Characterization of Mouse Carboxypeptidase N Small Active Subunit Gene Structure Kirstin W. Matthews and Rick A. Wetsel This information is current as J Immunol 2001; 166:6196-6202; ; of September 25, 2021. doi: 10.4049/jimmunol.166.10.6196 http://www.jimmunol.org/content/166/10/6196 Downloaded from References This article cites 26 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/166/10/6196.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on September 25, 2021 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Characterization of Mouse Carboxypeptidase N Small Active Subunit Gene Structure1 Kirstin W. Matthews* and Rick A. Wetsel2*† Carboxypeptidase N (CPN) is a plasma zinc metalloprotease comprised of two small subunits that have enzymatic activity, and two large subunits, which protect the enzyme from degradation. CPN cleaves the carboxyl-terminal amino acids arginine and lysine from biologically active peptides such as complement anaphylatoxins, kinins, and fibrinopeptides. To delineate the murine CPN small subunit coding region, gene structure, and chromosome location, cDNA and genomic clones were isolated, character- ized, and used in Northern and fluorescence in situ hybridization analyses. The results from this study demonstrate that the murine CPN small subunit gene is a single copy gene of ϳ29 kb that is transcribed in the liver into a 1793-bp mRNA with an open reading frame of 1371 nucleotides encoding 457 aa. The gene contains nine exons ranging in size from 455 bp (exon 1) to 100 bp (exon 7), and eight introns ranging in size from 6.2 kb (intron 2) to 1.4 kb (intron 4). All intron/exon junctions follow the normal consensus Downloaded from rule. The mouse CPN small subunit gene localized to chromosomal band 19D2, which is syntenic to human chromosome 10q23–25. Primer extension experiments using mouse liver mRNA indicate one major transcriptional initiation site and three minor sites. ,Sequence analysis of the 5؅-flanking region indicated a TATA-less promoter and numerous transcription factor binding sites which may confer liver-specific expression of the CPN small subunit gene. The Journal of Immunology, 2001, 166: 6196–6202. 3 arboxypeptidase N (CPN) is a zinc metalloprotease with CPN activity levels 21% of normal (9, 10). This individual http://www.jimmunol.org/ comprised of two small subunits (Mr 50,000 each) and exhibited chronic recurring angioedema characterized by swelling two large subunits (M 83,000 each) (1). The small sub- of the face and tongue and by red swellings on the extremities C r units contain the enzymatic activity for the protease, while the lasting ϳ24 h. The severe phenotype of a partial deficiency indi- large heavily glycosylated subunits protect the enzyme from being cates that CPN has an important role in vivo. degraded or filtrated from the bloodstream. These subunits form a The cDNA encoding the human CPN small and large subunits tetramer and are held together by noncovalent interactions. have been cloned (11, 12). Furthermore, the human large subunit CPN is produced by the liver (2) and, once secreted into the was localized to human chromosome 8p22–23, and the small sub- blood, it can cleave carboxyl-terminal arginine or lysine residues unit gene was mapped to chromosome 10 (13). In the current from biologically potent peptides released into the bloodstream, study, the cDNA and gene structure of mouse CPN small subunit by guest on September 25, 2021 such as kinins (3), kallidin (4), fibrinopeptides (5), and other sub- were identified. The CPN cDNA was isolated and found to encode strates (2). CPN also cleaves the carboxyl-terminal arginine from a protein 79% identical in amino acid sequence to the human CPN the complement anaphylatoxins C3a and C5a (3) (6). C3a and C5a small subunit. By Northern analysis, the gene expression was tis- are peptides generated from the activation of the complement cas- sue specific and detected only in the liver. The gene encoding for cade and can induce smooth muscle contraction, vasodilation, che- the murine CPN small subunit was 29 kb in length with nine exons motaxis of leukocytes, and the release of histamine from mast cells and located on mouse chromosome 19 band position D2, which is (7, 8). By removing the carboxyl-terminal arginine from the com- syntenic with the human CPN small subunit gene. plement anaphylatoxins, CPN greatly reduces C5a and C3a bio- logical