Molecular cloning and expression in COS-1 cells of pig kidney aminopeptidase P.
Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9), a key enzyme in the metabolism of the vasodilator bradykinin, has been cloned from a pig kidney cortex cDNA library following the use of the PCR to identify sub-libraries enriched in AP-P clones. The complete primary sequence of the enzyme has been deduced from a full-length cDNA clone. This predicts a protein of 673 amino acids with a cleavable N-terminal signal sequence and six potential N-linked glycosylation sites. A stretch of mainly hydrophobic amino acids at the C-terminus is predicted to co-ordinate the attachment of a glycosyl-phosphatidylinositol (GPI) membrane anchor. Although AP-P is a zinc metallopeptidase, the predicted primary sequence does not contain any recognizable zinc-binding motif. Transient expression of AP-P cDNA in COS-1 cells resulted in enzymic activity characteristic of AP-P, namely apstatin- and EDTA-sensitive hydrolysis of bradykinin and Gly-Pro-Hyp. The expressed protein was recognized as a polypeptide of M(r)91,000 under reducing conditions, following immunoblotting of COS-1 membranes with a polyclonal antibody raised against purified pig kidney AP-P. The presence of a GPI anchor on expressed AP-P was established by demonstrating release of the enzyme from a membrane fraction following treatment with bacterial phosphatidylinositol-specific phospholipase C and its corresponding conversion from an amphipathic to a hydrophilic form, as assessed by phase separation in Triton X-114. Sequence comparisons confirm that AP-P is a member of the proline peptidase family of hydrolytic enzymes and is unrelated in sequence to other brush-border membrane peptidases.
Hyde
R J
RJ
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Hooper
N M
NM
Turner
A J
AJ
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D007122:Immunoelectrophoresis; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013552:Swine
D002614:Chelating Agents; D010455:Peptides; D011480:Protease Inhibitors; D004492:Edetic Acid; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; C097277:apstatin