Cell surface localization of proteolysis of human endothelial angiotensin I-converting enzyme. Effect of the amino-terminal domain in the solubilization process.
Angiotensin-converting enzyme (ACE) belongs to the type I class of ectoproteins and is solubilized by Chinese hamster ovary cells transfected with the full-length human ACE cDNA. ACE release in Chinese hamster ovary cells involves a proteolytic cleavage occurring in the carboxyl-terminal region, between Arg-1137 and Leu-1138. The subcellular localization of ACE proteolysis was established by pulse-chase experiments, cell surface immunolabeling, and biotinylation of radiolabeled mature proteins. The proteolysis of ACE takes place primarily at the plasma membrane. The solubilization of ACE is less than 2% within 1 h, is increased 2.4-fold by phorbol esters, but is not influenced by ionophores. An ACE mutant lacking the transmembrane domain and the cytosolic part (ACE delta COOH), is secreted at a faster rate without a carboxyl-terminal cleavage, and phorbol esters or ionophores have no effect on its rate of production in the medium. Therefore, the proteolysis of ACE is dependent on the presence of the membrane anchor and suggests that the secretase(s) involved is also membrane-associated. An ACE mutant lacking the amino-terminal domain (ACECF) is secreted 10-fold faster compared with wild-type ACE. The solubilization of ACECF occurs at the plasma membrane and is stimulated 2.7-fold by phorbol esters, and the cleavage site is localized between Arg-1227 and Val-1228. The amino-terminal domain of ACE slows down the proteolysis and seems to act as a "conformational inhibitor" of the proteolytic process, possibly via interactions with the "stalk" of ACE and the secretase(s) itself.
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D018076:DNA, Complementary; D007703:Peptidyl-Dipeptidase A; D013755:Tetradecanoylphorbol Acetate