ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis–Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis–Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant Ki, further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.

The adipocyte-derived leucine aminopeptidase (A-LAP)/ER aminopeptidase-1 is a multi-functional enzyme belonging to the M1 family of aminopeptidases. It was reported that the polymorphism Lys528Arg in the human A-LAP gene is associated with essential hypertension. In this study, the role of Lys528 in the enzymatic activity of human A-LAP was examined by site-directed mutagenesis. Among non-synonymous polymorphisms tested, only Lys528Arg reduced enzymatic activity. The replacement of Lys528 with various amino acids including Ala, Met, His and Arg caused a significant decrease in the enzymatic activity. Molecular modeling of the enzyme suggested that Lys528 is located near the entrance of the substrate pocket. These results suggest that Lys528 is important for maximal activity of A-LAP by maintaining the appropriate structure of the substrate pocket of the enzyme. The reduced enzymatic activity of A-LAP may cause high blood pressure and the observed association between the polymorphism and hypertension.

An interferon-induced endoribonuclease, ribonuclease L (RNase L), is implicated in both the molecular mechanism of action of interferon and the fundamental control of RNA stability in mammalian cells. RNase L is catalytically active only after binding to an unusual activator molecule containing a 5 0 -phosphorylated 2 0 ,5 0 -linked oligoadenylate (2-5A), in the N-terminal half. Here, we report the crystal structure of the N-terminal ankyrin repeat domain (ANK) of human RNase L complexed with the activator 2-5A. This is the first structural view of an ankyrin repeat structure directly interacting with a nucleic acid, rather than with a protein. The ANK domain folds into eight ankyrin repeat elements and forms an extended curved structure with a concave surface. The 2-5A molecule is accommodated at a concave site and directly interacts with ankyrin repeats 2-4. Interestingly, two structurally equivalent 2-5A binding motifs are found at repeats 2 and 4. The structural basis for 2-5A recognition by ANK is essential for designing stable 2-5As with a high likelihood of activating RNase L.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme with an important role in processing antigenic peptides presented to class I major histocompatibility complex in the endoplasmic reticulum. In this study, we found that endoplasmic reticulum-retained ERAP1 was secreted from macrophages in response to activation by treatment with lipopolysaccharide (LPS) and interferon (IFN)-␥ and enhanced their phagocytic activity. Enhancement of the phagocytic activity of murine macrophage RAW264.7 cells induced by LPS/ IFN-␥ was inhibited by a potent aminopeptidase inhibitor, amastatin. The addition of recombinant wild-type but not inactive mutant ERAP1 to culture medium enhanced phagocytosis. These results suggest that enhancement of phagocytic activity is at least in part mediated by secreted ERAP1 through the generation of active peptides processed by the enzyme. Our data reveal ERAP1-mediated activation of macrophages for the first time and will provide new insights into the role of this enzyme in innate immunity.It is well known that endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme belonging to the M1 family of aminopeptidases with roles in the regulation of blood pressure, angiogenesis, ectodomain shedding of several cytokine receptors, and processing of antigenic peptides presented to MHC class I molecules (1-4). Its cDNA was initially cloned as adipocyte-derived leucine aminopeptidase (5). Based on its multifunctional properties, adipocyte-derived leucine aminopeptidase is also designated ERAP1, ERAAP (endoplasmic reticulum aminopeptidase associated with antigen presentation), PILSAP (puromycin-insensitive leucine-specific aminopeptidase), and ARTS-1 (aminopeptidase regulator of TNFR1 shedding) (6 -9) (in this paper, ERAP1 is used hereafter). Although it is evident that ERAP1 plays important roles in several pathophysiological processes, its subcellular localization is still under debate. Although several reports have presented evidence showing its localization in the ER (6, 7) or cytoplasm (10) as a soluble protein, others have shown it on the cell surface as a type II membrane-spanning protein (9).ERAP1 is a monomeric zinc-metallopeptidase that shows a preference for leucine when measured by synthetic substrates (5). On the other hand, it shows relatively broad substrate specificity toward natural peptide hormones, such as angiotensin II, kallidin, and neurokinin A, which may reflect its role in the processing of various precursors of antigenic peptides presented to MHC class I molecules (11). On the basis of a preference for substrates of a specific length and C-terminal hydrophobic amino acid, the "molecular ruler" mechanism was proposed for the processing of antigenic peptides by the enzyme (12).Because ERAP1 inactivates angiotensin II and converts kallidin to bradykinin, it was initially speculated that it might regulate blood pressure (11). Subsequently, by screening for polymorphisms in the human ERAP1 gene, Yamamoto et al. (13) identified an association of K5...