activities (8). Materials and Methods Currently, no known individual with a complete CPN deficiency Materials and reagents has been described. However, an individual has been identified Restriction enzymes and other molecular biology reagents were purchased from Roche Molecular Biochemicals (Indianapolis, IN) and used according ϩ *University of Texas-Houston Institute of Molecular Medicine for the Prevention of to manufacturer’s recommendations. Hybond N nylon membranes and 32 35 Human Diseases, and †Department of Biochemistry and Molecular Biology, Univer- radionucleotides [␣- P]dCTP and [ S]dATP were purchased from Am- sity of Texas-Houston Medical School, Houston, TX 77030 ersham (Arlington Heights, IL). [␥-32P]dATP was purchased from ICN Received for publication December 12, 2000. Accepted for publication March (Costa Mesa, CA). 5, 2001. Cloning of mouse CPN small subunit cDNA The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Two expression tag clones were identified from American Type Culture with 18 U.S.C. Section 1734 solely to indicate this fact. Collection (ATCC, Manassas, VA), 261J and 26K. ATCC-261J was 79% Ј 1 This work was supported by National Institutes of Health Grant AI25011 and by identical with the 3 end of human CPN small subunit cDNA and was American Heart Association National Grant-in-Aid 9950394 (both to R.A.W.). The purchased from ATCC. ATCC-26K was 75% identical with the 5Ј end of sequence data presented in this article have been submitted to the EMBL/GenBank human CPN small subunit. Although a portion of the 26-K sequence has Data Libraries under the accession numbers AF326477 and AF326478. been determined, the clone was lost at ATCC and could not be obtained for 2 Address correspondence and reprint requests to Dr. Rick A. Wetsel, Institute of further study. To obtain the entire sequence of the murine CPN small Molecular Medicine for the Prevention of Human Disease, University of Texas-Hous- subunit cDNA, a mouse B10.D2/nSnJ liver cDNA library was screened ton, 2121 West Holcombe Boulevard, Suite 907, Houston, TX 77030. E-mail address: (14). Approximately 500,000 plaques were plated, and duplicate filters [email protected] were screened using a random primed 32P-labeled ATCC-261J (used ac- 3 Abbreviations used in this paper: CPN, carboxypeptidase N; C/EBP, CCAAT/en- cording to manufacturer instructions; Roche) as a probe. Filters were hy- hancer binding protein; HNF-5, hepatocyte nuclear factor 5; TIS, transcription initi- bridized at 65°C in a 5ϫ SSC, 10ϫ Denhardt’s, and 1% SDS solution at ation site. pH 7.4. After 16 h, the filters were washed at 65°C in 0.2ϫ SSC and 1% Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 6197 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 1. Mouse CPN small subunit cDNA sequence. The cloning strategy for CPN with two ATCC clones (26K and 261J) and a cDNA library clone (pCPN.103K) (top). The nucleotide and translated amino acid sequences were determined from the cDNA clones, as described in the text (bottom). The coding region is depicted in capital letters with the initiating methionine defined as ϩ1. The 5Ј- and 3Ј-untranslated sequence are depicted in lower case. The 5Ј-untranslated sequence begins at the TIS, as determined by primer extension experiments. The resulting cDNA has 232 bp of 5Ј-untranslated, 1371-bp coding region, and 190-bp 3Ј untranslated. The CPN small subunit exons are denoted with slash marks at exon splice sites. The putative polyadenylation site (attaaa) is marked in bold. This sequence has been submitted to the GenBank/EMBL Data Bank with accession number AF326477. SDS and exposed to autoradiography film. Approximately 100 clones that For Southern analysis, ϳ25 ␮g of genomic DNA from 129SvJ mice was hybridized to the CPN probe were found after two additional rounds of digested with specified enzymes (see Results). The DNA was electropho- screening. Ten clones were plaque purified and sequenced on both strands. resed on a 0.8% agarose gel with radiolabeled molecular mass markers and then transferred to a nitrocellulose filter. The blots were hybridized in Northern blot and genomic Southern analysis modified Church and Gilbert buffer (500 mM sodium phosphate buffer (pH For Northern analysis, a blot containing 2 ␮g of mRNA from various 7.2), 10 mM EDTA, 7% SDS, and 1% BSA) individually with two probes. mouse tissues was obtained from Clontech (Palo Alto, CA).