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

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Citation Wang, Bruce, Guo-Ping Shi, Pin Mei Yao, Zhenqiang Li, Harold A. Chapman, and Dieter Brömme. 1998. “Human : 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¶ʈ, and Dieter Bro¨mmeʈ 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 -like cysteine prote- pressed, intracellular housekeeping responsible for ase, designated cathepsin F, has been cloned from a the general lysosomal breakdown. The L, B, ␭gt10-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, , the first known 58% homology with cathepsin W; about 42–43% with tissue-specific cysteine is primarily expressed in lym- Downloaded from cathepsins L, K, S, H, and O; and 38% with . 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). , 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 . phobic amino acids, facilitates the targeting of these 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 product in the juxtanuclear region of the cells. contains the 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 , 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 residues for cysteine proteases, by the payment of page charges. This article must therefore be hereby C-25 and N-175 (sense primer, 5Ј-tg(t/c) tgg gct tt(t/c) ag(t/c)-3Ј; anti- marked “advertisement” in accordance with 18 U.S.C. Section 1734 sense primer, 5Ј-(c/t/a/g)cc cca gct gtt (c/t)tt-3Ј) 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. ʈ 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 (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, 5Ј-cct aag ctt tca gcc atg att DNA sequence analysis demonstrated that this novel sequence con- tct tct ctg tcc caa aac c-3Ј 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, 5Ј-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-3Ј. The obtained PCR (Invitrogen) and labeled with [␣-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 ␭gt10 skeletal muscle cDNA library (1.5 ϫ 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 SKIIϩ 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 Ј regions of the mature protein. To isolate 5 sequences encoding the 37 °C in a humidified 5% CO2 atmosphere. Cathepsin F was transiently proregion of cathepsin F, two gene-specific oligonucleotides were de- expressed in the COS cells using LipofectamineTM (Life Technologies, signed that allowed the 5Ј rapid amplification of cDNA ends (RACE) to Inc.). Cells were harvested at 50–70% confluency from flasks by EDTA/ be performed (antisense for 5Ј-RACE, 5Ј-tag tca tcc tct gtc tcc agc-3Ј and trypsin (Sigma), washed twice with PBS (pH 7.4), seeded directly onto 5Ј-att gcc tgt gac tga gaa ggc-3Ј). These primers were used with Mara- sterilized three-field slides (BioGenex, San Ramon, CA), and cultured thon-Ready lung cDNA (CLONTECH) in conjunction with the Advan- for 12–15 h. The slides showing approximately 50% confluence were tage KlenTag polymerase kit as described in the user’s manual (CLON- used for transient transfection, which was carried out according to the TECH). Distinct 400–600-bp bands were gel-purified and cloned using manufacturer’s protocol. Briefly, slides were washed twice in PBS and the TA cloning system. DNA sequencing revealed that the fragments once in DMEM without any supplements. One ␮g of plasmid DNA from Downloaded from encoded the 5Ј part of the cathepsin F gene including two putative start a midikit preparation (Qiagen, Hilden, Germany) was resuspended in codons. The cDNA encoding the open reading frame starting with Met-1 100 ␮l of DMEM and mixed with 6 ␮l of Lipofectamine (which was (starting at nucleotide sequence position 321) and ending at the TGA earlier resolved in 100 ␮l of DMEM), and the mixture were incubated at stop codon was amplified by PCR from a ␭gt10 skeletal muscle cDNA room temperature for 30–40 min. After complex formation, 800 ␮lof library (CLONTECH) using Pfu polymerase and the following oligonu- DMEM were added, and cells were covered with 100–200 ␮l of trans- cleotide primers (sense, 5Ј-cg gaattc atg att tct tct ctg tcc caa aac-3Ј fection mixture/field. Cells were incubated in the CO incubator for 5–6 2 http://www.jbc.org/ containing an EcoRI site; antisense 5Ј-aa gcggccgc tca gtc cac cac cgc cga h, and then 700 ␮l of complete DMEM/field were added. After overnight gct ggc-3Ј containing a NotI site). The PCR product was ligated into the culture, the medium was replaced, and finally cells were cultured for vector pCR-ScriptAmp SK(ϩ) (Stratagene, La Jolla, CA). The insert of 48 h. the resulting vector, pCTSF1, was subsequently verified by sequencing. Slides with pcDNA3.1CF-T7 transiently transfected COS-7 cells Primer Extension—To determine the transcription start site, 20 ␮gof were washed twice with PBS (pH 7.4), and cells were fixed with fresh total RNA from human brain, human lung, and human vascular smooth 4% TBS (w/v) containing 0.02% Tween 20 (Fisher) for 10 min. After muscle cells, respectively, were isolated as described previously (17), three extensive washes, TBS/Tween cells were directly used for staining by guest on October 14, 2019 and mixed with 0.25 ␮gof[␥-P32]dATP-labeled oligonucleotide (5Ј-cc ata or subsequently dehydrated in ethanol (25, 50, 75, and 100% each at 15 ctg agc tgt gcc acg-3Ј). Mixtures were dried, and 50 ␮l of buffer contain- min) and stored at Ϫ20 °C until use. ing 1.5 M KCl, 0.1 M Tris, pH 8.3, 100 mM EDTA was added into each After blocking cells using goat serum in PBS (BioGenex) at room sample and denatured at 65 °C for 90 min. Denatured RNA-primer temperature for 45 min, cells were rinsed twice with TBS/Tween and ␮ ␮ mixtures were ethanol-precipitated and 13 l of distilled H2O, 5 lof then incubated with the anti-T7 monoclonal antibody (Novagen, Mad- reverse transcription buffer (Promega, Madison, WI), 2.5 ␮lof10mM ison, MI) overnight. After washing the cells with TBS containing 0.02% dNTP (Life Technologies, Inc.), 1.5 ␮l of bovine serum albumin (Pro- Tween 20 (3 ϫ 20 min), the cells were treated with anti-mouse IgG- mega), and 2 ␮l of Moloney murine leukemia virus (Life Technologies, tetramethyl rhodamine isothiocyanate (TRITC; both from Sigma) as Inc.) were added and incubated for 90 min at 42 °C followed by incu- recommended by the manufacturer. After washing the cells with TBS/ bation with 1 ␮lof0.5M EDTA and 1 ␮l of RNase A (10 mg/ml) at 37 °C Tween, the cells were mounted using Fluoromount-GTM (Southern Bio- for 30 min. Reaction mixtures were phenol/chloroform-extracted and technology Association Inc., Birmingham, AL) and viewed with a fluo- ethanol-precipitated, and then samples were dissolved into 10 ␮lof rescence microscope (Eclipse-E-800, Nikon). As control, COS-7 cells DNA sample buffer. 3-␮l aliquots of each reaction mixture were sepa- were stained with acridine orange to localize the acidic (lysosomal) rated on a 6% DNA sequencing gel, and the dried gel was exposed to compartments of the cells. COS-7 cells were seeded on three chamber X-Omat film (Eastman Kodak Co.). slides (1 ϫ 104 cells/ml) and grown overnight in DMEM. The cells were Northern Blot Analysis—An [␣-32P]dCTP-labeled 300-bp fragment of incubated with acridine orange (Sigma) at a final concentration of 20 cathepsin F was prepared as described above and used to probe multiple ␮M in DMEM for 15 min at 37 °C and finally washed in 1ϫ PBS. After tissue Northern blots containing mRNAs from various tissues (CLON- the washing, live cells were immediately viewed with the fluorescence TECH). The blots were washed in 2ϫ SSC, 0.05% SDS for 60 min at microscope. room temperature and for 60 min at 65 °C in 0.1ϫ SSC, 0.1% SDS. As Activation and Purification—The concentrated P. pastoris culture control DNA, a radioactive labeled actin probe (CLONTECH) was used. supernatant containing recombinant procathepsin F was adjusted to Construction of a Transfer Vector and Expression in Pichia pastoris— pH 4.5 using 3 M sodium acetate, pH 4.0, supplemented with dithio- The coding region of cathepsin F was excised from the vector pCTSF1 threitol (final concentration 0.5 mM) and 0.4 mg/ml porcine pepsin with EcoRI and NotI, ligated into the EcoRI and NotI sites of the (Sigma). The activation mixture was incubated in a shaker for5hat multicloning site of the pPIC-9 expression vector (Invitrogen), and 37 °C and 200 rpm. The activation was monitored using Z-Phe-Arg- subsequently linearized with BglII and electroporated into P. pastoris MCA as a fluorogenic substrate in 100 mM sodium acetate buffer, pH