It is becoming evident that several aminopeptidases belonging to the M1 family such as aminopeptidase A (APA), placental leucine aminopeptidase (P-LAP), and adipocyte-derived leucine aminopeptidase (A-LAP) play important roles in the regulation of blood pressure under both the physiological and pathological conditions. They share HEXXH(X)(18)E zinc-binding and GAMEN motifs essential for enzymatic activities. In this review, the current situation regarding the biochemical characteristics of these enzymes including enzymatic properties and modes of action is summarized.

Laeverin/aminopeptidase Q (APQ) is a cell surface protein specifically expressed on human embryo-derived extravillous trophoblasts that invades the uterus during placentation. The cDNA cloning of Laeverin/APQ revealed that the sequence encodes a protein with 990 amino acid residues, and Laeverin/ APQ contains the HEXXHX 18 E gluzincin motif, which is characteristic of the M1 family of aminopeptidases, although the exopeptidase motif of the family, GAMEN, is uniquely substituted for the HAMEN sequence. In this study, we expressed a recombinant human Laeverin/APQ using a baculovirus expression system, purified to homogeneity, and characterized its enzymatic properties. It was found that Laeverin/APQ had a broad substrate specificity toward synthetic substrate, although it showed a preference for Leu-4-methylcoumaryl-7-amide. Searching natural substrates, we found that Laeverin/APQ was able to cleave the N-terminal amino acid of several peptides such as angiotensin III, kisspeptin-10, and endokinin C, which are abundantly expressed in the placenta. In contrast to the case with other M1 aminopeptidases, bestatin inhibited the aminopeptidase activity of Laeverin/APQ much more effectively than other known aminopeptidase inhibitors. These results indicate that Laeverin/ APQ is a novel bestatin-sensitive leucine aminopeptidase and suggest that the enzyme plays important roles in human placentation by regulating biological activity of key peptides at the embryo-maternal interface.Aminopeptidases hydrolyze N-terminal amino acid of proteins or peptide substrates. Among them, the M1 family of zinc aminopeptidases (gluzincin) shares the consensus GAMEN and HEXXHX 18 E motifs essential for enzymatic activity and consists of 11 enzymes in human beings (1, 2). It is now becoming obvious that the M1 aminopeptidases are involved in many physiological events and are important for the maintenance of homeostasis. For instance, placental leucine aminopeptidase (P-LAP) 2 /oxytocinase plays a role in the progression of pregnancy by controlling the concentration of uterotonic and vasoactive hormones such as oxytocin and vasopressin to prevent premature delivery and pre-eclampsia (3). P-LAP is also referred to as insulin-regulated aminopeptidase, because it colocalizes with insulin-responsive glucose transporter 4 in the same vesicle in adipocyte and muscle cells and is translocated to plasma membrane by insulin stimulation, suggesting it has roles in the pathogenesis of diabetes (4). Recently, P-LAP/insulin-regulated aminopeptidase was also shown to be a specific receptor of angiotensin IV, further suggesting its significance in memory retention and retrieval (5). By searching databases for proteins homologous to P-LAP, we have cloned two novel aminopeptidases localized in the endoplasmic reticulum, adipocyte-derived leucine aminopeptidase/endoplasmic reticulum aminopeptidase-1, and leukocyte-derived arginine aminopeptidase/endoplasmic reticulum aminopeptidase-2 (6, 7). Subsequent studies indicated these to be final processing enzymes t...

Aminopeptidase A (APA) is a type II membrane-bound protein implicated in the regulation of blood pressure in the brain renin-angiotensin system. In this study, a recombinant soluble form of APA was expressed in a baculovirus system, purified to homogeneity, and characterized. By using synthetic substrates, it was shown that although the enzyme has a rather broad substrate specificity in the absence of Ca 2؉ , the preferential release of acidic amino acid residues was observed in the presence of Ca 2؉ . Moreover, Ca 2؉ up-or down-regulated the enzymatic activity depending on the substrate. By searching for natural substrates of APA, we found that peptides having acidic amino acids at their N terminus (angiotensin II, neurokinin B, cholecystokinin-8, and chromogranin A) were cleaved by the enzyme efficiently in the presence but not in the absence of Ca 2؉ . Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca 2؉ . These results suggest that Ca 2؉ increases the preference of the enzyme for the peptide substrates having N-terminal acidic amino acids. In addition, we found that angiotensin IV could bind to APA both in the presence and absence of Ca 2؉ and inhibited the enzymatic activity of APA competitively, suggesting that angiotensin IV acts as a negative regulator of the enzyme once generated from angiotensin II by the serial actions of aminopeptidases. Taken together, these results suggest that there exists a complex regulation of the enzymatic activity of APA, which may contribute to homeostasis such as regulation of blood pressure, maintenance of memory, and normal pregnancy by controlling the concentrations of peptide substrates.

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln(181) as important for enzymatic activity and substrate specificity. Replacement of Gln(181) by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln(181) is critical for the enzymatic activity and substrate specificity of ERAP-1.