• HEK293 cells;
  • insulin-regulated aminopeptidase;
  • metallopeptidase;
  • mutagenesis;
  • oxytocinase

Insulin-regulated aminopeptidase (IRAP) is a type II integral membrane protein belonging to the gluzincin family of metallopeptidases identified by the characteristic Zn2+-coordination sequence element, HEXXH-(18–64X)-E. A second conserved sequence element, the GXMEN motif, positioned 22–32 amino acids N-terminal to the Zn2+-coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importance of the G428AMEN and H464ELAH-(18X)-E487 motifs for the activity of IRAP, mutational analysis was carried out. cDNA encoding the full-length transmembrane form of human IRAP was expressed in HEK293 cells and recombinant wild-type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Mutational analysis using single amino-acid substitutions in the GAMEN motif (G428A, A429G, M430K, M430E, M430I, E431D and E431A) and in the Zn2+-binding motif (H464Y, E465D, E465Q, H468Y, E487D and E487Q) resulted in decreased or abolished aminopeptidase activity towards the leucine-para-nitroanilide substrate. The results show that conservation of residues within the GAMEN and Zn2+-binding motifs is important for IRAP enzyme activity.