Yoshikuni Goto

Cutting Edge: Coding Single Nucleotide Polymorphisms of Endoplasmic Reticulum Aminopeptidase 1 Can Affect Antigenic Peptide Generation In Vitro by Influencing Basic Enzymatic Properties of the Enzyme

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.

Reduced activity of the hypertension‐associated Lys528Arg mutant of human adipocyte‐derived leucine aminopeptidase (A‐LAP)/ER‐aminopeptidase‐1

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.

Secretion of Endoplasmic Reticulum Aminopeptidase 1 Is Involved in the Activation of Macrophages Induced by Lipopolysaccharide and Interferon-γ

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)-γ and enhanced their phagocytic activity. Enhancement of the phagocytic activity of murine macrophage RAW264.7 cells induced by LPS/IFN-γ 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.

Enzymatic Properties of Human Aminopeptidase A REGULATION OF ITS ENZYMATIC ACTIVITY BY CALCIUM AND ANGIOTENSIN IV

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 Ca2+, the preferential release of acidic amino acid residues was observed in the presence of Ca2+. Moreover, Ca2+ 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 Ca2+. Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca2+. These results suggest that Ca2+ 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 Ca2+ 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.

Biochemical and enzymatic properties of the M1 family of aminopeptidases involved in the regulation of blood pressure

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)18E 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.

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

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.

Laeverin/aminopeptidase Q, a novel bestatin-sensitive leucine aminopeptidase belonging to the M1 family of aminopeptidases.

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 HEXXHX18E 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.

Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A.

Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.

TLR-Mediated Secretion of Endoplasmic Reticulum Aminopeptidase 1 from Macrophages

Macrophages play an important role in host defense under several immunological, inflammatory, and/or infectious conditions. In our previous work, we demonstrated that endoplasmic reticulum aminopeptidase 1 (ERAP1) was secreted from macrophages in response to LPS and IFN-γ, and it enhanced their phagocytic activity. In this study, we analyzed the mechanism of LPS/IFN-γ–induced ERAP1 secretion. LPS/IFN-γ–induced secretion of the enzyme from the murine macrophage cell line RAW264.7 was suppressed by polymyxin B. Several agonists of TLRs, such as Pam3CSK4, FSL-1, and ODN1826, induced its secretion. In contrast, neutralizing Abs to IFN-β and TNF-α receptor type 1 suppressed its secretion. Using murine peritoneal macrophages derived from TNF-α and type 1 IFNR knockout mice, we confirmed the involvement of these two cytokines in ERAP1 secretion. In addition, secretion of ERAP1 from both RAW264.7 cells and murine peritoneal macrophages was induced by A23187 and thapsigargin and inhibited by BAPTA-AM and the calmodulin inhibitor W7. These results suggest that LPS/IFN-γ–induced secretion of ERAP1 is mediated by TLRs via induction of intermediate cytokines such as IFN-β and TNF-α, which in turn lead to enhanced cytosolic Ca2+ levels and calmodulin activation.

A Single Neonatal Injection of Ethinyl Estradiol Impairs Passive Avoidance Learning and Reduces Expression of Estrogen Receptor α in the Hippocampus and Cortex of Adult Female Rats.

Although perinatal exposure of female rats to estrogenic compounds produces irreversible changes in brain function, it is still unclear how the amount and timing of exposure to those substances affect learning function, or if exposure alters estrogen receptor α (ERα) expression in the hippocampus and cortex. In adult female rats, we investigated the effects of neonatal exposure to a model estrogenic compound, ethinyl estradiol (EE), on passive avoidance learning and ERα expression. Female Wistar-Imamichi rats were subcutaneously injected with oil, 0.02 mg/kg EE, 2 mg/kg EE, or 20 mg/kg 17β-estradiol within 24 h after birth. All females were tested for passive avoidance learning at the age of 6 weeks. Neonatal 0.02 mg/kg EE administration significantly disrupted passive avoidance compared with oil treatment in gonadally intact females. In a second experiment, another set of experimental females, treated as described above, was ovariectomized under pentobarbital anesthesia at 10 weeks of age. At 15–17 weeks of age, half of each group received a subcutaneous injection of 5 μg estradiol benzoate a day before the passive avoidance learning test. Passive avoidance learning behavior was impaired by the 0.02 mg/kg EE dose, but notably only in the estradiol benzoate-injected group. At 17–19 weeks of age, hippocampal and cortical samples were collected from rats with or without the 5 μg estradiol benzoate injection, and western blots used to determine ERα expression. A significant decrease in ERα expression was observed in the hippocampus of the estradiol-injected, neonatal EE-treated females. The results demonstrated that exposure to EE immediately after birth decreased learning ability in adult female rats, and that this may be at least partly mediated by the decreased expression of ERα in the hippocampus.