Angiotensin-converting enzyme (ACE) is a widely distributed metalloprotease which plays a key role in the control of blood pressure and fluid and electrolyte homeostasis. Although ACE exists primarily as a membrane - bound enzyme, a soluble form is present under normal conditions in blood plasma and other body fluids. To delineate the structural relationship between the cell-bound and the soluble forms of the protein and to determine the modes of their biosynthesis, we have used various expression systems of cloned ACE cDNA. Our studies indicate that ACE is anchored in the cellular plasma membrane through a hydrophobic domain near its C- terminal and its extracellular secretion is achieved by a proteolytic cleavage of this membrane anchoring domain. The responsible protease is a cellular enzyme that cleaves ACE at or near the plasma membrane in a regulated fashion. The exact cleavage site has also been determined. We hypothesize that recognition of specific structural features of ACE by a cell-membrane associated secretase activity is responsible for the extracellular release of this protein. To test this hypothesis, we will determine the role of its intracellular, transmembrane and extracellular domains in the process of cleavage-secretion, by deleting, mutating or exchanging each domain, with the corresponding domain of an uncleavable transmembrane protein. We will define the role of specific amino acid residues at and around the cleavage site by introducing suitable amino acid substitution or deletions in the region. We will determine the role of glycosylation in this process, design and express a cell-bound non- secretable form of ACE and determine its enzymatic properties. We also propose to purify and characterize the ACE-secretase activity which is responsible for this cleavage-secretion process. For this purpose we will develop a cell-free assay system for measuring the ACE-secretase activity, process. For this purpose we will develop a cell-free assay system for measuring the ACE-secretase activity, characterize its enzymatic properties, purify it to homogeneity and develop antibody to it, so t hat we can isolate its cDNA clone in the future. Thus, the proposed project will provide a better understanding of how cell-bound ACE is converted to athe soluble form, how the relative proportion of the two forms is physiologically regulated and whether this conversion has a cell- specificity thereby causing localized ACE action in some tissue. Above and beyond the ACE system, our proposed study, will be the prototype for an important and general mode of biological regulation and will identify the first member of a new class of membrane-bound proteases whose primary role is to selectively cleave and release many biologically important proteins.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL054297-02
Application #
2445299
Study Section
Cardiovascular and Renal Study Section (CVB)
Project Start
1996-08-01
Project End
2000-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Chattopadhyay, Saurabh; Karan, Goutam; Sen, Indira et al. (2008) A small region in the angiotensin-converting enzyme distal ectodomain is required for cleavage-secretion of the protein at the plasma membrane. Biochemistry 47:8335-41
Chattopadhyay, Saurabh; Santhamma, Kizhakkekara R; Sengupta, Saubhik et al. (2005) Calmodulin binds to the cytoplasmic domain of angiotensin-converting enzyme and regulates its phosphorylation and cleavage secretion. J Biol Chem 280:33847-55
Santhamma, Kizhakkekara R; Sadhukhan, Ramkrishna; Kinter, Michael et al. (2004) Role of tyrosine phosphorylation in the regulation of cleavage secretion of angiotensin-converting enzyme. J Biol Chem 279:40227-36
Santhamma, K R; Sen, I (2000) Specific cellular proteins associate with angiotensin-converting enzyme and regulate its intracellular transport and cleavage-secretion. J Biol Chem 275:23253-8
Sadhukhan, R; Santhamma, K R; Reddy, P et al. (1999) Unaltered cleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-alpha-converting enzyme-deficient mice. J Biol Chem 274:10511-6
Sadhukhan, R; Sen, G C; Ramchandran, R et al. (1998) The distal ectodomain of angiotensin-converting enzyme regulates its cleavage-secretion from the cell surface. Proc Natl Acad Sci U S A 95:138-43