Human Cathepsin F. Molecular Cloning, Functional Expression, Tissue Localization, and Enzymatic Characterization

Human Cathepsin F. Molecular Cloning, Functional Expression, Tissue Localization, and Enzymatic Characterization The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Wang, Bruce, Guo-Ping Shi, Pin Mei Yao, Zhenqiang Li, Harold A. Chapman, and Dieter Brömme. 1998. “Human Cathepsin F: MOLECULAR CLONING, FUNCTIONAL EXPRESSION, TISSUE LOCALIZATION, AND ENZYMATIC CHARACTERIZATION.” Journal of Biological Chemistry 273 (48): 32000–8. doi:10.1074/ jbc.273.48.32000. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41543161 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 48, Issue of November 27, pp. 32000–32008, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Human Cathepsin F MOLECULAR CLONING, FUNCTIONAL EXPRESSION, TISSUE LOCALIZATION, AND ENZYMATIC CHARACTERIZATION* (Received for publication, May 19, 1998, and in revised form, September 9, 1998) Bruce Wang‡§, Guo-Ping Shi§¶, Pin Mei Yao, Zhenqiang Li, Harold A. Chapman¶i, and Dieter Bro¨mmei From the Department of Human Genetics, Mount Sinai School of Medicine, CUNY, New York, New York 10029, the ¶Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, and ‡Incyte Pharmaceuticals, Palo Alto, California 94080 A cDNA for a novel human papain-like cysteine prote- pressed, intracellular housekeeping proteases responsible for ase, designated cathepsin F, has been cloned from a the general lysosomal protein breakdown. The cathepsins L, B, lgt10-skeletal muscle cDNA library. The nucleotide se- H, and probably O (1, 2) belong to this group. The other group quence encoded a polypeptide of 302 amino acids com- is characterized by a tissue restricted expression pattern and posed of an 88-residue propeptide and a 214-residue ma- by the assignment of specific functions correlated with their ture protein. Protein sequence comparisons revealed tissue distribution. For example, cathepsin S, the first known 58% homology with cathepsin W; about 42–43% with tissue-specific cysteine protease is primarily expressed in lym- Downloaded from cathepsins L, K, S, H, and O; and 38% with cathepsin B. phatic tissues (3, 4) and is responsible for the specific degradation Sequence comparisons of the propeptides indicated that of the invariant chain of MHC class II complexes in antigen- cathepsin F and cathepsin W may form a new cathepsin presenting cells (5, 6). Cathepsin K, which is predominantly subgroup. Northern blot analysis showed high expres- expressed in osteoclasts, is a major protease in bone resorption sion levels in heart, skeletal muscle, brain, testis, and (7–9), and, recently, cathepsin W as a cytotoxic lymphocyte- ovary; moderate levels in prostate, placenta, liver, and http://www.jbc.org/ specific protease has been reported (10). colon; and no detectable expression in peripheral leuko- cytes and thymus. The precursor polypeptide of human All presently known thiol-dependent cathepsins share com- recombinant cathepsin F, produced in Pichia pastoris, mon protein structures with a signal sequence of 16–18 amino was processed to its active mature form autocatalyti- acids, followed by a propeptide of 62–100 residues and then a cally or by incubation with pepsin. Mature cathepsin F catalytically active mature region of about 220–230 amino acids (1). The signal sequence, containing stretches of hydro- was highly active with comparable specific activities by guest on October 14, 2019 toward synthetic substrates as reported for cathepsin L. phobic amino acids, facilitates the targeting of these proteins The protease had a broad pH optimum between 5.2 and into secretory pathways via the endoplasmic reticulum (11). 6.8. Similar to cathepsin L, its pH stability at cytosolic The propart is involved in the folding of the precursor protein, pH (7.2) was short, with a half-life of approximately 2 in the temporary inhibition of the protease in its precursor min. This may suggest a function in an acidic cellular form, and in transport of the proenzyme to the endosomal/ compartment. Transient expression of T7-tagged ca- lysosomal compartment using mannose 6-phosphate N-glyco- thepsin F in COS-7 cells revealed a vesicular distribution sylation sites (12, 13). Finally, the mature, catalytically active, of the gene product in the juxtanuclear region of the cells. enzyme contains the catalytic triad consisting of Cys-25, His- However, contrary to all known cathepsins, the open 159, and Asn-175 (papain numbering) and is folded into a reading frame of the cathepsin F cDNA did not encode a two-domain structure (14). In addition, thiol-dependent cathe- signal sequence, thus suggesting that the protease is tar- psins have been characterized as lysosomal enzymes, since geted to the lysosomal compartment via an N-terminal they have signal sequences and potential N-glycosylation