Laeverin/Aminopeptidase Q, a Novel Bestatin-sensitive Leucine Aminopeptidase Belonging to the M1 Family of Aminopeptidases
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m702650200
ISSN1083-351X
AutoresMasato Maruyama, Akira Hattori, Yoshikuni Goto, Masamichi Ueda, Michiyuki Maeda, Hiroshi Fujiwara, Masafumi Tsujimoto,
Tópico(s)Signaling Pathways in Disease
ResumoLaeverin/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. 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. 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 HEXXHX18E motifs essential for enzymatic activity and consists of 11 enzymes in human beings (1Hooper N.M. FEBS Lett. 1994; 31: 1-6Crossref Scopus (672) Google Scholar, 2Tsujimoto M. Hattori A. Biochim. Biophys. Acta. 2005; 1751: 9-18Crossref PubMed Scopus (143) Google Scholar). 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) 2The abbreviations used are: P-LAP, placental leucine aminopeptidase; ADAM, a disintegrin and metalloproteinase; Ang, angiotensin; APA, aminopeptidase A; APN, aminopeptidase N; APQ, aminopeptidase Q; EVT, extravillous trophoblast; MCA, 4-methylcoumaryl-7-amide; sLaeverin, a soluble form of Laeverin; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; HPLC, high-performance liquid chromatography. /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 (3Nomura S. Ito T. Yamamoto E. Sumigama S. Iwase A. Okada M. Shibata K. Ando H. Ino K. Kikkawa F. Mizutani S. Biochim. Biophys. Acta. 2005; 1751: 19-25Crossref PubMed Scopus (41) Google Scholar). 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 (4Keller S.R. Scott H.M. Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1995; 270: 23612-23618Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). 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 (5Albiston A.L. McDowall S.G. Matsacos D. Sim P. Clune E. Mustafa T. Lee J. Mendelsohn F.A. Simpson R.J. Connolly L.M. Chai S.Y. J. Biol. Chem. 2001; 276: 48623-48626Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). 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 (6Hattori A. Matsumoto H. Mizutani S. Tsujimoto M. J. Biochem. (Tokyo). 1999; 125: 931-938Crossref PubMed Scopus (109) Google Scholar, 7Tanioka T. Hattori A. Masuda S. Nomura Y. Nakayama H. Mizutani S. Tsujimoto M. J. Biol. Chem. 2003; 278: 32275-32283Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Subsequent studies indicated these to be final processing enzymes that trim precursors to antigen peptides presented to major histocompatibility complex class I molecules (8Saric T. Chang S-C. Hattori A. York I.A. Markant S. Rock K.L. Tsujimoto M. Goldberg A.L. Nat. Immunol. 2002; 3: 1169-1176Crossref PubMed Scopus (425) Google Scholar, 9Saveanu L. Carroll O. Lindo V. Del Val M. Lopez D. Lepelletier Y. Greer F. Schomburg L. Fruci D. Niedermann G. van Endert P.M. Nat. Immunol. 2005; 6: 689-697Crossref PubMed Scopus (353) Google Scholar). Adipocyte-derived leucine aminopeptidase is also reported to be involved in the regulation of angiogenesis, the shedding of cytokine receptor, and blood pressure (10Miyashita H. Yamazaki T. Akada T. Niizeki O. Ogawa M. Nishikawa S. Sato Y. Blood. 2002; 99: 3241-3249Crossref PubMed Scopus (58) Google Scholar, 11Cui X. Hawari F. Alsaaty S. Lawrence M. Combs C.A. Geng W. Rouhani F.N. Miskinis D. Levine S.J. J. Clin. Invest. 2002; 110: 515-526Crossref PubMed Scopus (193) Google Scholar, 12Cui X. Rouhani F.N. Hawari F. Levine S.J. J. Biol. Chem. 2003; 278: 28677-28685Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Hattori A. Kitatani K. Matsumoto H. Miyazawa S. Rogi T. Tsuruoka N. Mizutani S. Natori Y. Tsujimoto M. J. Biochem. (Tokyo). 