GS115 host cells using standard procedures (Invitrogen, San Diego, 5.5, containing 2.5 mM EDTA-Na2 and 2.5 mM dithiothreitol. The acti- CA). Using the EcoRI site in the multicloning site of pPIC-9 for the vated supernatant was cleared again by centrifugation, loaded on a 5Ј-end insertion of cathepsin F results in a fusion protein between the HiTrap SP column (Amersham Pharmacia Biotech), and eluted with a S. cerevisiae ␣-mating factor prepropeptide and full-length cathepsin F 0–1 M NaCl gradient in 20 mM sodium acetate, pH 5.5, containing 1 mM with an additional insertion of the tripeptide Tyr-Val-Glu derived from dithiothreitol and 1 mM EDTA. Active cathepsin F was eluted between the multicloning site (instruction manual for the pPIC-9 vector, Invitro- 0.2 and 0.3 M NaCl. gen, San Diego, CA). HisϩMutS clones that produced recombinant hu- To compare the autocatalytic pH-dependent activation of procathep- man cathepsin F were selected and tested for productivity. A P. pastoris sin F with that of pepsin-mediated activation, unpurified recombinant clone that was expressing a high level of cathepsin F activity was precursor was incubated at pH 4.2, pH 5.0 (10 mM sodium acetate selected for fermentation as described by Invitrogen. After centrifuga- buffer, 2.5 mM EDTA/dithiothreitol), and pH 6.0 (100 mM potassium tion of the fermentor harvest, the clear supernatant was concentrated phosphate buffer, 2.5 mM EDTA/dithiothreitol) in the absence or pres- (60-fold) and diafiltered on a YM10 membrane (Amicon) with 50 mM ence of 0.4 mg/ml porcine pepsin (Sigma). The activation mixture was sodium acetate, 2.5 mM EDTA, 2.5 mM dithiothreitol, pH 5.5. incubated in a shaker at 37 °C and 200 rpm. At appropriate time 32002 Human Cathepsin F intervals, aliquots were withdrawn, and the activation of the protease was monitored as described above and by Western blot analyses. Western Analyses—A polyclonal antibody (MS25) against the human cathepsin F peptide, GHMQSANFSAEK, was raised in rabbits and purified on a Protein A column (Pierce). For Western analyses, an antibody dilution of 1:10,000 was used, and signals were detected using POD-labeled anti-rabbit IgG and the ECL™ substrate (Amersham Pharmacia Biotech). In order to determine N-linked glycosylation of recombinant cathepsin F, unpurified precursor protein from the culture medium supernatant was incubated for1hatpH5.5with endoglyco- sidases F and H (Boehringer Mannheim) as recommended by the manufacturer. Cathepsin F Assays with Methylcoumarylamide Substrates—Initial rates of substrate hydrolysis were monitored in 1-cm cuvettes at 25 °C in a Perkin-Elmer fluorimeter at excitation and emission wavelengths of 380 and 450 nm, respectively. Recombinant human cathepsin F was assayed at a constant enzyme concentration (1–18 nM)in50mM potas- sium phosphate buffer, pH 6.5, containing 2.5 mM dithioerythritol and

