Human laeverin/aminopeptidase Q (LVRN/APQ) is a novel member of the M1 family of zinc aminopeptidases and is specifically expressed on the cell surface of human extravillous trophoblasts. Multiple sequence alignment of human M1 aminopeptidase revealed that the first Gly residue within the conserved exopeptidase motif of the M1 family, GXMEN motif, is uniquely substituted for His in human LVRN/APQ. In this study, we evaluated the roles of nonconserved His 379 , comprising the exopeptidase motif in the enzymatic properties of human LVRN/APQ. We revealed that the substitution of His 379 with Gly caused significant changes in substrate specificity both toward fluorogenic substrates and natural peptide hormones. In addition, the susceptibilities of bestatin, a sensitive inhibitor for human LVRN/ APQ, and natural inhibitory peptides were decreased in the H379G mutant. A molecular model suggested a conformational difference between wild-type and H379G human LVRN/APQs. These results indicate that His 379 of the enzyme plays essential roles in its distinctive enzymatic properties and contributes to maintaining the appropriate structure of the catalytic cavity of the enzyme. Our data may bring new insight into the biological significance of the unique exopeptidase motif of LVRN/APQ obtained during the evolution of primates.The M1 family of mono-zinc aminopeptidases is widely distributed in bacteria, fungi, plants, and animals (1, 2). They hydrolyze peptide bonds linking to the N-terminal amino acids of peptide or protein substrates. The human M1 family consists of 11 enzymes that participate in many important physiological events, such as reproduction (3), angiogenesis (4 -6), antigen presentation (7-10), blood pressure control (11-13), and memory retention (14), and thus play essential roles in the maintenance of homeostasis.M1 aminopeptidases share two conserved domains: HEXXH(X) 18 E gluzincin motif and the exopeptidase motif, which is conserved as a GAMEN sequence in most members. The function of each conserved residue within these motifs has been elucidated using site-directed mutagenesis. For instance, two His residues and the second Glu within the HEXXH(X) 18 E motif coordinate the zinc atom essential for catalytic activity. The first Glu in the motif polarizes to the water molecule that coordinates the zinc atom and promotes nucleophilic attack of the carbonyl carbon of the peptide bond forming a tetrahedral intermediate in water (15). The conserved Glu within the exopeptidase motif, the GAMEN sequence, was shown to be involved in recognition of the ␣-amino group of substrates (16,17).Laeverin (LVRN) 4 was originally identified as a cell-surface protein specifically expressed on human extravillous trophoblasts (18). The cDNA cloning of human LVRN revealed that it contains both consensus motifs and is a novel member of the family. Another group predicted the existence of the LVRN gene in the human genome through a genomic search and named it aminopeptidase Q (APQ) (19); however, its exopeptidase motif is uniquely c...

Aminopeptidases hydrolyze the N-terminal amino acid of proteins or peptide substrates. Among them, the M1 family of zinc aminopeptidases (gluzincins) shares the consensus GAMEN and HEXXH(X) 18 E motifs essential for enzymatic activity. This family, which consists of 11 enzymes in humans, [1][2][3] plays important roles in several pathophysiological processes, such as angiogenesis, cell cycle regulation, reproduction, memory retention, blood pressure control, and antigen presentation to major histocompatibility complex (MHC) class I molecules. [4][5][6][7][8][9][10][11][12] Laeverin was originally identified as a cell-surface protein specifically expressed on human extravillous trophoblasts. 13) cDNA cloning of human laeverin revealed that it contains both consensus motifs and thus is a novel member of the M1 family. Another group also predicted the existence of the 'leaverin' gene in the human genome through a genomic search and named it aminopeptidase Q (APQ).14) Recently, we established a large-scale production system of the enzyme and characterized its enzymatic properties in detail. 15) We found that the enzyme degraded several placenta-derived peptide hormones, such as angiotensin III, endokinin C, and kisspeptin-10. Furthermore because its exopeptidase motif GAMEN is uniquely composed of the HAMEN sequence, we examined the enzymatic significance of His-379 of human laeverin/APQ by site-directed mutagenesis and found that this residue contributes to the unique properties of the enzyme.16) Considering the susceptibility of these peptides and their specific expression in the placenta, we speculated that laeverin/APQ plays important roles in the maintenance of normal pregnancy in humans.In our previous work, we identified a residue affecting enzymatic activity and the substrate specificity of endoplasmic reticulum aminopeptidase (ERAP)1, a member of the M1 aminopeptidase family. 17) In particular, replacement of Gln-181 with Asp caused a marked change in the substrate specificity of ERAP1. On the other hand, replacement with Ala led to almost complete loss of enzymatic activity. To further analyze the residue affecting enzymatic activity and substrate specificity of the M1 family of aminopeptidases, we examined in the present study the effect of replacing Gln-238 of human laeverin/APQ with Ala. We found that Q238A laeverin/APQ retained substantial enzymatic activities with marked change of its substrate specificity, indicating the significance of this residue for the enzymatic characteristics of laeverin/APQ. Data presented in this study imply that direct electrostatic interaction is not involved in the mutant enzyme's preference for basic amino acids, which was suggested in the previous work. 17) MATERIALS AND METHODS Molecular Modeling of Laeverin/APQThe published X-ray crystallographic structure of Thermoplasma acidophilum Tricorn-interacting factor F3 (TIFF3) (Protein Data Bank code 1Z5H) 18) was used as a template for modeling the catalytic site of ERAP1 with the three-dimensional Jigsaw Protein Server (h...