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Carboxypeptidase N Is Not Protein in the Mouse, Whereas Pro

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Carboxypeptidase N Is Not Protein in the Mouse, Whereas Pro Pro-Carboxypeptidase R is an Acute Phase Protein in the Mouse, Whereas Carboxypeptidase N Is Not This information is current as Tomoo Sato, Takashi Miwa, Hiroyasu Akatsu, Noriyuki of September 23, 2021. Matsukawa, Kyoko Obata, Noriko Okada, William Campbell and Hidechika Okada J Immunol 2000; 165:1053-1058; ; doi: 10.4049/jimmunol.165.2.1053 http://www.jimmunol.org/content/165/2/1053 Downloaded from References This article cites 26 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/165/2/1053.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Pro-Carboxypeptidase R is an Acute Phase Protein in the Mouse, Whereas Carboxypeptidase N Is Not1,2 Tomoo Sato,*† Takashi Miwa,*† Hiroyasu Akatsu,† Noriyuki Matsukawa,† Kyoko Obata,* Noriko Okada,* William Campbell,† and Hidechika Okada3* Carboxypeptidase R (EC 3.4.17.20; CPR) and carboxypeptidase N (EC 3.4.17.3; CPN) cleave carboxyl-terminal arginine and lysine residues from biologically active peptides such as kinins and anaphylatoxins, resulting in regulation of their biological activity. Human proCPR, also known as thrombin-activatable fibrinolysis inhibitor, plasma pro-carboxypeptidase B, and pro- carboxypeptidase U, is a plasma zymogen activated during coagulation. CPN, however, previously termed kininase I and ana- phylatoxin inactivator, is present in a stable active form in plasma. We report here the isolation of mouse proCPR and CPN cDNA clones that can induce their respective enzymatic activities in culture supernatants of transiently transfected cells. Potato car- boxypeptidase inhibitor can inhibit carboxypeptidase activity in culture medium of mouse proCPR-transfected cells. The expres- Downloaded from sion of proCPR mRNA in murine liver is greatly enhanced following LPS injection, whereas CPN mRNA expression remains unaffected. Furthermore, the CPR activity in plasma increased 2-fold at 24 h after LPS treatment. Therefore, proCPR can be considered a type of acute phase protein, whereas CPN is not. An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators generated at the site of Gram-negative bacterial infection and may consequently prevent septic shock. In view of the ability of proCPR to also inhibit fibrinolysis, an excess of proCPR induced by LPS may contribute to hypofibrinolysis in patients suffering from disseminated intravascular coagulation caused by sepsis. The Journal of Immunology, 2000, 165: http://www.jimmunol.org/ 1053–1058. t has been well established that human carboxypeptidase N sin-like enzymes such as thrombin (11), thrombin/thrombomodu- (CPN),4 which consists of two small active subunits and two lin complex (14), and plasmin (11), thereby acquiring enzymatic I large glycosylated subunits, is an important inactivator of activity by which it cleaves carboxyl-terminal arginine and lysine several potent peptides, including anaphylatoxins, kinins, and fi- residues from various peptides. This activity can be completely brinopeptides (1–4). CPN is present in the active form in plasma inhibited by potato carboxypeptidase inhibitor (PCI) at concentra- (5, 6). Mathews et al. (7) has reported familial partial carboxypep- tions at which CPN is not affected (15). Because the activity of by guest on September 23, 2021 tidase N deficiency resulting in angioedema/chronic urticaria, hay CPR is similar to that of CPN, this enzyme has been implicated in fever/asthma, or both. Over the last decade, a new basic car- the inactivation of several natural potent peptides, including ana- boxypeptidase that is generated from its zymogen during coagu- phylatoxins and bradykinin, as well as in the removal of arginine lation was identified independently by a number of groups (8–12). and lysine residues from partially plasmin-degraded fibrin (8, 15, We designated this enzyme carboxypeptidase R (CPR) and its zy- 16). To elucidate the in vivo roles of proCPR and CPN, the es- mogen, proCPR. The activated form appeared to remove arginine tablishment of an animal model is essential. For this purpose we residues (R) more rapidly than lysine residues (K) at the carboxyl identified cDNAs of mouse proCPR and CPN and studied the ef- terminus of substrates compared with CPN (5, 11, 13). It has also fects of LPS on the expression of these enzymes during the in- been termed plasma carboxypeptidase B (11), carboxypeptidase U flammatory process. (CPU) (8), and thrombin-activatable fibrinolysis inhibitor (TAFI) (12). It is known that human proCPR can be hydrolyzed by tryp- Materials and Methods Probes for screening a