Molecular characterization of a puromycin-insensitive leucyl-specific aminopeptidase, PILS-AP

Abstract

The family M1 of Zn-dependent aminopeptidases comprises members of closely related enzymes which are known to be involved in a variety of physiologically important processes. On the basis of two highly conserved peptide motifs, we have identified a new member of this family by PCR amplification and cDNA-library screening. The longest ORF encodes a protein of 930 residues. It contains the HEXXH(X)18E Zn-binding motif and displays high homology to the other M1 family members except for its N-terminus for which a signal sequence of 20 residues can be predicted. This interpretation was supported by expressing fusion proteins formed with green fluorescent protein which localized to intracellular vesicles in COS-7 and BHK cells. Northern-blot analysis revealed ubiquitous expression of a major 3.1-kb transcript. For enzymatic studies, the complete protein was expressed in Sf9 insect cells. When aminoacyl β-naphthylamides were used as substrates, efficient hydrolysis was only observed for Leu (and to a lesser extent Met). The activity was inhibited by chelators of bivalent cations and by other known aminopeptidase inhibitors, but surprisingly puromycin was without effect. This newly identified puromycin-insensitive leucyl-specific aminopeptidase is a signal-sequence-bearing member of family M1 and may be another example of the small subset of substrate-specific peptidases.