2000; 128: 755-762Crossref PubMed Scopus (97) Google Scholar, 14Yamamoto N. Nakayama J. Yamakawa-Kobayashi K. Hamaguchi H. Miyazaki R. Arinami T. Hum. Mutat. 2002; 19: 251-257Crossref PubMed Scopus (82) Google Scholar). In addition, several physiological and/or pathological functions in the brain, including the regulation of blood pressure and apoptosis, are assigned to M1 aminopeptidases such as aminopeptidase A (APA), aminopeptidase N (APN), thyrotropin-releasing hormone-degrading enzyme, and puromycin-sensitive aminopeptidase. Because of the pathological significance of M1 family aminopeptidases, it is important to characterize their enzymatic properties in detail (15Zini S. Fournie-Zaluski M.C. Chauvel E. Roques B.P. Corvol P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11968-11973Crossref PubMed Scopus (291) Google Scholar, 16Reaux A. Fournie-Zaluski M.C. David C. Zini S. Roques B.P. Corvol P. Llorens-Cortes C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13415-13420Crossref PubMed Scopus (158) Google Scholar, 17Schomburg L. Turwitt S. Prescher G. Lohmann D. Horsthemke B. Bauer K. Eur. J. Biochem. 1999; 265: 415-422Crossref PubMed Scopus (44) Google Scholar, 18Constam D.B. Tobler A.R. Rensing-Ehl A. Kemler I. Hersh L.B. Fontana A. J. Biol. Chem. 1995; 270: 26931-26939Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 19Tobler A.R. Constam D.B. Schmitt-Graff A. Malipiero U. Schlapbach R. Fontana A. J. Neurochem. 1997; 68: 889-897Crossref PubMed Scopus (57) Google Scholar, 20Osada T. Ikegami S. Takiguchi-Hayashi K. Yamazaki Y. Katoh-Fukui Y. Higashinakagawa T. Sakaki Y. Takeuchi T. J. Neurosci. 1999; 15: 6068-6078Crossref Google Scholar). Laeverin was originally identified as a cell surface CHL2 antigen expressed in human extravillous trophoblasts (EVTs) (21Fujiwara H. Higuchi T. Yamada S. Hirano T. Sato Y. Nishioka Y. Yoshioka S. Tatsumi K. Ueda M. Maeda M. Fujii S. Biochem. Biophys. Res. Commun. 2004; 313: 962-968Crossref PubMed Scopus (29) Google Scholar). It was shown that the CHL2 antigen was specifically expressed in the outer layer of the chorion laeve in the human fetal membrane and on the migrating human EVTs in the maternal decidual tissues but not on fetal amniotic epithelial cells and maternal decidual cells by immunohistochemistry. The cDNA cloning predicted that the CHL2 antigen is a type II membrane-spanning protein and has the zinc binding motif, HEXXHX18E motif and HAMEN sequence similar to the GAMEN motif, suggesting that the protein is a novel gluzincin M1 family of aminopeptidases. This novel protein was named Laeverin after its restricted expression in chorion laeve, and it is expected that its enzymatic action will be important for the EVT function during pregnancy. Another group (22Puente X.S. Sanchez L.M. Overall C.M. Lopez-Otin C. Nat. Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (758) Google Scholar) has predicted the existence of a novel aminopeptidase from the human Laeverin gene by using a genomic search and proposed to name it aminopeptidase Q (APQ). However, the enzymatic activity of Laeverin/APQ still remains to be explored because of its limited availability. In this study, we established a large scale production system of recombinant protein of human Laeverin/APQ. The availability of purified protein made it possible to characterize its structure and enzymatic properties in detail. This is the first report describing the