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Cathepsin Z, a Novel Human Cysteine Proteinase with a Short Propeptide Domain and a Unique Chromosomal Location*

  • Iñigo Santamarı́a
    Footnotes
    Affiliations
    Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Gloria Velasco
    Affiliations
    Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Alberto M. Pendás
    Affiliations
    Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Antonio Fueyo
    Affiliations
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  • Carlos López-Otı́n
    Correspondence
    To whom correspondence should be addressed: Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Tel.: 34-85-104201; Fax: 34-85-103564 or 34-85-232255;
    Affiliations
    Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Author Footnotes
    * This work was supported by Grant SAF97-0258 from the Comisión Interministerial de Ciencia y Tecnologı́a, Grant BMH4-CT96–0017 from EU-BIOMED II, and grants from Glaxo-Wellcome, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF032906.
    ‡ Recipient of a fellowship from Ministerio de Educación y Ciencia (Spain).
Open AccessPublished:July 03, 1998DOI:https://doi.org/10.1074/jbc.273.27.16816
      We have identified and characterized a novel human cysteine proteinase of the papain family. A full-length cDNA for this enzyme was cloned from a human brain cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA codes for a polypeptide of 303 amino acids, tentatively called cathepsin Z, that exhibits structural features characteristic of cysteine proteinases. Fluorescent in situ hybridization experiments revealed that the human cathepsin Z gene maps to chromosome 20q13, a location that differs from all cysteine proteinase genes mapped to date. The cDNA encoding cathepsin Z was expressed inEscherichia coli as a fusion protein with glutathioneS-transferase, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, used as a substrate for cysteine proteinases. Northern blot analysis demonstrated that cathepsin Z is widely expressed in human tissues, suggesting that this enzyme could be involved in the normal intracellular protein degradation taking place in all cell types. Cathepsin Z is also ubiquitously distributed in cancer cell lines and in primary tumors from different sources, suggesting that this enzyme may participate in tumor progression as reported for other cathepsins. Finally, on the basis of a series of distinctive structural features, including diverse peptide insertions and an unusual short propeptide, together with its unique chromosomal location among cysteine proteinases, we propose that cathepsin Z may be the first representative of a novel subfamily of this class of proteolytic enzymes.
      The cysteine proteinases belonging to the papain family represent a major component of the lysosomal proteolytic system and play an essential role in protein degradation and turnover (
      • Bond J.S.
      • Butler P.E.
      ,
      • Chapman H.A.
      • Riese R.J.
      • Shi G.P.
      ). In addition, these proteolytic enzymes appear to play an extracellular role in a number of normal and pathological conditions including bone remodeling (
      • Tezuka K.
      • Tezuka Y.
      • Maejima A.
      • Sato T.
      • Nemoto K.
      • Kamioka H.
      • Hakeda Y.
      • Kumegawa M.
      ), prohormone activation (
      • Krieger T.J.
      • Hook V.Y.H.
      ), rheumatoid arthritis (
      • Mort J.S.
      • Recklies A.D.
      • Poole A.R.
      ), Alzheimer's disease (
      • Golde T.E.
      • Estus S.
      • Younkin L.H.
      • Selkoe D.J.
      • Younkin S.G.
      ), pulmonary emphysema (
      • Mason R.W.
      • Johnson D.A.
      • Barrett A.J.
      • Chapman H.A.
      ), and cancer invasion and metastasis (
      • Sloane B.F.
      ). To date, nine human cysteine proteinases of the papain family have been isolated and characterized at the amino acid sequence level: cathepsin B (
      • Chan S.J.
      • San Segundo B.
      • McCormick M.B.
      • Steiner D.F.
      ), cathepsin L (
      • Gal S.
      • Gottesman M.M.
      ,
      • Joseph L.J.
      • Chang L.C.
      • Stamenkovich D.
      • Sukhatme V.P.
      ), cathepsin H (
      • Fuchs R.
      • Machleidt W.
      • Gassen H.G.
      ,
      • Ritonja A.
      • Popovic T.
      • Kotnik M.
      • Machleidt W.
      • Turk V.
      ), cathepsin S (
      • Wiederanders B.
      • Brömme D.
      • Kirschke H.
      • von Figura K.
      • Schmidt B.
      • Peters C.
      ,
      • Shi G.P.
      • Munger J.S.
      • Meara J.P.
      • Rich D.H.
      • Chapman H.A.
      ), cathepsin C (
      • Paris A.
