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 enzy me in the metabolism of the vasodilator bradykinin, has been cloned fr om a pig kidney cortex cDNA library following the use of the PCR to id entify sub-libraries enriched in AP-P clones. The complete primary seq uence of the enzyme has been deduced from a full-length cDNA clone. Th is predicts a protein of 673 amino acids with a cleavable N-terminal s ignal sequence and six potential N-linked glycosylation sites. A stret ch of mainly hydrophobic amino acids at the C-terminus is predicted to co-ordinate the attachment of a glycosyl-phosphatidylinositol (GPI) m embrane anchor. Although AP-P is a zinc metallopeptidase, the predicte d primary sequence does not contain any recognizable zinc-binding moti f. Transient expression of AP-P cDNA in COS-1 cells resulted in enzymi c activity characteristic of AP-P, namely apstatin- and EDTA-sensitive hydrolysis of bradykinin and Gly-Pro-Hyp. The expressed protein was r ecognized as a polypeptide of M(r) 91000 under reducing conditions, fo llowing immunoblotting of COS-1 membranes with a polyclonal antibody r aised against purified pig kidney AP-P. The presence of a GPI anchor o n expressed AP-P was established by demonstrating release of the enzym e from a membrane fraction following treatment with bacterial phosphat idylinositol-specific phospholipase C and its corresponding conversion from an amphipathic to a hydrophilic form, as assessed by phase separ ation in Triton X-114. Sequence comparisons confirm that APP is a memb er of the proline peptidase family of hydrolytic enzymes and is unrela ted in sequence to other brush-border membrane peptidases.