biochemical and enzymatic properties of the novel M1 aminopeptidase, Laeverin/APQ. Expression and Purification of a Soluble Form of Human Laeverin/APQ in a Baculovirus System—cDNA encoding the full-length human Laeverin/APQ-(1–990), with the His6 tag at its C terminus, was generated by PCR. The amplified fragment was cloned into the EcoRI-XhoI site of pFastBac1 vector (Invitrogen). Then resultant plasmid was introduced into DH10Bac cells to produce recombinant bacmid DNA containing the Laeverin/APQ cDNA. Next, Sf9 insect cells were transfected with the bacmid DNA using Cellfectin reagent (Invitrogen), and after 72-h incubation, recombinant baculoviruses were harvested. For the expression of recombinant human Laeverin/APQ, Sf9 cells (2.0 × 106/ml) infected with the recombinant baculovirus (multiplicity of infection =∼1–3) were cultured for 72 h in 3 liters of SFM-900 III medium (Invitrogen) at 27 °C supplied with 8.0 ppm of O2 (Cellmaster-1700, Wakenyaku, Kyoto, Japan). The conditioned medium containing the soluble form of Laeverin/APQ (sLaeverin/APQ) was collected by centrifugation, and then applied to a hydroxyapatite column (2.5 × 10 cm, Nacalai Tesque, Kyoto, Japan) equilibrated in 50 mm Tris/HCl buffer (pH 7.5) and eluted with 100 mm sodium phosphate buffer (pH 7.5). The eluate were applied to a chelating-Sepharose (GE Healthcare Bio-Science, Piscataway, NJ) column (1.0 × 10 cm) preloaded with Ni2+ and then eluted with 200 mm imidazole. The active fractions were collected, concentrated, and subjected to further characterization. De-glycosylation of sLaeverin/APQ—After the heat denature (100 °C, 5 min), purified sLaeverin/APQ (100 μg/ml) was incubated with peptide:N-glycosidase F (30 μg/ml, New England Biolabs, Beverly, MA) in 50 mm sodium phosphate buffer (pH 7.5) at 37 °C for 2 h. Measurement of Aminopeptidase Activity of Laeverin/APQ—The aminopeptidase activity of recombinant human sLaeverin/APQ was determined with various fluorogenic substrates, aminoacyl-4-metheylcoumaryl-7-amides (aminoacyl-MCAs). The reaction mixture containing various concentrations of aminoacyl-MCA and the enzyme in 0.5 ml of 25 mm Tris/HCl buffer (pH 7.0) was incubated at 37 °C for 10 min. The amount of 7-amino-4-methylcoumarin released was measured by spectrofluorophotometry (F-2000, Hitachi) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The kinetic parameters were calculated from Lineweaver-Burk plots. The results are represented by Km, kcat, and kcat/Km values. All measurements were performed in triplicate. Cleavage of Peptide Hormones by sLaeverin/APQ—Peptide hormones (25 μm, Peptide Institute, Osaka, Japan) were incubated with the enzyme (2 μg/ml) at 37 °C in 25 mm Tris/HCl buffer (pH 7.0). The reaction was terminated by adding of 2.5% (v/v) formic acid. The reactants and products were separated on a reversed-phase column, COSMOSIL (4.6 × 250 mm, Nacalai Tesque) using an automated HPLC system (AT-10, Shimadzu, Kyoto, Japan). Peptides generated from angiotensins, kallidin, and endokinin C were isocratically eluted with the following buffers at a flow rate of 0.5 ml/min: for peptides from angiotensins or kallidin, 19% acetonitrile containing 0.086% trifluoroacetic acid; for peptides from endokinin C, 30% acetonitrile containing 0.084% trifluoroacetic acid. Peptides generated from dynorphin A1–8 were loaded onto the column equilibrated in 10% acetonitrile containing 0.088% trifluoroacetic acid and eluted with a linear gradient of 10% acetonitrile containing 0.088% trifluoroacetic acid to 30% acetonitrile