      • Strukelj B.
      • Pungercar J.
      • Renko M.
      • Dolenc I.
      • Turk V.
      ), cathepsin O (
      • Velasco G.
      • Ferrando A.A.
      • Puente X.A.
      • Sánchez L.M.
      • López-Otı́n C.
      ), cathepsin K (
      • Inaoka T.
      • Bilbe G.
      • Ishibashi O.
      • Tezuka K.
      • Kumegawa M.
      • Kokubo T.
      ,
      • Bromme D.
      • Okamoto K.
      • Wang B.B.
      • Biroc S.
      ), cathepsin W (
      • Linnevers C.
      • Smeekens S.P.
      • Bromme D.
      ), and cathepsin L2 (
      • Santamarı́a I
      • Velasco G.
      • Cazorla M.
      • Fueyo A.
      • Campo E.
      • López-Otı́n C.
      ). All of them contain an essential cysteine residue in their active site but differ in tissue distribution and in some enzymatic properties, including substrate specificities and pH stability. Furthermore, several groups have reported the existence of additional cysteine proteinases including cathepsins M, N, P, and T, which were originally identified because of their degrading activity on specific substrates such as aldolase, collagen, proinsulin, or tyrosine aminotransferase, but whose characterization at the molecular level has not yet been reported (
      • Pontremoli S.
      • Melloni E.
      • Salamino F.
      • Sparatore B.
      • Michetti M.
      • Horecker B.L.
      ,
      • Maciewicz R.
      • Etherington D.
      ,
      • Docherty K.
      • Carroll R.J.
      • Steiner D.F.
      ,
      • Gohda E.
      • Pitot H.C.
      ).
      According to structural and functional data, it is well established that the different cysteine proteinases of the papain family are synthesized as preproenzymes, which are processed to the corresponding proenzymes and targeted to the lysosomes by the mannose 6-phosphate signal attached to them. The enzymes are further processed to mature forms consisting of either a single polypeptide chain or a two-chain form composed of heavy and light chains linked by a disulfide bond (
      • Berti P.J.
      • Storer A.C.
      ). However, in some cases, the precursors of these lysosomal enzymes escape from this processing pathway and continue along the secretory route, entering storage granules and being finally released into the extracellular space (
      • Kornfeld S.
      • Mellman I.
      ). In fact, a series of reports have shown that several lysosomal cysteine proteinases are released by tumor cells from different sources, supporting the concept that secreted lysosomal enzymes may play important roles in the developmnent of malignant processes (
      • Sloane B.F.
      • Dunn J.R.
      • Honn K.V.
      ,
      • Recklies A.D.
      • Poole A.R.
      • Mort J.S.
      ,
      • Gal S.
      • Gottesman M.M.
      ). Furthermore, lysosomal cysteine proteinases have been found to be secreted by activated macrophages, by osteoclasts, or by fibroblasts from patients with I-cell disease, thereby extending the pattern of physiological and pathological conditions in which these enzymes may be involved (
      • Chapman H.A.
      • Riese R.J.
      • Shi G.P.
      ,
      • Kornfeld S.
      • Mellman I.
      ). Based on the hypothesis that a number of different cysteine proteinases could be responsible for the wide variety of biological functions ascribed to this protein family, we have been interested in examining the possibility that additional yet uncharacterized family members could be produced by human tissues. This search for new human cysteine proteinases led us to identify cathepsin O, originally cloned from a breast carcinoma but widely distributed in human tissues (
      • Velasco G.
      • Ferrando A.A.
      • Puente X.A.
      • Sánchez L.M.
      • López-Otı́n C.
      ). We have also described the cloning and characterization of human bleomycin hydrolase, a cytosolic cysteine proteinase distantly related to other members of the papain family and involved in chemotherapy resistance (
      • Ferrando A.A.
      • Velasco G.
      • Campo E.
      • López-Otı́n C.
      ,
      • Ferrando A.A.
      • Pendás A.M.
      • Llano E.
      • Velasco G.
      • Lidereau R.
      • López-Otı́n C.
      ). Finally, we have recently reported the finding of cathepsin L2, a cysteine proteinase structurally related to cathepsin L, but showing a unique tissue distribution (
      • Santamarı́a I
      • Velasco G.
      • Cazorla M.
      • Fueyo A.
      • Campo E.
      • López-Otı́n C.
      ). In this study, we report the identification, chromosomal location, and structural and enzymatic characterization of a novel member of this family of enzymes, which has been tentatively called cathepsin Z.

