Molecular investigation of aminopeptidase N expression in the winter flounder, Pleuronectes americanus

Abstract

The primary structure of a polypeptide with significant similarity to human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of two overlapping cDNA clones derived from winter flounder (Pleuronectes americanus, Walbaum) intestinal RNA and a partial genomic clone derived from winter flounder DNA. The deduced amino acid sequence (975 amino acids) is comprised of an N-terminal cytosolic portion of seven amino acid residues, followed by a 24-residue transmembrane anchor region, a 38-residue serine/threonine-rich junction and a 906-residue enzymatic domain that protrudes from the apical membrane of the enterocyte. A highly conserved signature sequence characteristic of zinc-dependent metallopeptidases (HExxH) is present, as well as seven putative glycosylation sites (NxS/T). Fourteen introns are present in the 7.53 kb portion of the gene that was cloned and sequenced. Reverse transcriptase polymerase chain reaction assays using primers spanning intron/exon boundaries were used to determine the timing of expression of this gene in larval winter flounder. This represents the first aminopeptidase N sequence to be determined from a teleost fish and underscores the utility of molecular biological information in the investigation of larval fish digestion.