Aminopeptidase-A. I. cDNA cloning and expression and localization in rat tissues

Aminopeptidase-A (APA) is an ectoenzyme that selectively hydrolyzes acidic residues from the amino terminus of oligopeptides, including biologically a ctive [Asp(1)]ANG II and [Asp(1)]CCK-8. We sought to characterize rat APA b y cDNA cloning and expression and to determine its tissue distribution by i n situ hybridization and immunohistochemistry. Sequence analysis of overlap ping cDNA clones isolated from rat kidney cDNA libraries indicated that the full-length cDNA encoded a 945-amino acid protein with a predicted molecul ar mass of 108 kDa; the size was confirmed by in vitro translation of a ful l-length cDNA construct. Transient transfection of the full-length cDNA con struct in mammalian cells yielded a protein similar to 140 kDa in size, a s ize that agrees with the immunoblots of APA from rat tissue and is consiste nt with APA being known as a glycosylated protein. Tissue APA activity and mRNA expression were highest in the kidney and ileum. Localization of APA b y in situ hybridization and immunohistochemistry indicated that, with the e xception Of the kidney and ileum, where APA was localized to the luminal br ush border of proximal tubules and enterocytes, respectively, APA was assoc iated with either capillaries or the lining of sinusoids. Areas known to be physiological targets for ANG II, including glomeruli, the zona glomerulos a, and anterior pituitary, had high levels of APA. The localization pattern suggests that APA may subserve multiple functions, i.e., a generalized rol e in peptide scavenging and perhaps a more specific role in metabolism of c irculating or locally produced ANG II or CCK-8.