Identification of Human Aminopeptidase O, a Novel Metalloprotease with Structural Similarity to Aminopeptidase B and Leukotriene A4 Hydrolase

Abstract

We have cloned and characterized a human brain cDNA encoding a new metalloprotease that has been called aminopeptidase O (AP-O). AP-O exhibits a series of structural features characteristic of aminopeptidases, including a conserved catalytic domain with a zinc-binding site (HEXXHX 18 E) that allows its classification in the M1 family of metallopeptidases or gluzincins. The structural complexity of AP-O is further increased by the presence of an additional C-terminal domain 170 residues long, which is predicted to have an ARM repeat fold originally identified in the Drosophila segment polarity gene product Armadillo. This ARM repeat domain is also present in aminopeptidase B, aminopeptidase B-like, and leukotriene A 4 hydrolase and defines a novel subfamily of aminopeptidases that we have called ARM aminopeptidases. Northern blot analysis revealed that AP-O is mainly expressed in the pancreas, placenta, liver, testis, and heart. Human AP-O was produced in Escherichia coli, and the purified recombinant protein hydrolyzed synthetic substrates used for assaying aminopeptidase activity. This activity was abolished by general inhibitors of metalloproteases and specific inhibitors of aminopeptidases. Recombinant AP-O also cleaved angiotensin III to generate angiotensin IV, a bioactive peptide of the renin-angiotensin pathway with multiple actions on diverse tissues, including brain, testis, and heart. On the basis of these results we suggest that AP-O could play a role in the proteolytic processing of bioactive peptides in those tissues where it is expressed.Aminopeptidases are exopeptidases that have the ability to catalyze the hydrolysis of amino acid residues from the N terminus of peptide or protein substrates. These enzymes are widely distributed in multiple organisms from bacteria to human and play essential roles in protein maturation and regulation of the metabolism of bioactive peptides (1, 2). Furthermore, alterations in the function and regulation of aminopeptidases underlie several human diseases, including cancer and cardiovascular disorders (3-5). Among the different families of human aminopeptidases, there has been a growing interest in the analysis of the biological and pathological relevance of members of the M1 family of zinc metallopeptidases or gluzincins (6, 7). To date, 12 members of this family have been identified in human tissues (www.uniovi.es/degradome) (8). Aminopeptidase A (APA or ENPEP) is a type II membranebound protease that cleaves N-terminal amino acid residues from peptides such as cholecystokinin-8 and angiotensin II (9, 10) and contributes to the control of blood pressure and angiogenesis (11-13). Aminopeptidase N (APN, 1 ANPEP, or CD13) is also a membrane-bound exopeptidase associated with angiogenesis regulation (14 -16) that acts as a receptor for the coronavirus and tumor-homing peptides (17, 18). Aminopeptidase Q (APQ or laeverin) is structurally related to APN and participates in regulation of the extravillous trophoblast invasion of maternal decidual tissues (8, 19). The thyr...

Identification and Characterization of Human Archaemetzincin-1 and -2, Two Novel Members of a Family of Metalloproteases Widely Distributed in Archaea

“…We next performed a kinetic analysis of the proteolytic reaction catalyzed by the catalytic domains of AMZ1 and AMZ2 with their preferred substrates (Ala-AMC and Arg-AMC, respectively). The fitting of the resulting data to the Michaelis-Menten equation yielded k cat /K m values of 46 M Ϫ1 s Ϫ1 and 22 M Ϫ1 s Ϫ1 for catalytic domain proteins of AMZ1 and AMZ2, respectively, which are similar to the value reported for recombinant aminopeptidase O produced in the same expression system (30). We have also tried to perform similar experiments with the fulllength proteins produced in bacterial systems.…”

MicroRNA-24 Is a Novel Regulator of Aldosterone and Cortisol Production in the Human Adrenal Cortex

“…31,32 Although the role of aminopeptidase-O is less well defined, it can cleave angiotensin III to angiotensin IV. 30 Angiotensin III, but not angiotensin IV, stimulates aldosterone production in the adrenal gland by a mechanism thought to involve type 2 angiotensin II receptors. 33 Reduced transcription at this locus in APA could, therefore, stimulate aldosterone production via post-transcriptional CYP11B2 regulation while simultaneously reducing angiotensin III cleavage.…”

“…30 A closely related enzyme, aminopeptidase-A, plays a crucial role in blood pressure regulation and is integral to the renin-angiotensin system, cleaving angiotensin II to angiotensin III. 31,32 Although the role of aminopeptidase-O is less well defined, it can cleave angiotensin III to angiotensin IV.…”

Homology Modeling and Site-directed Mutagenesis of Pyroglutamyl Peptidase II

“…nopeptidase O is an exception, with an Ala that substitutes the glutamate residue (43). The second residue involved in the ␣-N-terminus recognition (APA-Glu-215, APN-Gln-213, or LTA4H-Gln-136) was Glu or Gln for aminopeptidases, in contrast to Ser in PPII sequences (Ser-269 in rat) (Fig.…”