PROTEOLYTIC RELEASE OF HUMAN ANGIOTENSIN-CONVERTING ENZYME - LOCALIZATION OF THE CLEAVAGE SITE

Angiotensin-converting enzyme (EC 3.4.15.1, ACE) is a transmembrane pr otein with a short carboxyl-terminal cytoplasmic domain, a 17-amino ac id hydrophobic anchor domain, and a large N-terminal extracellular reg ion containing two catalytically homologous domains. An active soluble form of ACE circulates in human plasma and is produced in culture med ium of Chinese hamster ovary (CHO) cells transfected with the full-len gth human ACE cDNA. The mechanism of ACE release in CHO cells involves a post-translational proteolytic cleavage occurring in the carboxyl-t erminal region. The carboxyl terminus of the secreted recombinant ACE, AGQR, was established by carboxyl-terminal microsequencing and corres ponds to a cleavage site between Arg-1137 and Leu-1138. Two independen t studies confirmed this proposed cleavage site: amino acid analysis o f a carboxyl-terminal peptide derived from soluble ACE and immunochara cterization of membrane-bound and soluble ACE with antibodies raised a gainst three peptides located along the carboxyl-terminal ACE sequence . In order to assess the importance of. Arg-1137, this amino acid was mutated to a glutamine residue. This mutation did not prevent the secr etion of ACE, suggesting that the solubilizing enzyme can accommodate this change or can use an alternative cleavage site. Finally, the prod uction of soluble ACE in CHO cells appears to be proportional to the l evel of cellular ACE, implying that the solubilizing enzyme is not a l imiting factor. In addition, the carboxyl-terminal sequence of the hum an plasma ACE was identified as AGQR, thus supporting the fact that a similar mechanism could operate in human vascular cells.