containing 0.084% trifluoroacetic acid in 20 min at a flow rate of 0.5 ml/min, and peptides from kisspeptin-10 were eluted with a linear gradient of 20% acetonitrile containing 0.086% trifluoroacetic acid to 40% acetonitrile containing 0.082% trifluoroacetic acid in 20 min at a flow rate of 0.5 ml/min. The molecular masses of peptides were determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry with a REFLEX mass spectrometer (Bruker-Franzen Analytik) using α-cyano-4-hydroxycinnamic acid as the matrix. Gel Filtration Chromatography—The molecular mass of recombinant Laeverin/APQ was measured by gel filtration chromatography. The sample was loaded onto a TSK G-3000SW (7.5 × 300 mm) ODS column (TOSOH, Tokyo, Japan) and eluted with 50 mm Tris/HCl buffer (pH7.5) containing 100 mm Na2SO4 at flow rate of 0.5 ml/min. Materials—Asp-, Gln-, Glu-, Gly-, Ile-, Val-, and S-benzyl-Cys-MCAs were purchased from BACHEM AG (Bubendorf, Switzerland). Ala-, Arg-, Leu-, Lys-, Met-, and Phe-MCAs were from Peptide Institute (Osaka, Japan). Bestatin was obtained from Nacalai Tesque, and p-(4-amidinophenyl)methanesulfonyl fluoride was from Wako Pure Chemical Industries (Tokyo, Japan). Dynorphin A-(1–8), amastatin, and E64 were purchased from Sigma, and actinonin was from LKT laboratories (St. Paul, MN). Leupeptin, pepstatin, and all peptide hormones except for dynorphin A1–8 were from the Peptide Institute. Production of a Recombinant Human Laeverin/APQ—To examine the enzymatic properties of Laeverin/APQ in detail, we tried to produce a recombinant human Laeverin/APQ using a baculovirus expression system. To facilitate purification, we introduced the His6 tag at its C terminus as described previously in the production of recombinant human APA. Although it was predicted that full-length Laeverin/APQ would be expressed as a type II membrane-integral protein, the recombinant protein was mainly detected in culture medium as a secretory protein rather than in cell lysate of virus-infected Sf9 cells (data not shown). Consequently, we purified the soluble form of human Laeverin/APQ (sLaeverin/APQ) to homogeneity from the culture medium by serial chromatography on columns of hydroxyapatite and Ni2+-chelating Sepharose. From 3 liters of culture medium, ∼1.0 mg of sLaeverin/APQ was obtained. The purified sLaeverin/APQ gave a single band with a molecular mass of ∼120 kDa on SDS-PAGE under both reducing and non-reducing conditions (Fig. 1A). From N-terminal amino acid sequence analysis, a homogenous sequence of 10 amino acid residues (65KPTPTPKPSS74) was obtained, suggesting that the enzyme was proteolytically processed on the cell membrane during the preparation process. Because there are 15 potential N-glycosylation sites in the sequence of sLaeverin/APQ, we next examined whether it was N-glycosylated or not. As shown in Fig. 1B, purified sLaeverin/APQ was sensitive to peptide:N-glycosidase F, and the molecular weight of de-glycosylated sLaeverin/APQ shifted to 106 kDa, which was identical to the expected value, indicating that sLaeverin/APQ was N-glycosylated as expected. Because homodimer formation is a characteristic feature of the membrane-bound M1 family of aminopeptidases (23Matsumoto H. Rogi T. Yamashiro K. Kodama S. Tsuruoka N. Hattori A. Takio K. Mizutani S. Tsujimoto M. Eur. J. Biochem. 2000; 267: 46-52Crossref PubMed Scopus (102) Google Scholar, 24Goto Y. Hattori A. Ishii Y. Mizutani S. Tsujimoto M. J. Biol. Chem. 2006; 281: 23503-23513Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 25Danielsen E.M. Biochemistry. 