cDNA library and Northern blot analysis *Department of Molecular Biology, Nagoya City University Medical School, We obtained two partial mouse sequences similar to that of human plasma Nagoya, Japan; and †Choju Medical Institute, Fukushimura Hospital, Toyohashi, pro-carboxypeptidase B (proCPR) using Entrez (http://www.ncbi.nlm.nih- Japan .gov/Entrez/) from the National Center of Biotechnology Information. The GenBank accession numbers are AA244760 and AA254734. In the same Received for publication December 6, 1999. Accepted for publication May 1, 2000. manner we obtained data on three partial mouse sequences from clones The costs of publication of this article were defrayed in part by the payment of page similar to the human small subunit of CPN. The numbers for these charges. This article must therefore be hereby marked advertisement in accordance sequences are AA238838, AA285540, and AA396985. The sets of primers with 18 U.S.C. Section 1734 solely to indicate this fact. generated based on the above were: mCPR86ϩ,5Ј-CTGCTCTTCCAA 1 This work was supported by a research grant from the Organization for Pharma- GAACCTCC-3Ј; mCPR292Ϫ,5Ј-CCACGTTGTTCATCAGAACG-3Ј; ceutical Safety and Research. mCPR533ϩ,5Ј-GAATCCATGCCAGAGAATGG-3Ј; mCPR740Ϫ,5Ј- ϩ 2 The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/ TTGTTCTTGTGAGCAGAGCG-3Ј; mCPN108 ,5Ј-CAAGGTGCA Ϫ GenBank nucleotide sequence databases under accession numbers AB021968 and CAACCAATGC-3Ј; mCPN510 ,5Ј-GAAGTAGGTGTTGAGATCCGG-3Ј; AB021969. mCPN609ϩ,5Ј-GATTCGCTCCTTGAACTTCG-3Ј; and mCPN1118Ϫ,5Ј- Ј 3 Address correspondence and reprint requests to Dr. H. Okada, Department of Mo- GTTCACCTGAAGTGACGTCG-3 . RT-PCR was performed with these sets lecular Biology, Nagoya City University Medical School, Mizuho-cho, Nagoya 467- of primers using mouse liver total RNA. PCR products were subcloned with 8601, Japan. E-mail address: [email protected] a TA cloning kit (Invitrogen, San Diego, CA). The presence of these inserts 4 Abbreviations used in this paper: CPN, carboxypeptidase N; mCPN, mouse CPN; was confirmed by sequencing. Clones were digested with several restric- CPR, carboxypeptidase R; TAFI, thrombin-activatable fibrinolysis inhibitor; CP, car- tion enzymes followed by electrophoresis on a 1.0% agarose gel. The in- boxypeptidase; EST, expressed sequence tags; TM, thrombomodulin; DIC, dissemi- serts were purified using a Prep-A-Gene kit (Bio-Rad, Hercules, CA) and nated intravascular coagulation; PCI, potato carboxypeptidase inhibitor. the probes were labeled CPR1, CPR2, CPN1, and CPN2, respectively. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 1054 MOUSE proCPR IS AN ACUTE PHASE PROTEIN Molecular cloning and base sequence analysis (Bio-Rad). The mCPN BamHI fragment was also subcloned into BamHI sites of pDR2EF1, and this form was designated m-CPN/pDR2EF1. The Dr. Mayumi Nonaka of our laboratory provided the cDNA library, which ϩ presence and fidelity of m-proCPR/pDR2EF1 and m-CPN/pDR2EF1 were was constructed using the ZAP II vector with poly(A) RNA isolated from confirmed by restriction enzyme digestion and sequencing, respectively. adult BALB/c mouse liver. Recombinants (1 ϫ 106) were plated at 50,000 plaques/137-mm plate. Screening was conducted under stringent condi- Transient expression of the cloned cDNAs in COS cells tions by the plaque hybridization method using each of the [␣-32P]dCTP- labeled probes (CPR1, CPR2, CPN1, and CPN2) described above. Hybrid- COS-7 cells derived from the kidney of an African green monkey were 2 ization was performed at 65°C for 16 h in a buffer containing 50 mM Tris cultured in 25-cm flasks in DMEM containing 10% FBS, 2 mM L-glu- (pH 7.4), 10ϫ Denhardt’s solution, 1 M NaCl, 10 mM EDTA, 0.1% SDS, tamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 ␮g/ml streptomy- and 0.1 mg/ml denatured salmon sperm DNA (Wako, Osaka, Japan). Fil- cin, and 1 ␮g/ml fungizone in a 37°C humidified incubator (95% air/5% ϫ ters were washed twice with 2 SSC (0.3 M NaCl and 30 mM sodium CO2 atmosphere). The m-proCPR/pDR2EF1 and m-CPN/pDR2EF1 ex- citrate, pH 7.0) containing 0.1% SDS at 65°C for 30 min and once with pression vectors were transfected into COS-7 cells using Lipofectamine 0.2ϫ SSC containing 0.1% SDS at room temperature for 60 min. The dried reagent (Life Technologies) in a 24-well culture plate. When the cells were filters were then exposed for 16 h at Ϫ80°C to x-ray films (X-OMAT AR, 60–80% confluent, the conditioned medium in each well was replaced Eastman Kodak, Rochester, NY). The pBluescript phagemids were excised with 300 ␮l of Opti-MEM I Reduced Serum Medium (Life Technologies) from positive clones using R408 helper phage (Stratagene, La Jolla, CA).
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