2.5 mM-Na2EDTA and variable substrate concentrations. Fluorogenic substrates, Z-FR-MCA and Z-RR-MCA, were purchased from Bachem International (Bubendorf, Switzerland); Z-LR-MCA and Z-VR-MCA were synthesized as described using standard procedures in peptide chemistry (18). The kinetic constants, Vmax and Km, were obtained by nonlinear regression analysis using the program Enzfitter (19). The active site concentration of cathepsin F was determined with morpho- Downloaded from line urea-Leu-homophenylalanine-vinyl sulfone-Ph using the method described for E-64 as active site titrant (20). Morpholine urea-Leu- homophenylalanine-vinyl sulfone-Ph is a potent and irreversible inhib- FIG.1. Determination of human cathepsin F transcription itor of cysteine proteases (21) (kindly provided by J. T. Palmer (Axys start site by primer extension. 20 ␮g of total RNA from human brain Pharmaceutical, South San Francisco, CA). (lane 1), human lung (lane 2), and human vascular smooth muscle cells pH Activity Profile and pH Stability—Initial rates of substrate hy- (lane 3) were used for primer extension, and a cDNA sequencing reac- drolysis were monitored as described above. The pH activity profile of tion was used as molecular size marker. The antisense primer for this http://www.jbc.org/ human cathepsin F was obtained at 1 ␮M substrate (Z-FR-MCA) con- reaction started from nucleotides 450–469 (see cDNA for detail). Ͻ centration ([S] Km, where the initial rate, vo, is directly proportional to the kcat/Km value). The following buffers were used for the pH activity 2 profile: 100 mM sodium citrate (pH 2.8–5.6) and 100 mM sodium phos- cathepsin, we assigned the name cathepsin F to the gene and phate (pH 5.8–8.0). All buffers contained 1 mM EDTA and 0.4 M NaCl its gene product. to minimize the variation in ionic strength. A three-protonation model Three putative translation initiation codons were identified

(22) was used for least square regression analysis of the pH activity in the 5Ј region of the isolated cDNA starting at nucleotides 50, by guest on October 14, 2019 data. The data were fitted to the following equation. 213, and 321. Primer extension experiments using total RNA indicated that the major transcription initiation starts at posi- ͑ ͒ ϭ ͑ ͒ ͓͑ ϩ͔ ϩ ϩ ͓ ϩ͔͒ kcat/Km obs kcat/Km / H /K1 1 K2/ H (Eq. 1) tion 219 with tct tct ctg . . . , thereby eliminating the potential The pH stabilities of active cathepsins F and L were determined in atg start codons at positions 50 and 213 (Fig. 1). Interestingly, 100 mM sodium acetate buffer, pH 5.0 (lysosomal pH) and in potassium a second minor start site was found upstream of the potential phosphate buffer, 7.2 (cytosolic pH). At appropriate time intervals, atg start codon at position 119 in mRNA isolated from smooth aliquots of the incubation mixture were withdrawn, and the activity muscle cells, a site not found in either brain or lung mRNA was measured using the fluorogenic substrate assay described above. (Fig. 1). No start site that would include the potential atg start codon at position 50 was identified. In contrast to related lyso- RESULTS AND DISCUSSION somal cysteine proteases like cathepsins L, K, S, and H, the Human Cathepsin F cDNA—A novel 323-bp PCR fragment Met-1 in cathepsin F is not succeeded by a typical hydrophobic was obtained from a human alveolar lung macrophage library signal sequence (25) (neither were the methionines coded by using degenerate primers derived from regions surrounding atg codons beginning at nucleotides 50 and 213). Since cathep- the conserved active site residues, cysteine and asparagine, of sin F is missing a signal sequence, it is unlikely that the the papain superfamiliy (23). The PCR fragment displayed protease is able to enter the endoplasmic reticulum via the several motifs typical for papain-like cysteine proteases; how- SRP-dependent route and may not be targeted to lysosomes. In ever, it was not identical to the sequences of human cathepsins addition, neither an endoplasmic reticulum retention signal B, L, H, S, K, C, O, and W. Using this fragment, approximately sequence (26, 27) nor peptide sequences for targeting cytosolic 700,000 clones of a ␭gt10 skeletal muscle cDNA library were proteins to lysosomes (28) have been identified in the open screened, and four positive clones were isolated. Since all four reading frame of cathepsin F. Studies to determine the intra- clones were incomplete at the 5Ј-end of the coding region, the cellular localization of cathepsin F are presently in progress. missing 5Ј-end was obtained using the 5Ј-RACE cloning system Based on these studies, full-length cDNA of human cathep- and specific primers obtained from the initial 323-bp fragment sin F encodes a 302-amino acid protein (Fig. 2) with a calcu- and the incomplete clones. The complete 3Ј-untranslated end, lated molecular mass of 33,860 Da. The open reading frame including the polyadenine tail, was obtained by searching the starts after a typical translation initiation sequence (Ϫ3, A expressed sequence tag data base using peptide sequences ob- (29)) at nucleotide 321 with an atg codon. A guanidine residue tained from ␭gt10 skeletal muscle clones. An expressed se- present in the ϩ4-position in the cathepsin F cDNA sequence quence tag clone (AA564691) was identified to contain the has also been described to be optimal for the binding of ribo- 3Ј-untranslated region of the protease. In addition, several expressed sequence tag clones (AI042401, H15749, and 2 H40022) were identified to contain additional 5Ј sequences It should be noted that the name cathepsin F was previously as- signed to a 50–70-kDa proteoglycan-degrading activity (24). However, (nucleotides 1–118). Since the nucleotide and the derived pro- this cathepsin F activity was characterized neither on a gene nor on a tein sequence clearly represented a novel putative papain-like protein level, and it is unrelated to the protease described in this report. Human Cathepsin F 32003

