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Reticulum Mainly Localized in the Endoplasmic Predominantly Human Cathepsin W, a Cysteine Protease Predominantly Expressed in NK Cells, Is Mainly Localized in the Endoplasmic Reticulum This information is current as of September 24, 2021. Thomas Wex, Frank Bühling, Heike Wex, Dagmar Günther, Peter Malfertheiner, Ekkehard Weber and Dieter Brömme J Immunol 2001; 167:2172-2178; ; doi: 10.4049/jimmunol.167.4.2172 http://www.jimmunol.org/content/167/4/2172 Downloaded from References This article cites 44 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/167/4/2172.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 24, 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Human Cathepsin W, a Cysteine Protease Predominantly Expressed in NK Cells, Is Mainly Localized in the Endoplasmic Reticulum1 Thomas Wex,2,3*† Frank Bu¨hling,2§ Heike Wex,*‡ Dagmar Gu¨nther,¶ Peter Malfertheiner,† Ekkehard Weber,¶ and Dieter Bro¨mme3* Human cathepsin W (also called lymphopain) is a recently described papain-like cysteine protease of unknown function whose gene expression was found to be restricted to cytotoxic cells. Here we demonstrate that cathepsin W is expressed predominantly in NK cells and, to a lesser extent, in CTLs. Quantitative RT-PCR revealed that NK cells contained ϳ21 times more cathepsin W transcript than CTLs. The predominant expression of cathepsin W in NK cells was further confirmed by Western blot analysis and immunohistochemistry. IL-2-mediated stimulation of NK cells and CTLs revealed a stronger up-regulation of the cathepsin Downloaded from W gene and protein expression in NK cells (7-fold) than in CTLs (2-fold). Transfection experiments of HeLa cells and biochemical analyses revealed that cathepsin W is exclusively “high mannose-type” glycosylated and is mainly targeted to the endoplasmic reticulum (ER). Interestingly, the ER localization of cathepsin W was also found in NK cells, in which colocalization studies revealed an overlapping staining of cathepsin W and Con A, an ER-specific lectin. Furthermore, subcellular fractionation of cathepsin W-expressing cells confirmed the ER localization and showed that cathepsin W is membrane associated. Based on the results of this study, cathepsin W might represent a putative component of the ER-resident proteolytic machinery. The constitutive http://www.jimmunol.org/ expression in NK cells and the stronger up-regulation of cathepsin W by IL-2 in NK cells than CTLs suggest that cathepsin W is not just a marker of cytotoxic cells but is, rather, specifically expressed in NK cells. The Journal of Immunology, 2001, 167: 2172–2178. atural killer cells and CD8ϩ CTLs represent two major found in both NK cells and CTLs. Here, binding of a target cell by cell populations involved in the initial immune response a CTL or NK cell stimulates a Ca2ϩ-dependent degranulation pro- N against tumors, viral infections, and allografts (1–3). cess in the effector cell that leads to a release of a “death cocktail,” However, despite the common ability of NK cells and CTLs to lyse causing the subsequent lysis of target cells (5, 8). by guest on September 24, 2021 target cells, there are differences between both cell types. Whereas The most prominent components of this death mixture are per- NK cells exist as preactivated cytotoxic cells capable of mediating forin and a family of serine proteases termed granzymes, which are their effector function without depending on presented antigenic both known to be specifically expressed in CTLs and NK cells (9). peptides (MHC class I independent) (2, 3), CTLs mostly have to be Besides the granzymes, there are also cysteine proteases involved activated by Ag-derived peptides bound to the MHC class I com- in the secretory pathway. Dipeptidyl peptidase I (synonymous with plex, and therefore, they are considered to act MHC class I de- cathepsin C) was shown to be the only enzyme capable of pro- pendently (3, 4). In general, two different cytotoxic pathways are cessing progranzymes into their active forms by removing a Glu- known (reviewed in Ref. 5). The first, the perforin-independent Glu dipeptide from the proforms (10, 11). Furthermore, the mem- 2ϩ lytic pathway, occurs in Ca -free conditions and was found pre- bers of the caspase family mediate the downstream events that dominantly in CTLs. This way is triggered through cell surface finally lead to the death of the target cell (12). Interestingly, some receptors such as Fas/Apo-1 (CD95) and TNFR (CD97) (6, 7). The other cysteine protease-related proteins such as the CTL Ag 2-␤ second, the secretory mechanism, is perforin dependent and was (13), the cysteine protease inhibitor leukocystatin (14), and cathep- sin W (15) were found to be predominantly expressed in cytotoxic *Department of Human Genetics, Mount Sinai School of Medicine, New York, NY cells, suggesting specific functions of these proteins. The potential 10029; Departments of †Gastroenterology, Hepatology, and Infectious Diseases and involvement of other cysteine proteases in cytotoxic processes is ‡Pediatric Hematology and Oncology, and §Institute of Immunology, Otto-von-Gu- ericke University, Magdeburg, Germany; and ¶Medical Faculty, Institute of Physio- further supported by data showing the blocking of TCR-induced logical Chemistry, Martin Luther University Halle-Wittenberg, Wittenberg, Germany death of T cells by papain-like-specific protease inhibitors (16). Received for publication February 29, 2000. Accepted for publication June 7, 2001. Recently, the cDNA encoding a novel papain-like cysteine pro- The costs of publication of this article were defrayed in part by the payment of page tease, designated cathepsin W (also referred to as lymphopain), charges. This article must therefore be hereby marked advertisement in accordance was cloned and further characterized by our group and others (15, with 18 U.S.C. Section 1734 solely to indicate this fact. 17). Protein sequence alignments showed that this enzyme is a 1 This work was supported by a fellowship (We/2170/2-1) and in part by the SFB 387 (project B7) of the Deutsche Forschungsgemeinschaft (Germany). member of the papain superfamily, and that all major structural features such as the positions of the amino acids forming the cat- 2 T.W. and F.B. contributed equally to this work. alytic triad, the putative cleavage sites for the signal sequence, and 3 Address correspondence and reprint requests to Dr. Thomas Wex, Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke Univer- the propeptide are in accordance with other members of this pro- sity, Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany. E-mail address: tease family (15). The in vivo gene expression of cathepsin W [email protected] or Dr. Dieter Bro¨mme, Department of Hu- man Genetics, Mount Sinai School of Medicine, Box 1498, Fifth Avenue at 100th studied by Northern blot analyses revealed a high abundance of the Street, New York, NY 10029. E-mail address: [email protected] corresponding mRNA in peripheral cytotoxic cells and related cell Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 2173 ϩ lines only, whereas other hematopoetic cells such as CD4 T cells, Triton X-100 (three times for 20 min), the cells were treated with anti- B cells (CD19ϩ), and monocytes (CD14ϩ) did not contain signif- mouse IgG-FITC or anti-mouse IgG-tetramethylrhodamine isothiocyanate icant amounts of cathepsin W mRNA (15, 17). (TRITC), which were both obtained from Sigma (St. Louis, MO) and used as recommended by the manufacturer. After five washing steps with PBS/T In this study, we describe the cellular and subcellular localiza- containing 0.05% Triton X-100, cells were mounted using Fluoromount-G tion and regulation of endogenous cathepsin W, which is predom- (Southern Biotechnology Associates, Birmingham, AL) and viewed with a inantly expressed in NK cells and was found to be up-regulated by fluorescence microscope (Eclipse E-800; Nikon, Melville, NY). IL-2. Furthermore, we provided evidence that endogenous cathep- sin W might be mainly localized in the endoplasmic reticulum Electrophoresis and Western blot analysis 4 (ER), a novel compartment for papain-like proteases. In general, SDS-PAGE was conducted by following the method of Lae- mmli (20). Briefly, samples were heated in reducing sample buffer at 95°C Materials and Methods for 5 min and subjected to SDS-PAGE (Fisher Scientific). Electrophoresis Expression of recombinant cathepsin W was performed at 50 V for 30 min and 120 V for another 1.5 h using the Mini Gel system (Bio-Rad, Hercules, CA). Prestained protein benchmarker Previously cloned cathepsin W cDNA (15) was used as a template for PCR (Life Technologies) was used as the molecular mass standard. Proteins amplifications with Pfu polymerase (Stratagene, La Jolla, CA) using the were electroblotted onto nitrocellulose membrane (Fisher Scientific) at 35 following primers that contained either a HindIII or XbaI restriction site for Vfor3h(4°C) using the buffer system that contained 25 mM Tris, 192 cloning (primer 1, 5Ј-AAA AAG CTT ACC GGC ATG GCA CTG ACT mM glycine, and 20% (v/v) methanol.
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