Histidine 379 of Human Laeverin/Aminopeptidase Q, a Nonconserved Residue within the Exopeptidase Motif, Defines Its Distinctive Enzymatic Properties

Abstract

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...

Promoting Roles of Embryonic Signals in Embryo Implantation and Placentation in Cooperation with Endocrine and Immune Systems

“…Members of the M1 family of aminopeptidases share a common peptide-binding site (GXMEN) and peptidase activity motif (HEXXHX18E). Primate laeverin has a unique peptide-binding motif (HXMEN) where the first glycine (Gly) residue is substituted with histidine (His) [143], inducing significant changes in substrate specificity toward natural peptide hormones [144]. Laeverin is specifically expressed on EVT in the placenta from early and term pregnancy.…”

Involvement of Phenylalanine 297 in the Construction of the Substrate Pocket of Human Aminopeptidase B

“…Figure A shows a phylogenetic tree of the M1 family of human aminopeptidases based on their amino acid sequence identities. Twelve proteins belonging to the family have been identified in the human genome, and their enzymatic properties have been characterized. ,− It seems that APB, LTA4H, and RNPEP-L1 (arginyl aminopeptidase-like 1) comprise a distinct subfamily together with APO and PSA. ,, To identify whether there was a single residue responsible for the unique characteristics and enzymatic activity of APB, we compared the amino acid sequences of M1 aminopeptidases around the GXMEN motif (Figure B). As expected, alignment of M1 aminopeptidases around this motif indicated that APB has a sequence highly identical with those of LTA4H and RNPEP-L1, whereas it is less similar to other M1 aminopeptidases.…”

<i>Lvrn</i> expression is not critical for mouse placentation

“…The consensus peptide recognition sequence of LVRN aminopeptidase is HXMEN. In rodents, Histidine has been substituted with Glycine and altered LVRN’s substrate specificity [27]. Moreover while human LVRN is specifically expressed in extravillous trophoblast [12], Lvrn mRNA was ubiquitously detected in mice (Fig.…”