1990; 29: 305-308Crossref PubMed Scopus (32) Google Scholar), we measured the molecular weight of sLaeverin/APQ by gelfiltration column chromatography. The purified enzyme was eluted between ferritin (440 kDa) and catalase (232 kDa) (data not shown). These results suggest that sLaeverin/APQ is a non-sulfide-linked homodimeric protein. Characterization of Aminopeptidase Activity of Laeverin/APQ—We then measured the relative hydrolytic activity of sLaeverin/APQ toward various synthetic substrates, aminoacyl-MCAs (Fig. 2). Among the substrates tested, Leu-MCA was hydrolyzed by sLaeverin/APQ most efficiently, followed by Met-, Arg-, and Lys-MCA. Although showing lower activity, the hydrolysis of Phe-, S-benzyl-Cys-, Ala-, and Gln-MCA by sLaeverin/APQ was also observed, indicating a broad substrate specificity of the enzyme. The optimal pH was determined to be 7.0 using Leu-MCA as a substrate (data not shown). We next performed kinetic studies on the aminopeptidase activities of sLaeverin/APQ toward several synthetic substrates (Table 1). The calculated Km, kcat, and kcat/Km values from a Lineweaver-Burk plot of sLaeverin/APQ for the most efficient substrate Leu-MCA were 78.2 ± 5.5 μm, 14.4 ± 0.6 s–1, and 184 ± 4.6 μm–1 s–1, respectively. The catalytic efficiency (kcat/Km) of sLaeverin/APQ for Met-MCA was ∼23% of the value for Leu-MCA due to a significantly lower kcat value. In contrast, the Km values of sLaeverin/APQ for Lys- and Arg-MCA (41.5 ± 5.1 μm and 34.9 ± 1.7 μm) indicated higher affinity to these substrates than Leu-MCA. However, because the turnover numbers of the enzyme for both basic substrates were much less than that for Leu-MCA, the hydrolytic efficiencies for these substrates were calculated to be less than that for Leu-MCA. A similar inversion phenomenon in the kinetics parameters between neutral residues and basic residues was observed in the kinetic parameters of APN, which shows the highest homology (∼36%) with Laeverin/APQ among M1 family members (26Turner A.J. Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 2nd. Ed. Academic Press Inc., San Diego, CA2004: 289-294Crossref Scopus (16) Google Scholar).TABLE 1Kinetic parameters of sLaeverin/APQ toward various aminoacyl-MCASubstrateKmaThe values are mean ± S.D. (n = 3).kcataThe values are mean ± S.D. (n = 3).kcat/Km × 103aThe values are mean ± S.D. (n = 3).μms-1μm-1·s-1Leu-MCA78.2 ± 5.514.4 ± 0.6184 ± 4.6Met-MCA77.0 ± 5.43.3 ± 0.242.4 ± 0.8Lys-MCA41.5 ± 5.12.7 ± 0.264.3 ± 3.6Arg-MCA34.9 ± 1.72.9 ± 0.183.6 ± 3.3a The values are mean ± S.D. (n = 3). Open table in a new tab We next examined the effects of various divalent cations on the aminopeptidase activity of sLaeverin/APQ (data not shown). We found that Zn2+, an effective inhibitor of several M1 aminopeptidases, suppressed the Leu-MCA hydrolytic activity effectively in a dose-dependent manner (IC50 = 6.6 ± 1.1 μm). Cu2+ (IC50 = 19.6 ± 3.6 μm), Co2+ (IC50 = 84.9 ± 12.2 μm), and Ni2+ (IC50 = 28.2 ± 3.7 μm) also showed moderate inhibitory effects on the enzymatic activity of sLaeverin/APQ, which is consistent with the other M1 aminopeptidases so far tested. In our previous work, Ca2+ was shown to modulate the enzymatic activity of APA (24Goto Y. Hattori A. Ishii Y. Mizutani S. Tsujimoto M. J. Biol. Chem. 2006; 281: 23503-23513Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). However, no significant effect of Ca2+ on the enzymatic activity of sLaeverin/APQ was observed up to 3 mm. The effects of various known protease inhibitors on the enzymatic activity