      EXPERIMENTAL PROCEDURES

      Materials

      Restriction endonucleases and other reagents used for molecular cloning were from Boehringer Mannheim (Mannheim, Germany). Human brain and prostate cDNA libraries, constructed in λgt11, and Northern blots containing poly(A)+ RNAs prepared from different human tissues and cancer cell lines were purchased from CLONTECH (Palo Alto, CA). Double-stranded DNA probes were radiolabeled with [α-32P]dCTP (3000 Ci/mmol) from Amersham (Amersham, UK) using a commercial random-priming kit purchased from Pharmacia LKB (Uppsala, Sweden). Oligonucleotides were synthesized by the phosphoramidite method in an Applied Biosystems DNA synthesizer (model 392A) and used directly after synthesis. Synthetic peptides Z-Phe-Arg-AMC, Z-Arg-Arg-AMC, and Z-Arg-AMC were from Bachem (Bubendorf, Switzerland), and proteinase inhibitor E-64
      The abbreviations used are: E-64,trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane; bp, base pair(s); kb, kilobase(s); EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PAC, P1 artificial chromosome; Z-Phe-Arg-AMC, benzyloxycarbonyl-l-phenylalanyl-l-arginine-7-amido-4-methylcoumarin.
      was from Sigma.

      Screening of a Human Brain cDNA Library

      A computer search of the GenBankTM data base for sequences with homology to human cysteine proteinases led us to identify a sequence derived from an ovarian tumor cDNA clone (AA283747)
      Deposited by L. Hillier, N. Clark, T. Dubuque, K. Elliston, M. Hawkins, M. Holman, M. Hultman, T. Kucaba, M. Le, G. Lennon, M. Marra, J. Parsons, L. Rifkin, T. Rohlfing, F. Tan, E. Trevaskis, R. Waterston, A. Williamson, P. Wohldmann, and R. Wilson, WashU-Merck EST project.
      and showing a significant similarity with sequences previously determined for other cathepsins. After looking for additional human expressed sequence tags (ESTs) similar to this one, we found approximately 20 overlapping ESTs, spanning around 1350 bp, and useful to design a specific probe for the gene encoding this putative novel cysteine proteinase. This DNA fragment was obtained by PCR amplification of cDNA from a commercially available human brain cDNA library as follows: total λ-phage DNA from this brain cDNA library was screened for the presence of the hypothetical cysteine proteinase using two specific primers 5′-CACCCGGAACCAGCACAT (primer Z1) and 5′-CGATGGGGTCCCCAATG (primer Z2) derived from the overlapping ESTs. The PCR reaction was performed in a GeneAmp 2400 PCR system from Perkin-Elmer for 35 cycles of denaturation (94 °C, 15 s), annealing (56 °C, 15 s), and extension (72 °C, 1 min). The PCR product, 708 bp long, was phosphorylated with T4 polynucleotide kinase and cloned into anEcoRV-cut pBS vector. The cloned cDNA was sequenced and found to be identical with the expected sequence. This cDNA was then excised from the vector, radiolabeled, and used to screen a human brain cDNA library, according to standard procedures (
      • Maniatis T.
      • Fritsch E.F.
      • Sambrook J.
      Molecular Cloning: A Laboratory Manual.
      ). Hybridization to the radiolabeled probe was carried out for 18 h in 6 × SSC (1× = 150 mm NaCl, 15 mmsodium citrate, pH 7.0), 5 × Denhardt's (1× = 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 0.1% SDS, and 100 μg/ml denatured herring sperm DNA at 55 °C. The membranes were washed twice for 1 h at 55 °C in 2 × SSC, 0.1% SDS and exposed to XAR-5 film (Kodak) at −70 °C with intensifying screens. Following plaque purification, several cloned inserts were excised by EcoRI digestion and the resulting fragments subcloned into the EcoRI site of pUC18.