FIG.2.Nucleotide sequence and de- duced amino acid sequence of human cathepsin F cDNA. The amino acid se- quence is shown in single letter code above the nucleotide sequence. The main Met1 and the active site residues Cys113, His249, and Asn269 are indicated in bold- face type, the potential N-glycosylation

sites are underlined, and the polyadenyl- Downloaded from ation site is double underlined.Anarrow- head indicates the putative cleavage site between the prodomain and catalytic do- main of human cathepsin F. Two transla- tion initiation codons have been identified in the open reading frame of the cathepsin

F cDNA (see Fig. 1). The putative protein http://www.jbc.org/ sequence between the first and third atg codon is printed in italic type. Methio- nines upstream of Met1 are designated as Met2 and Met3. Conserved amino acid res- idues in the prodomain forming the ERF- NIN/ERFNAQ motif are double under- lined. The original cDNA fragment obtained by PCR using degenerate prim- by guest on October 14, 2019 ers is dotted.

somes (29). None of the two other putative translation start shown to form disulfide bridges in cathepsin K (31). Therefore, sites have both of these residues in positions Ϫ3 and ϩ4. The the overall fold of cathepsin F may be similar to cathepsin K. open reading frame ends at nucleotide 1269 with a tga stop An arrowhead in Fig. 2 marks the putative cleavage site, codon followed by a consensus polyadenylation site (aataaa) Leu88-Ala89, between the proregion and the mature region of beginning at nucleotide 1555. The open reading frame of ca- the protease. Typically for cathepsins, the second amino acid thepsin F had a propeptide and a mature region containing the residue adjacent to the processing site is a proline. conserved putative active site residues, Cys113, His249, and To date, eight human cathepsins of the papain family have Asn269. In addition, the sequence of mature cathepsin F with a been cloned and sequenced (cathepsins B, L, H, S, K, C, O, and calculated molecular mass of 23,592 Da contained all other W). Multiple sequence alignments of the full-length precursor conserved residues in cysteine proteases including Gln87 (oxy- cathepsins clearly demonstrate that cathepsin F is a new mem- anion binding pocket), Trp271, and Trp275, as well as Gly153 and ber of this protease family (Fig. 3). Cathepsin F shares a rela- Gly154 (30). Besides its catalytically active cysteine residue, tively low degree of protein sequence identity with other cys- Cys113, mature cathepsin F had six additional cysteine residues teine proteases, at best being 42% (58% homology) identical (Cys110, Cys144, Cys151, Cys184, Cys242, and Cys290), which also with cathepsin W and only 22% (37.8% homology) identical were conserved in related cathepsins. The homologous cysteine with cathepsin B (Table I). The putative propeptide of cathep- residue pairs 110–151, 144–184, and 242–290 have been sin F consists of 88 amino acids. Although the propeptides of 32004 Human Cathepsin F Downloaded from http://www.jbc.org/ by guest on October 14, 2019

FIG.3.Multiple amino acid sequence alignment of human cathepsin F with human cathepsins S, L, K, H, B, and W. The amino acid sequences have been extracted from the GenBankTM and SwissProt data base (human cathepsin W precursor, accession no. AF055903; human precursor, accession no. P09668; human cathepsin K (O2) precursor, accession no. S79895; human cathepsin L precursor, accession no. P07711; human precursor, accession no. P43234; human cathepsin S precursor, accession no. P25774; human cathepsin B precursor, accession no. P07858), and the multiple alignment was performed using the Clustal W multiple sequence alignment of MacVector (Oxford Molecular Group, PLC). Identical amino acid residues are darkly shaded, similar amino acids are lightly shaded, and unrelated residue have a white background. papain-like cysteine proteases are less conserved than their 11–27% sequence identity (27–42% homology) (Table I). In catalytic domains, Karrer et al. (32) identified a motif that contrast to cathepsin F, cathepsin W had an N-terminal hydro- distinguished two subfamilies, cathepsin B- and cathepsin L- phobic sequence that was typical for signal sequences. like proteases. The majority of papain-like cysteine proteases Tissue Distribution of Cathepsin F—Human cathepsin F ex- possess the so-called ER(F/W)NIN motif and belong to the pression, detected as a single transcript on Northern blot anal- cathepsin-L-like enzymes. In contrast, cathepsin F and the ysis, was high to moderate in most of the tested tissues and recently identified cathepsin W (10) appear to form a third organs (Fig. 4). High levels of expression were observed in subgroup with the motif ERFNAQ (double underlined in Fig. heart, brain, skeletal muscle, testis, and ovary; moderate levels 2), which may have diverged from the cathepsin-L-like subfam- were expressed in kidney, pancreas, placenta, liver, and colon; ily. The putative propeptides of cathepsins F and W share and low levels were observed in lung, spleen, and small intes- approximately 41% protein sequence identity (54.5% homolo- tine. In addition to the major transcript, very low levels of gy), whereas the propeptides of all other cathepsins share only expression of a 100–200-base pair larger transcript were ob- Human Cathepsin F 32005

TABLE I Protein sequence homologies of cathepsin F with the cathepsins L, K, S, B, H, O, and W The first number reflects sequence identity, and the number in parenthesis shows . The sequences were analyzed with the program PCGENE using the Myers and Miller algorithm.