of sLaeverin/APQ are shown in Fig. 3. It was obvious that bestatin, a competitive aminopeptidase inhibitor, was the most potent inhibitor of sLaeverin/APQ (Ki = 0.96 ± 0.33 μm). The specific APN inhibitors phebestin and actinonin (Ki = 3.02 ± 0.61 μm and 259 ± 30.7 μm, respectively) and the specific inhibitor of APA amastatin (Ki = 34.5 ± 10.0 μm) were less active. As in the case with other M1 aminopeptidases, 1,10-phenanthroline, a metal chelator, inhibited sLaeverin/APQ activity at 0.01–1 mm. Although leupeptin (a serine protease inhibitor) and pepstatin (an asparatic protease inhibitor) suppressed sLaeverin/APQ activity significantly only at high concentration (1 mm), E64 (a cysteine protease inhibitor) and p-(4-amidinophenyl)methanesulfonyl fluoride (a serine protease inhibitor) had only small inhibitory effects at 1 mm. Taken together, these data revealed that Laeverin/APQ is indeed a novel M1 aminopeptidase having enzymatic properties characteristic to the M1 family (7Tanioka T. Hattori A. Masuda S. Nomura Y. Nakayama H. Mizutani S. Tsujimoto M. J. Biol. Chem. 2003; 278: 32275-32283Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 13Hattori A. Kitatani K. Matsumoto H. Miyazawa S. Rogi T. Tsuruoka N. Mizutani S. Natori Y. Tsujimoto M. J. Biochem. (Tokyo). 2000; 128: 755-762Crossref PubMed Scopus (97) Google Scholar, 23Matsumoto H. Rogi T. Yamashiro K. Kodama S. Tsuruoka N. Hattori A. Takio K. Mizutani S. Tsujimoto M. Eur. J. Biochem. 2000; 267: 46-52Crossref PubMed Scopus (102) Google Scholar, 24Goto Y. Hattori A. Ishii Y. Mizutani S. Tsujimoto M. J. Biol. Chem. 2006; 281: 23503-23513Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Cleavage of Natural Peptides by Laeverin/APQ—We then searched for sLaeverin/APQ-mediated degradation of natural peptide hormones to estimate the physiological roles of the enzyme. To identify possible substrates, we examined the competition of 18 human natural peptides with Leu-MCA by measuring its hydrolysis (Table 2). As for angiotensins, which are often good substrates of M1 aminopeptidases, Ang III and IV inhibited the hydrolytic activity of sLaeverin/APQ efficiently, but Ang II was less effective. Bradykinin and substance P showed moderate inhibitory activities. Furthermore, strong inhibition of the sLaeverin/APQ-mediated Leu-MCA hydrolysis was observed by kallidin (Lys-bradykinin), endokinin C, dynorphin A1–8, and kisspeptin-10. In contrast, nine other peptides (Met-enkephalin, endokinin D, Arg-vasopressin, parathyroid hormone-(69–84), cholecystokinin-8, neurokinin A, neurokinin B, oxytocin, and delta sleep-inducing peptide) were less inhibitory at 100 μm ( 50% inhibition of the hydrolysis of the fluorogenic substrate at 100 μm in this assay could be assumed to be substrates of the enzyme. Therefore, we examined the cleavage of candidate peptides by detecting degraded peptides after separating on reverse-phase HPLC. As shown in Fig. 4A, although Ang II slightly inhibited the Leu-MCA hydrolytic activity of sLaeverin/APQ at 100 μm, no significant degradation of the hormone was observed up to 6 h, indicating that Ang II is not a substrate of Laeverin/APQ. On the other hand, sLaeverin/APQ-mediated cleavage of Ang III (Fig. 4B, peak a) to Ang IV (Fig. 4B, peak b) was clearly detected within 10 min (Fig. 4B). However, further degradation did not occur after longer incubation (6 h). Therefore, we examined whether or not Ang IV can be a substrate of sLaeverin/APQ. As expected from the Ang III hydrolysis profile, the degradation of Ang IV by sLaeverin/APQ was barely detectable (Fig. 4C), suggesting that Ang IV was not a substrate of the enzyme. Ang IV was shown to inhibit APA (24Goto Y. Hattori A. Ishii Y. Mizutani S. Tsujimoto M. J. Biol. Chem. 2006; 281: 23503-23513Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We therefore tested whether or not Ang IV is an inhibitor of Laeverin/APQ and found that it is a competitive inhibitor of Laeverin/APQ with a Ki value of 8.96 ± 1.07 μm (data not shown). Because sLaeverin/APQ showed a moderate preference for basic amino acid residues toward synthetic substrates (Fig. 2), we next examined the degradation of several candidate peptides having basic amino acids at the N terminus (i.e. kallidin (Lys-bradykinin), endokinin C, bradykinin, and substance P). As shown in Fig. 5A, kallidin was a good substrate, and release of the N-terminal lysine residue from kallidin (Lys-bradykinin) was detected within 3 min in this assay. Consequently, kallidin (Fig. 5A, peak a) was converted to bradykinin (Fig. 5A, peak b) completely within 30 min. As in the case with kallidin, rapid release of the N-terminal lysine residue from endokinin C and generation of de-[Lys]endokinin C (36%) (Fig. 5B, peak b) were also detected within 10 min. Of note, the transient accumulation of de-[Lys]endokinin C was observed at 30 min (Fig. 5B, peak b), and degradation to de-[Lys-Lys]endokinin C (Fig. 5B, peak c) seemed to be much slower than the first cleavage, suggesting that the second lysine residue is a poorer substrate of Laeverin/APQ than the first one. It is plausible that subsite interactions are required for the enzymatic action of Laeverin/APQ. It has been reported that M1 aminopeptidases are not able to release an N-terminal amino acid residue adjacent to a proline residue (7Tanioka T. Hattori A. Masuda S. Nomura Y. Nakayama H. Mizutani S. Tsujimoto M. J. Biol. Chem. 2003; 278: 32275-32283Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 13Hattori A. Kitatani K. Matsumoto H. Miyazawa S. Rogi T. Tsuruoka N. Mizutani S. Natori Y. Tsujimoto M. J. Biochem. (Tokyo). 2000; 128: 755-762Crossref PubMed Scopus (97) Google Scholar, 23Matsumoto H. Rogi T. Yamashiro K. Kodama S. Tsuruoka N. Hattori A. Takio K. Mizutani S. Tsujimoto M. Eur. J. Biochem. 2000; 267: 46-52Crossref PubMed Scopus (102) Google Scholar, 24Goto Y. Hattori A. Ishii Y. Mizutani S. Tsujimoto M. J. Biol. Chem. 2006; 281: 23503-23513Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Therefore we examined the susceptibilities of bradykinin and substance P, which both have Arg-Pro sequence at N termini to Laeverin/APQ. We found that, although both peptides bind to Laeverin/APQ, they were not cleaved by the enzyme at all and acted as competitive inhibitors (Ki = 57.8 ± 11.0 and 4.63 ± 0.49 μm, respectively) (data not shown). Because Laeverin/APQ also showed a moderate preference to Phe-MCA, we next examined the cleavage of kisspeptin-10 and dynorphin A1–8, of which the N-terminal amino acid residues are tyrosine (aromatic amino acid). Fig. 6A shows the hydrolytic profile of kisspeptin-10, which is abundant in the placenta and suppresses the cell motility strongly by binding to a G-protein-coupled receptor, GPR54 (27Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. Kitada C. Usuki S. Kurokawa T. Onda H. Nishimura O. Fujino M. Nature. 2001; 31: 613-617Crossref Scopus (1195) Google Scholar). Release of the N-terminal tyrosine residue of kisspeptin-10 was detected within 3 min, and ∼30% of kisspeptin-10 was degraded to de-[Tyr]kisspeptin-10 (
Referência(s)