Expression and characterization of recombinant human angiotensin I-converting enzyme. Evidence for a C-terminal transmembrane anchor and for a proteolytic processing of the secreted recombinant and plasma enzymes.
Chinese hamster ovary (CHO) cells have been transfected with either a full-length cDNA encoding human angiotensin I-converting enzyme (kininase II; EC 3.4.15.1) (ACE) or a mutated cDNA, in which the last C-terminal 47 amino acids, including the putative transmembrane domain, are not translated. Cell lines expressing high levels of the wild-type ACE or the mutant were established. The cells transfected with the wild-type cDNA (CHO-ACE) express a membrane-bound ectoenzyme with an intracellular C terminus, as shown by indirect immunofluorescence using an antiserum (28A7) raised against a synthetic peptide corresponding to the deduced C terminus of ACE. This enzyme is structurally, immunologically, and enzymatically identical to human kidney ACE. In addition, CHO-ACE cells also produce a secreted form of the enzyme. Neither this secreted form nor the enzyme purified from human plasma is recognized by the antiserum 28A7, indicating that they undergo a truncation in the C-terminal region. On the other hand, the transfected cells expressing the C-terminally truncated mutant (CHO-ACE delta COOH) do not retain ACE in the plasma membrane, but secrete it into the medium. These results indicate that ACE is anchored to the plasma membrane by the predicted C-terminal transmembrane domain, and the secreted form is derived from the membrane-bound form by a post-translational proteolytic cleavage of the C-terminal region.
Wei
L
L
U. 36 Institut National de la Santé et de la Recherche Médicale, Collège de France, Paris.
Alhenc-Gelas
F
F
Soubrier
F
F
Michaud
A
A
Corvol
P
P
Clauser
E
E
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D004247:DNA; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A