Homology and identity to cathepsin F LKSBHOW % Full sequence 31.1 (41.7) 31.8 (43.7) 29.1 (43) 22.2 (37.8) 34.8 (47.7) 30.5 (43.4) 42.1 (58) Mature sequence 40.2 (50.9) 37.9 (46.3) 35 (46.7) 33.6 (46.2) 38.3 (50.9) 33.6 (46.2) 40.7 (58) Proregion 25 (36.4) 14.8 (33) 13.6 (31.8) 11.3 (27.4) 26.1 (39.7) 27.7 (42.2) 40.9 (54.4)

FIG.4.Northern blot analysis of hu- man cathepsin F in human tissues and tumor cell lines. Multiple human tissue and tumor cell line nitrocellulose blots were hybridized with a 32P-labeled human cathepsin F probe. See text for Downloaded from details. http://www.jbc.org/

FIG.5. Expression and localization of cathepsin F in COS-7 cells. A, COS-7

cells were transiently transfected with by guest on October 14, 2019 pcDNA3.1 containing a cDNA insert of T7-tagged procathepsin F. The cDNA in- sert contained two translation initiation codons coding for Met1 and Met2. Cells were fixed and stained with anti-T7 monoclonal antibody and anti-mouse IgG- tetramethyl rhodamine isothiocyanate as described under “Experimental Proce- dures.” B, COS-7 cells stained with acri- dine orange.

served in pancreas, testis, ovary, HeLaS3, and G-361 cells. This with Met-1 is the primary translation product but that an is in accordance with the observation of a minor transcription expression of a longer polypeptide beginning with Met-2 is also start site in selected tissues (Fig. 1). Thymus and peripheral possible (Figs. 1 and 2). Immunostaining clearly revealed an leukocytes did not reveal detectable levels of expression. expression of cathepsin F in vesicles predominantly present in Of note was the high level of expression in brain, which has the juxtanuclear region of the cell (Fig. 5A). No staining was been not observed for other cathepsins. In addition, Northern observed with secondary antibody only or mouse IgG. Treat- analysis of a variety of tumor cell lines revealed high levels of ment of COS-7 cells with acridine orange as marker for acidic cathepsin F expression in HeLa cells as well as melanoma compartments revealed a comparable juxtanuclear staining G-361 and A549 cells. Low level expression was observed in pattern typical for lysosomes (Fig. 5B). The vesicular, lysoso- K562 cells, whereas the expression of cathepsin F in various mal-like distribution of the gene product is unexpected for a leukemic and lymphoblastic cell lines was below the detection protein expressed without a signal sequence. Interestingly, the level (Fig. 4). The ubiquitous expression of cathepsin F is sim- staining pattern of cathepsin F is similar to that observed for a ilar to those of cathepsins B, L, and H but is in contrast to the truncated form of cathepsin B, which also is missing its signal tissue-specific expression of cathepsins S, K, and W. sequence and a part of its prodomain (33). Mehtani et al. (33) Expression of Cathepsin F in COS-7 Cells—T7-tagged hu- could demonstrate that truncated cathepsin B is associated man cathepsin F was detected by immunofluorescence in tran- with the cytosolic surface of membrane organelles. Whether siently transfected COS-7 cells when expressed from the cyto- cathepsin F is located inside the observed vesicles or facing the megalovirus promoter in pcDNA3.1. The cDNA insert of the cytosolic side of vesicular membranes is presently under expression vector contained the first two putative atg start investigation. codons of cathepsin F coding for Met-1 and Met-2. Primer Expression, Activation, and Purification of Recombinant Hu- extension experiments suggested that cathepsin F beginning man Cathepsin F—To generate a functional protease for in 32006 Human Cathepsin F

FIG.6. Activation of procathepsin F. Left, time-dependent activation of hu- man cathepsin F at 37 °C in 100 mM so- dium acetate buffer, pH 4.2, in 100 mM sodium acetate buffer, pH 5.0, and 100 mM potassium phosphate buffer, pH 6.0, and at pH 4.2 with pepsin (0.4 mg/ml). Aliquots were withdrawn from the prein- cubation mixture at the indicated time points, and the activity was monitored with 10 ␮M Z-LR-MCA. Right, Western analyses of procathepsin F autocatalytic processing at pH 4.2 and pepsin-mediated processing at pH 4.2. Aliquots of recombi- nant human procathepsin F containing supernatant of a 3-day P. pastoris culture were incubated with pepsin as described above. The times of digestion are as indi- cated. Molecular mass standards (kDa) are indicated on the right. Mature cathep- sin F and procathepsin F were detected by Western blot analysis using a cathepsin F surface peptide targeted antibody (see “Experimental Procedures”). Downloaded from vitro studies, human cathepsin F was expressed in yeast using the P. pastoris expression system and the ␣-pheromone signal sequence for extracellular targeting and thus for facilitating protein purification. The expression level of the protease in the