sites signal peptide-independent lysosomal targeting pathway. and generally have pH optima in the acidic pH range. In this report, we describe a newly identified member of the papain family, designated cathepsin F, which is ubiquitously Cathepsins of the papain family can be divided into two expressed in human tissues but which in contrast to the known functional groups. One group comprises ubiquitously ex- cathepsins (L, B, S, H, K, and W) lacks a signal sequence. EXPERIMENTAL PROCEDURES * This research was supported in part by National Institutes of Health Grants AR 39191, AR 41331, and HL44712. In addition, this Cloning of Human Cathepsin F cDNA—In an effort to identify novel research was supported by Axys Pharmaceuticals (South San Fran- cysteine proteases, degenerate oligonucleotides, designed to regions cisco, CA). The costs of publication of this article were defrayed in part around the characteristic active site residues for cysteine proteases, by the payment of page charges. This article must therefore be hereby C-25 and N-175 (sense primer, 59-tg(t/c) tgg gct tt(t/c) ag(t/c)-39; anti- marked “advertisement” in accordance with 18 U.S.C. Section 1734 sense primer, 59-(c/t/a/g)cc cca gct gtt (c/t)tt-39) were used to amplify solely to indicate this fact. cDNAs from alveolar lung macrophage transcribed mRNAs (15). The The nucleotide sequence(s) reported in this paper has been submitted resulting PCR1 products (approximately 500 bp) were subcloned into TM to the GenBank /EBI Data Bank with accession number(s) AF071748 the vector pCR®II (Invitrogen, San Diego, CA). 150 colonies were con- and AF071749. § These two authors contributed equally to this work. i To whom correspondence should be addressed: Dept. of Human 1 The abbreviations used are: PCR, polymerase chain reaction; bp, Genetics, Mount Sinai School of Medicine, Box 1498, Fifth Avenue at base pair(s); E-64, L-3-carboxytrans-2,3-epoxypropionyl-leucylamido-(4- 100th St., New York, NY 10029. Tel.: 212-824-7540; Fax: 212-849-2508; guanidino)butane; Z-, benzyloxycarbonyl; -MCA, 4-methyl-7-coumaryl- E-mail: [email protected] (for D. Bro¨mme) or Brigham and amide; RACE, rapid amplification of cDNA ends; PBS, phosphate-buff- Woman’s Hospital and Harvard Medical School, Boston, MA 02115. ered saline; DMEM, Dulbecco’s modified Eagle’s medium; TBS, Tris- E-mail: [email protected] (for H. A. Chapman). buffered saline. 32000 This paper is available on line at http://www.jbc.org Human Cathepsin F 32001 firmed for insert with EcoRI digestion and grouped with diagnostic Construction of a pcDNA3.1-based Expression Vector for Cathepsin F digestion using HinfI and DdeI, respectively. 30 colonies were selected and Expression in COS-7 Cells—The cDNA encoding the open reading for DNA sequence analysis based on the diagnostic digestions. In addi- frame of cathepsin F starting with Met-2 was amplified by PCR using tion to cathepsins S, K, L, and H, one novel sequence containing 323 bp pCTSF1 as template, Pfu polymerase (Stratagene, La Jolla, CA), and was found based on the alignment with other known cysteine proteases. the following oligonucleotide primers: sense, 59-cct aag ctt tca gcc atg att DNA sequence analysis demonstrated that this novel sequence con- tct tct ctg tcc caa aac c-39 containing a HindIII site; antisense containing tained the original antisense primer sequence but not the sense primer. a T7 Tag (underlined) and a XbaI site, 59-aa tct aga gct cat ccc atc tgc tgt The 323-bp fragment was amplified and cloned into the vector pCR®II cct cca gtc ata ctg gcc atg tcc acc acc gcc gag ctg g-39. The obtained PCR (Invitrogen) and labeled with [a-32P]dCTP (300 Ci/mmol; NEN Life product was digested with XbaI and HindIII, gel-purified using Gene- Science Products) using a random prime DNA labeling system (Boeh- Clean (Bio 101, Inc., Vista, CA), and subcloned into the expression ringer Mannheim). The labeled insert was used to screen 700,000 clones vector pcDNA 3.1 (Invitrogen, Carlsbad, CA). The insert and the flank- of a lgt10 skeletal muscle cDNA library (1.5 3 106 independent clones; ing regions of the resulting vector, pcDNA 3.1 CF-T7, were sequenced in CLONTECH, Palo Alto, CA) as described (16), and four positive clones both directions. were isolated. Purified phage DNA was isolated using the Trap Plus Green monkey COS-7 cells were maintained in Dulbucco’s modified system (CLONTECH), and cDNA inserts were cloned into the EcoRI Eagle’s medium supplemented with 20 mM glutamine, 100 units of site of plasmid pBluescript SKII1 phagemid (Stratagene, La Jolla, CA). penicillin, 100 units of streptomycin (all from Fisher), and 10% heat- The cDNA clones isolated were not full-length and encoded overlapping inactivated fetal bovine serum (Gemini Bio-Products, Calabasas, CA) at 9 regions of the mature protein.

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