      Nucleotide Sequencing

      Selected DNA fragments were inserted in the polylinker region of phage vector M13mp19 and sequenced by the Sanger method (
      • Sanger F.
      • Nicklen S.
      • Coulson A.R.
      ), using either M13 universal primer or cDNA specific primers and the Sequenase Version 2.0 kit (U. S. Biochemicals). Sequence ambiguities were solved by substituting dITP for dGTP in the sequencing reactions. All nucleotides were identified in both strands. Computer analysis of DNA and protein sequences was performed with the GCG software package of the University of Wisconsin Genetics Computer Group (
      • Devereux J.
      • Haeberli P.
      • Smithies O.
      ).

      Fluorescent in SituHybridization

      A high density gridded human P1 artificial chromosome (PAC) genomic library (kindly supplied by the Human Genome Mapping Resource Center) was screened by filter hybridization with the full-length cathepsin Z cDNA as probe. Three independent clones were identified enclosing the cathepsin Z gene as demonstrated by PCR and Southern blot analysis. DNA from one of these PAC clones was obtained with the standard alkaline lysis method and then used for fluorescent in situ hybridization mapping. To do that, 2 μg of the PAC DNA was nick translated with biotin-16-dUTP and hybridized to normal male metaphase chromosomes obtained from phytohemagglutinin-stimulated cultured lymphocytes, as described previously (
      • Pendás A.M.
      • Matilla A.
      • Urı́a J.A.
      • Freije J.P.
      • Fueyo A.
      • Estivill X.
      • López-Otı́n C.
      ). Biotinylated probe was detected using two avidin-fluorescein layers. Chromosomes were diamidine-2-phenylindole dihydrochloride-banded, and images were captured in a Zeiss axiophot fluorescent microscope equipped with a CCD camera (Photometrics).

      Expression in E. coli

      To prepare an expression vector suitable for production of recombinant cathepsin Z in E. coli, we first generated a 727-bp DNA fragment containing the coding sequence for the mature human cathepsin Z by PCR amplification of the isolated full-length cDNA with primers 5′-ATGCTGCCCAAGAGCTGGGAC and 5′-CGATGGGGTCCCCAAATG. The PCR reaction was carried out for 30 cycles of denaturation (95 °C, 30 s), annealing (56 °C, 30 s), and extension (72 °C, 1 min) using the ExpandTM Long Template PCR System (Boehringer Mannheim) to reduce error frequency. The PCR product was phosphorylated with T4 polynucleotide kinase, repared with Klenow fragment, and ligated to the expression vector pGEX-3X (Pharmacia LKB), previously treated withSmaI and alkaline phosphatase. The resulting plasmid, called pGEX-3X CathZ, was transformed into E. coli strain BL21(DE3), and the transformed cells were grown in LB broth containing 100 μg/ml ampicillin at 37 °C for about 16 h, diluted 1/100 with the same medium, and grown to an A 600 of 1.0. Then, isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm, and the incubation was continued for 3 h. Cells were collected by centrifugation, washed, and resuspended in 0.05 volumes of phosphate-buffered saline, lysed by using a French press, and centrifuged at 20,000 ×g for 20 min at 4 °C. The soluble extract was treated with glutathione-Sepharose 4B and eluted with glutathione elution buffer (10 mm reduced glutathione in 50 mmTris-HCl, pH 8.0) following manufacturer's instructions.