culture supernatant following activation with pepsin was mon- http://www.jbc.org/ itored by its Z-FR-MCA-hydrolyzing activity inhibitable with 100 ␮M E-64. Wild-type P. pastoris does not produce an endog- enous Z-FR-MCA-hydrolyzing activity. The culture superna- tant was collected after 3 days of fermentation and was 20-fold concentrated by ultrafiltration. Activation of the inactive pre- FIG.7. Recombinant human cathepsin F is N-glycosylated. Treatment with endoglycosidases H and F results in a decrease in cursor was achieved by autoactivation at pH 4.2 or could be by guest on October 14, 2019 accelerated by the addition of pepsin (Fig. 6). Autoactivation molecular mass of the proenzyme by approximately 9 kDa and a de- crease in mass of the mature enzyme by 5 kDa, indicating that both the was very weak at pH 5.0 and practically abolished at pH 6.0 proregion and the mature part of recombinant cathepsin F are N-linked (Fig. 6). Pepsin-mediated activation had the advantage that it glycosylated. The enzyme was activated autocatalytically at pH 4.2 for resulted at the same time in the efficient proteolytic removal of 48 h. most of the contaminating high molecular protein components present in the culture supernatant. Complete activation was ated as fusion protein containing the strong yeast ␣-pheromone achieved after3hat37°Cwith 0.4 mg/ml porcine pepsin (Fig. secretory signal sequence. Thus, the fusion protein was tar- 6). Autocatalytic and pepsin-mediated processing of the 44-kDa geted to the secretory pathway, and subsequent N-linked gly- precursor proceeded via an intermediate of 38 kDa to the ma- cosylation occurred. Since human wild-type cathepsin F lacks ture active form of approximately 34 kDa (Fig. 6) (molecular the signal peptide, translocation into the endoplasmic reticu- masses are based on the Amersham Pharmacia Biotech protein lum via the signal recognition particle route is unlikely, as is standard and vary with different standards). the processing of the N-glycosylation sites. No evidence is pres- The deduced amino acid sequence of cathepsin F contained ently available as to whether these sites are used for in vivo four putative N-glycosylation sites, one located in the propart glycosylation. (Asn13-Arg-Thr) and three in the mature region (Asn185-Phe- 196 258 Activated cathepsin F was purified at room temperature on Ser; Asn -Asp-Ser; Asn -Arg-Thr) (underlined in Fig. 2). HiTrap SP-Sepharose. Active cathepsin F was eluted as a sin- Recombinant propeptides and mature cathepsin F polypep- gle peak at a salt concentration of 0.2–0.3 M NaCl using a tides, which were produced in yeast using the ␣-pheromone gradient of 0–1 M NaCl in 20 mM sodium acetate buffer, pH 5.5. signal peptide for extracellular secretion, were glycosylated The resulting protein had approximately 80–90% purity on a and had molecular masses of 44 and 34 kDa, respectively. After SDS-polyacrylamide gel (data not shown) and was used for the treatment with endoglycosidases H and F, the molecular physicokinetic characterization of the protease. masses decreased to approximately 35 and 29 kDa, respec- S2P2 Subsite Specificity of Recombinant Human Cathepsin tively. This suggests that both the proregion and the mature F—The substrate binding region of proteases is defined as an protein are N-linked glycosylated and that the glycosylation of arrangement of binding pockets or subsites (S) for several the prosite is approximately 4 kDa and that of the mature sites peptide substrate amino acid residues (P) contiguous to both about 5 kDa. It is likely that more than one of the three sides of the scissile bond. Seven such sites have been described putative N-glycosylation sites of the mature polypeptide were for the plant papain (34). However, based on used. The determined molecular masses for the recombinant x-ray crystallographic studies of papain-like proteases, such as cathepsin F polypeptides were in very good accord with those of cruzain and cathepsins B, L, and K (31, 35–37) only the S other recombinant cathepsins such as cathepsins L, K, and S 2 3 subsite, which binds the second amino acid residue upstream of (Fig. 7). As stated above, recombinant cathepsin F was gener- the scissile bond, forms a real pocket, whereas the other sub- sites are more or less shallow enclaves on the surface of the 3 D. Bro¨mme, unpublished data. protease. The S2 binding pocket defines the primary substrate Human Cathepsin F 32007

TABLE II Kinetic parameters for the Z-XR-MCA hydrolysis by recombinant human cathepsin F

Substrate kcat Km kcat/Km

Ϫ1 Ϫ1 Ϫ1 s ␮MMs Z-FR-MCA 2.5 Ϯ 0.1 0.44 Ϯ 0.02 5,682,000 (100)a Z-LR-MCA 1.3 Ϯ 0.1 0.23 Ϯ 0.09 5,652,000 (99) Z-VR-MCA 1.3 Ϯ 0.2 1.24 Ϯ 0.44 1,048,000 (18) Z-RR-MCA 0.2 Ϯ 0.04 2.87 Ϯ 0.81 70,000 (1.2)

a Relative activities of the kcat/Km values in reference to the best substrate (Z-FR-MCA ϭ 100%) are shown in parentheses. Downloaded from

FIG.8.kcat/Km values for the hydrolysis of Z-XR-MCA by cathep- sins F, L, K, S, and B. Values were normalized to the best substrate, Ϫ Ϫ equal to 1 as follows: Cathepsin F (Z-FR-MCA), 5,682,000 M 1 s 1; Ϫ Ϫ cathepsin K (Z-LR-MCA), 257,900 M 1 s 1; cathepsin S (Z-LR-MCA), Ϫ Ϫ Ϫ Ϫ http://www.jbc.org/ 243,000 M 1 s 1; cathepsin L (Z-FR-MCA), 5,111,000 M 1 s 1; cathepsin Ϫ Ϫ B (Z-FR-MCA), 460,000 M 1 s 1. Data for cathepsins K, L, and B are from Ref. 8.

