PRODUCTION AND PROPERTIES OF A RECOMBINANT SOLUBLE FORM OF AMINOPEPTIDASE-A

Aminopeptidase A is a homodimeric membrane-bound zinc metallopeptidase anchored at the plasma membrane by a 22-amino-acid hydrophobic segmen t. The anchor segment separates a small N-terminal cytoplasmic domain from a large ectodomain that contains the active site. Site-directed m utagenesis was performed to investigate the role of the cytoplasmic do main of aminopeptidase A in membrane anchoring and routing of the enzy me. Expression in COS-7 cells of a mutant lacking the N-terminal cytop lasmic domain resulted in the efficient secretion of a catalytically a ctive enzyme in the medium. The soluble mutated aminopeptidase A, puri fied from the medium of a stable cell line, exhibited similar biochemi cal features to those of the wild-type enzyme. Pulse/chase metabolic l abeling experiments revealed that the soluble form is generated intrac ellularly at an early stage of biosynthesis, suggesting that the signa l peptide/membrane anchor domain of aminopeptidase A is removed in the endoplasmic reticulum through the action of the signal peptidase.