Proteolytic release of human angiotensin-converting enzyme. Localization of the cleavage site.
Angiotensin-converting enzyme (EC 3.4.15.1, ACE) is a transmembrane protein with a short carboxyl-terminal cytoplasmic domain, a 17-amino acid hydrophobic anchor domain, and a large N-terminal extracellular region containing two catalytically homologous domains. An active soluble form of ACE circulates in human plasma and is produced in culture medium of Chinese hamster ovary (CHO) cells transfected with the full-length human ACE cDNA. The mechanism of ACE release in CHO cells involves a post-translational proteolytic cleavage occurring in the carboxyl-terminal region. The carboxyl terminus of the secreted recombinant ACE, AGQR, was established by carboxyl-terminal microsequencing and corresponds to a cleavage site between Arg-1137 and Leu-1138. Two independent studies confirmed this proposed cleavage site: amino acid analysis of a carboxyl-terminal peptide derived from soluble ACE and immunocharacterization of membrane-bound and soluble ACE with antibodies raised against 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 secretion of ACE, suggesting that the solubilizing enzyme can accommodate this change or can use an alternative cleavage site. Finally, the production of soluble ACE in CHO cells appears to be proportional to the level of cellular ACE, implying that the solubilizing enzyme is not a limiting factor. In addition, the carboxyl-terminal sequence of the human plasma ACE was identified as AGQR, thus supporting the fact that a similar mechanism could operate in human vascular cells.
Beldent
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Institut National de la Santé et de la Recherche Médicale, Unit 36, Collège de France, Paris.
Michaud
A
A
Wei
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L
Chauvet
M T
MT
Corvol
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P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis
D011994:Recombinant Proteins; D007703:Peptidyl-Dipeptidase A