specificity of cathepsins. The S2P2 subsite specificity of cathep- sin F was determined with synthetic substrates of the type

Z-XF-MCA (where X represents Phe, Leu, Val, or Arg) and by guest on October 14, 2019 compared with the relative activities of cathepsins L, K, B, and

S. The specificity of cathepsin F resembled those of cathepsins FIG.9.pH activity profile for recombinant human cathepsin F. L, S, and K. All four cathepsins prefer bulky hydrophobic The calculated pH optimum was 5.9. Cathepsin F is active at values residues (phenylalanine and leucine) over the smaller that are at least 90% of its maximum activity from pH 5.2 to 6.8. ␤-branched valine, and a positively charged arginine residue in

P2 results in a very poor substrate hydrolysis. Only cathepsin B of cathepsin F, the enzyme’s stability was determined at pH 5.0 was able to efficiently hydrolyze Z-RR-MCA (38). However, in (lysosomal pH) and pH 7.2 (cytosolic pH). Cathepsins F and L contrast to cathepsins L, S, and K, which preferred either were both stable at pH 5.0 for 4 h but both very unstable at leucine or phenylalanine in the P2 position, cathepsin F ac- neutral pH. However, cathepsin F exhibited a slightly higher cepted both residues equally well (Fig. 8). This may indicate a pH stability at pH 7.2 with short incubation times. Whereas wide and less restricted S2 subsite pocket in cathepsin F. lysosomal cathepsin L had a half life of less than 1 min at pH The catalytic efficiency (kcat/Km) of cathepsin F toward the 7.2 and 37 °C, the half-life of cathepsin F was more than twice studied synthetic substrates was high and comparable with as long (2.2 min). Compared with previously described cytosolic that of cathepsin L (Table II). The high second-order rate cysteine proteases such as the (41) and bleomycin constants for both enzymes were mainly due to the submicro- (42), cathepsin F activity at neutral pH was very molar Km values. It should be noted that cathepsin L is the short lived. The determination of the enzyme’s precise subcel- catalytically most active known lysosomal cysteine protease lular localization will shed light on the contradiction between a (1). possible nonlysosomal localization (suggested by the lack of the pH Activity Profile and pH Stability—Recombinant human signal peptide) and the low pH stability of the protease at cathepsin F had a pH optimum between 5.2 and 6.8 (90% of cytosolic pH. maximal activity). The enzyme was characterized by a rela- In conclusion, human cathepsin F is a new member of the tively broad pH activity profile with flanking pK values of 4.5 papain-protease family that is ubiquitously expressed in most and 7.2 (Fig. 9). The descending limb of the profile is incom- tissues. Multiple sequence alignments indicate that cathepsin plete due to the instability of the protease at pH values above F belongs with cathepsin W to a new cathepsin subgroup. Ϫ pH 7.2. The width of the pH profile (pK2 pK1), which reflects Contrary to all other related cysteine proteases, cathepsin F the stability of the ion pair formed by the active site residues does not possess a signal sequence, which suggests either a cysteine and histidine (39), was 2.7 for cathepsin F. This value nonlysosomal subcellular localization or an alternative mech- is 0.6 units smaller than that of the neutral pH-stable cathep- anism for targeting to the lysosome. Its catalytic efficiency sin S (40). No value for human cathepsin L could be calculated toward synthetic peptide substrates is comparable with cathep- since the instability of the protease at neutral pH did not allow sin L, with a primary substrate specificity similar to that of determination of a pK2 value. cathepsins L, S, and K. The widespread tissue distribution and The pH stability of cathepsin F was compared with that of high catalytic activity may indicate a housekeeping function of cathepsin L. Considering a potential nonlysosomal localization cathepsin F essential in most cells. In addition, cathepsin F 32008 Human Cathepsin F must be considered as a novel antitarget for drug discovery 19. Leatherbarrow, R. J. (1987) Enzfitter, Elsevier Biosoft, Cambridge, UK 20. Barrett, A. J., and Kirschke, H. (1981) Methods Enzymol. 80, 535–561 efforts directed against cysteine proteases such as cathepsins 21. Palmer, J. T., Rasnick, D., Klaus, J. L., and Bro¨mme, D. (1995) J. Med. Chem. K, B, L, and S. 38, 3193–3196 22. Khouri, H. E., Vernet, T., Menard, R., Parlati, F., Laflamme, P., Tessier, D. C., Acknowledgment—We thank Dr. R. J. Desnick (Mount Sinai School Gour-Salin, B., Thomas, D. Y., and Storer, A. C. (1991) Biochemistry 30, of Medicine, New York) for critical reading of the manuscript. 8929–8936 23. Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 461–486 REFERENCES 24. Dingle, J. T., Blow, A. M., Barrett, A. J., and Martin, P. E. (1977) Biochem. J. 167, 775–785 1. Kirschke, H., Barrett, A. J., and Rawlings, N. D. (1995) Protein Profiles 25. von Heijne, G. (1995) Exper. Suppl. (Basel) 73, 67–76 (Sheterline, P., ed) Vol. 2, Academic Press, London 26. Nilsson, T., and Warren, G. (1994) Curr. Opin. Cell Biol. 6, 517–521 2. Velasco, G., Ferrando, A. A., Puente, X. S., Sanchez, L. M., and Lopez-Otin, C. 27. Urade, R., Oda, T., Ito, H., Moriyama, T., Utsumi, S., and Kito, M. (1997) (1994) J. Biol. Chem. 269, 27136–27142 J. Biochem. (Tokyo) 122, 834–842 3. Turnsek, T., Kregar, I., and Lebez, D. (1975) Biochim Biophys Acta 403, 28. Dice, J. F. (1990) Trends Biochem. Sci. 15, 305–308 514–520 29. Kozak, M. (1986) Cell 44, 283–292 4. Kirschke, H., Schmidt, I., and Wiederanders, B. (1986) Biochem. J. 240, 30. Berti, P. J., and Srorer, A. C. (1995) Biochemistry 246, 273–283 455–459 31. McGrath, M. E., Klaus, J. L., Barnes, M. G., and Bro¨mme, D. (1997) Nat. 5. Riese, R. J., Wolf, P., Bro¨mme, D., Natkin, L. R., Villadangos, J. A., Ploegh, Struct. Biol. 4, 105–109 H. L., and Chapman, H. A. (1996) Immunity 4, 357–366 32. Karrer, K. M., Peiffer, S. L., and DiTomas, M. E. (1993) Proc. Natl. Acad. Sci. 6. Villadangos, J. A., Riese, R. J., Peters, C., Chapman, H. A., and Ploegh, H. L. U. S. A. 90, 3063–3067 (1997) J. Exp. Med. 186, 549–560 33. Mehtani, S., Gong, Q., Panella, J., Subbiah, S., Peffley, D. M., and Frankfater, 7. Tezuka, K., Tezuka, Y., Maejima, A., Sato, T., Nemoto, K., Kamioka, H., Hakeda, Y., and Kumegawa, M. (1994) J. Biol. Chem. 269, 1106–1109 A. (1998) J. Biol. Chem. 273, 13236–13244 8. Bro¨mme, D., Okamoto, K., Wang, B. B., and Biroc, S. (1996) J. Biol. Chem. 271, 34. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 2126–2132 157–162 9. Gelb, B. D., Shi, G. P., Chapman, H. A., and Desnick, R. J. (1996) Science 273, 35. Kamphuis, I. G., Drenth, J., and Baker, E. N. (1985) J. Mol. Biol. 182, 317–329 1236–1238 36. Musil, D., Zucic, D., Turk, D., Engh, R. A., Mayr, I., Huber, R., Popovic, T., 10. Linnevers, C., Smeekens, S. P., and Bro¨mme, D. (1997) FEBS Lett. 405, Turk, V., Towatari, T., Katunuma, N., and Bode, W. (1991) EMBO J. 10, 253–259 2321–2330 11. von Heijne, G. (1994) Subcell. Biochem. 22, 1–19 37. McGrath, M. E., Eakin, A. E., Engel, J. C., McKerrow, J. H., Craik, C. S., and Downloaded from 12. Fox, T. E., de Miguel, J. S., and Storer, A. C. (1992) Biochemistry 31, Fletterick, R. J. (1995) J. Mol. Biol. 247, 251–259 12571–12576 38. Knight, C. G. (1980) Biochem. J. 189, 447–453 13. von Figura, K. (1991) Curr. Opin. Cell Biol. 3, 642–646 39. Menard, R., Khouri, H. E., Plouffe, C., Laflamme, P., Dupras, R., Vernet, T., 14. Brocklehurst, K. B., Willenbrook, F. S., and Salih, E. (1987) New Compr. Tessier, D. C., Thomas, D. Y., and Storer, A. C. (1991) Biochemistry 30, Biochem. 16, 39–158 5531–5538 15. Shi, G. P., Chapman, H. A., Bhairi, S. M., DeLeeuw, C., Reddy, V. Y., and 40. Bro¨mme, D., Bonneau, P. R., Lachance, P., Wiederanders, B., Kirschke, H., Weiss, S. J. (1995) FEBS Lett. 357, 129–134 Peters, C., Thomas, D. Y., Storer, A. C., and Vernet, T. (1993) J. Biol. Chem.

