE89 cells and from rabbit lung. We demonstrate that soluble ACE-secretase from both sources failed to cleave its substrate in solution, suggesting a requirement for anchoring to the membrane.
Highlights
Many proteins exist in both membrane-bound and soluble forms as a result of post-translational proteolytic processing
Secretion can be enhanced by treatment of cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, indicating the existence of cellular mechanisms that regulate the conversion of cell-bound ACET to its soluble form
The secretases studied so far share certain general properties, such as stimulation by PMA and sensitivity to hydroxamic acid-based metalloprotease inhibitors. These common properties suggest that the shedding phenomena may be mediated by the same cellular components since mutant cell lines have been isolated that are defective in hydroxamic acid inhibitor-susceptible, PMA-stimulated cleavage and secretion of many proteins, including TGF-␣, TNF-␣, L-selectin, interleukin-6 receptor ␣-subunit, and -APP [14]
Summary
Materials—Lisinopril (N␣-[1-(S)-carboxy 3-phenylpropyl]-L-lysyl-Lproline) was provided by Merck Sharp and Dohme Research Laboratories (Rahway, NJ). Rabbit lung membranes were prepared as described previously [5], and the ACE-secretase activity was solubilized from these membranes by detergent extraction, as described above for ACE89 cell membranes. Assay for ACE-secretase Activity—In the deglycosylation-PAGE assay (Fig. 3A), detergent extracts of ACE89 cell membranes (0.25 ml) were incubated with or without 300 l of lisinopril-Sepharose [6] or concanavalin A (ConA)-agarose (Sigma, 200 l) in a final volume of 1.5 ml for 1 h at 37 °C. For the detergent-extraction assay (Table IV), membranes or Triton X-100 extract of the membranes prepared from rabbit lung were incubated with or without lisinopril-Sepharose, as described above, at 4 °C or 37 °C for 1 h. Uncleaved and cleaved ACEP proteins were physically separated by the temperature-induced phase-separation method utilizing Triton X-114, as described previously [5], and ACEP enzyme activity was measured in both the detergent and the aqueous phase. The corrected value for cleaved ACEP was calculated by subtracting this latter value from the total ACEP activity determined in the aqueous phase
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