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

Goto, Yoshikuni; Tanji, Hiroe; Hattori, Akira; Tsujimoto, Masafumi

doi:10.1042/bj20080965

Cited by 36 publications

(34 citation statements)

References 33 publications

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“…Though our modeling is tentative and the specific interactions may depend on the nature of a certain substrate, such a model would explain substrate binding in case of a disordered active site. Such a mechanism would also explain the marginal cleavage of shorter peptides, although it has been shown that ERAP1 is able to hydrolyse synthetic substrates (25), albeit with greatly reduced affinity.…”

Section: Resultsmentioning

confidence: 99%

“…The pocket is further bordered by the side chain of Met 319 , the aliphatic part of Glu 183 and the carboxamide group of Gln 181 . A previous study showed that mutating Gln 181 to glutamate increased the affinity of ERAP1 for basic N-terminal residues (25). The end of the pocket is formed by the aliphatic part of the side chain of Arg 430 and its bottom is sealed via an electrostatic interaction of the guanidinium group of Arg 430 with the carboxylate of Glu 865 of domain IV, which stacks against the imidazole ring of His 160 from domain I.…”

Section: Resultsmentioning

confidence: 99%

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Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Kochan

Krojer

Harvey

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

235

286

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Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-ðXÞ 18 -E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528 R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.antigen presentation | ERAP1 mechanism | MHC restriction

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Kochan

Krojer

Harvey

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

235

286

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“…Although wild-type enzyme caused a 1.4-fold increase in the activity, enhancement was marginal when RAW264.7 cells were treated with enzymatically inactive E354Q ERAP1, the Zn 2ϩ binding site of which was disrupted. In addition, Q181D ERAP1, which showed different substrate specificity from the wild-type enzyme and cleaved basic amino acids preferentially, also had only a marginal effect (16); thus, the enzymatic activity of wild-type ERAP1 is required for the enhancement of phagocytosis. We then examined the effects of other M1 aminopeptidases, such as placental leucine aminopeptidase (P-LAP)/insulin-regulated aminopeptidase/oxytocinase, laeverin/aminopeptidase Q (APQ), and aminopeptidase A (APA), on the uptake of IgG-coated latex beads (17)(18)(19).…”

Section: Resultsmentioning

confidence: 99%

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

Goto

Ogawa

Hattori

et al. 2011

Journal of Biological Chemistry

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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...

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“…Although bestatin inhibited the aminopeptidase activities of wild-type and H379F LVRN/APQ rather efficiently, H379G, H379K, and H379L LVRN/APQs were less susceptible to the inhibitor, suggesting that an aromatic ring of His 379 is important for the bestatin sensitivity of wild-type LVRN/APQ. On the other hand, amastatin inhibited the aminopeptidase activity of H379G LVRN/APQ (K i ϭ 17.3 Ϯ 5.4 M) more effectively than adipocyte-derived leucine aminopeptidase/endoplasmic reticulum aminopeptidase-1 (K i ϭ 41.8 Ϯ 10.3 M), to which amastatin is much more effective than bestatin (24). These results indicated that His 379 was responsible for the unique inhibitor profile of wild-type LVRN/APQ.…”

Section: Hismentioning

confidence: 60%

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

Maruyama

Arisaka

Goto

et al. 2009

Journal of Biological Chemistry

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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...

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

Cited by 36 publications

References 33 publications

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

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

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

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