16. Bro¨mme, D., and Okamoto, K. (1995) Biol. Chem. Hoppe-Seyler 376, 379–384 268, 4832–4838 http://www.jbc.org/ 17. Sukhova, G. K., Shi, G. P., Simon, D. I., Chapman, H. A., and Libby, P. (1998) 41. Inomata, M., Nomoto, M., Hayashi, M., Nakamura, M., Imahori, K., and J. Clin. Invest. 102, 576–583 Kawashima, S. (1984) J. Biochem. (Tokyo) 95, 1661–1670 18. Bro¨mme, D., Steinert, A., Friebe, S., Fittkau, S., Wiederanders, B., and 42. Bro¨mme, D., Rossi, A. B., Smeekens, S. P., Anderson, D. C., and Payan, D. G. Kirschke, H. (1989) Biochem. J. 264, 475–481 (1996) Biochemistry 35, 6706–6714 by guest on October 14, 2019 Human Cathepsin F: MOLECULAR CLONING, FUNCTIONAL EXPRESSION, TISSUE LOCALIZATION, AND ENZYMATIC CHARACTERIZATION Bruce Wang, Guo-Ping Shi, Pin Mei Yao, Zhenqiang Li, Harold A. Chapman and Dieter Brömme J. Biol. Chem. 1998, 273:32000-32008. doi: 10.1074/jbc.273.48.32000

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