      Enzyme Activity Assays

      The enzymatic activity of purified cathepsin Z produced in E. coli was measured using 20 μm Z-Phe-Arg-AMC, Z-Arg-Arg-AMC, or Z-Arg-AMC as substrates and following the procedure described by Barrett and Kirschke (
      • Barrett A.J.
      • Kirschke H.
      ) with minor modifications. Assays were performed at 30 °C, in 100 mm sodium acetate buffer, pH 5.5, containing 8 mm dithiothreitol, 2 mm EDTA, and 0.05% Brij 35. Substrate hydrolysis was monitored in a Cytofluor 2350 fluorometer (Millipore) at excitation and emission wavelengths of 360 and 460 nm, respectively. For inhibition assays, the reaction mixture was preincubated with 20 μm E-64 at 30 °C for 15 min, and the remaining activity was determined using the fluorogenic substrate Z-Phe-Arg-AMC as above.

      Northern Blot Analysis

      Northern blots containing 2 μg of poly(A)+ RNA of different human tissue specimens and cancer cell lines or 20 μg of total RNA from diverse tumor tissues were prehybridized at 42 °C for 3 h in 50% formamide, 5 × SSPE (1 × = 150 mm NaCl, 10 mmNaH2PO4, 1 mm EDTA, pH 7.4), 10 × Denhardt's, 2% SDS, and 100 μg/ml denatured herring sperm DNA. After prehybridization, filters were hybridized with the 708-bp radiolabeled probe corresponding to the core region of the cathepsin Z cDNA. After hybridization, filters were washed with 0.1 × SSC, 0.1% SDS for 2 h at 50 °C and exposed to autoradiography. RNA integrity and equal loading were assessed by hybridization with an actin probe.

      RESULTS

      Isolation and Characterization of a Human Brain cDNA Encoding a Novel Member of the Papain Family of Cysteine Proteinases

      In an attempt to identify novel members of the papain family of cysteine proteinases, we carried out a computer search of the human EST data base, looking for sequences with similarity to previously described family members. This analysis led to the identification of a series of overlapping ESTs that, after assembly, encoded a protein sequence with a significant degree of similarity to the C-terminal region of the different cysteine proteinases of the papain family characterized to date. A cDNA containing part of the nucleotide sequence corresponding to these assembled ESTs was obtained by PCR amplification of total λ-phage DNA prepared from a commercially available human brain cDNA library. The amplified DNA fragment (708 bp) was cloned, and its identity was confirmed by nucleotide sequence analysis. The cloned fragment was then radiolabeled and used as a probe to hybridize the same human brain cDNA library utilized for the previous PCR amplification experiment. Upon screening of approximately 1 × 106 plaque-forming units, a total of four independent clones showing positive hybridization with the probe were identified. Then, DNA was isolated from each of these positive clones, and their nucleotide sequence was determined by standard procedures. Analysis of the resulting sequences revealed that all of them seemed to derive from the same gene, which would encode a putative novel human cathepsin. However, none of these isolated cDNA clones contained in their nucleotide sequence the coding information corresponding to the N-terminal region of this novel human enzyme. To obtain this sequence, we screened a human prostate cDNA library using as a probe a 85-bp DNA fragment, prepared by PCR amplification and corresponding to the most 5′-region present in the isolated brain cDNA clones. Upon screening of approximately 1 × 106 plaque-forming units, DNA was isolated from a single clone showing positive hybridization with the 85-bp probe, and its nucleotide sequence was determined. Sequence analysis of this positive clone allowed us to extend by approximately 300 bp the 5′-sequence determined for the brain cDNA clones as well as to confirm the remaining part of the nucleotide sequence derived from these clones. Computer analysis of the 5′-extended sequence revealed an open reading frame 909 bp long, starting with an ATG codon at position 241 and ending with a TAA codon at position 1150 (Fig. 1). Assuming that translation starts at this first ATG, the identified open reading frame codes for a protein of 303 amino acids and a predicted molecular weight of 33,881.
      Figure thumbnail gr1
      Figure 1Nucleotide sequence and deduced amino acid sequence of human cathepsin Z cloned from a brain cDNA library. The amino acid sequence is shown insingle-letter code below the nucleotide sequence. The active site residues characteristic of cysteine proteinases areunderlined. Arrows indicate the putative cleavage sites between the signal sequence and the propeptide as well as between the propeptide and the mature enzyme.
      Pairwise comparisons for sequence similarities between the identified amino acid sequence and those determined for the different human cysteine proteinases of the papain family characterized to date showed that the percentage of identities ranged from 34% with cathepsin C to 26% with cathepsin B. Despite this overall limited sequence identity, the deduced amino acid sequence from the human cDNA isolated in this work shows all the structural features characteristic of the different members of the papain family (Fig. 2). Thus, the presence of a N-terminal sequence rich in hydrophobic residues suggests the presence of the signal peptide which targets these enzymes to the secretory pathway (
      • Bond J.S.
      • Butler P.E.
      ,
      • Chapman H.A.
      • Riese R.J.
      • Shi G.P.
      ,
      • Berti P.J.
      • Storer A.C.
      ). Interestingly, this 20-residue hydrophobic peptide ends in a sequence Ala-Gly-Ala that matches perfectly the Ala-X-Ala motif found at the processing site of eukaryotic preproteins (
      • von Hejne G.
      ). Assuming that the signal peptidase cleaves after this consensus sequence, the resulting protein would be composed of 283 amino acid residues. The multiple alignment of this sequence with those determined for human cysteine proteinases belonging to the papain family also allows us to identify a proregion and a mature proteinase sequence as well as to define the putative cleavage site between both protein domains. In fact, as shown in Fig. 2, the N-terminal of most mature cysteine proteinases can be precisely aligned, the second amino acid being proline in all cases. By analogy with them, the active processed form of the putative novel cysteine proteinase would start at the leucine residue located at position 62, with a predicted molecular weight of 27,280. On the other hand, the amino acid sequence alignment shown in Fig. 2 also allows us to identify the putative active site Cys residue (at position 92) of the novel protein sequence as well as other residues important for the catalytic properties of cysteine proteinases, including the His-241 and Asn-261 residues, which would complete the catalytic triad of these enzymes (
      • Berti P.J.
      • Storer A.C.
      ). A more detailed analysis of the amino acid sequences surrounding these three essential residues revealed a high degree of similarity between the novel sequence and that of the remaining cysteine proteinases. Thus, the N-terminal region contains the conserved tryptophan residue and hydrophobic segment immediately adjacent to the cysteine active site as well as the glutamine residue (at position 87) of the putative oxyanion hole characteristic of the structure of these proteolytic enzymes (
      • Kamphuis I.G.
      • Drenth J.
      • Baker E.N.
      ,
      • Musil D.
      • Zucic D.
      • Turk D.
      • Engh R.A.
      • Mayr I.
      • Huber R.
      • Popovic T.
      • Turk V.
      • Towatari T.
      • Katunuma N.
      • Bode W.
      ,
      • Coulombe R.
      • Grochulski P.
      • Sivaraman J.
      • Ménard R.
      • Mort J.S.
      • Cygler M.
      ). On the other hand, the C-terminal region contains a series of conserved aromatic residues and the tripeptide Asn-Ser-Trp located in the active site. The predicted protein sequence also contains two potential sites of N-glycosylation located in the mature region (Asn-Tyr-Thr at positions 184 and 224). Because all mammalian cysteine proteinases characterized to date are glycosylated, it is presumed that at least one of these residues has attached the mannose 6-phosphate marker required for lysosomal targeting. Taking together all structural characteristics, we can conclude that the isolated cDNA codes for a novel human cysteine proteinase of the papain family that we propose to call cathepsin Z, because to our knowledge this name is not still used to define any other member of the family.
      Figure thumbnail gr2
      Figure 2Multiple amino acid sequence alignment of cathepsin Z with other human cysteine proteinases. The amino acid sequences of previously known human cysteine proteinases were extracted from the SwissProt data base, and the multiple alignment was performed with the PILEUP program of the GCG package (
      • Devereux J.
      • Haeberli P.
      • Smithies O.
      ). Numberingcorresponds to the sequence of cathepsin Z. The N-terminal extension characteristic of cathepsin C has not been included in the multiple alignment. Residues which are common to all sequences areshadowed. Gaps are indicated by hyphens.

      Chromosomal Mapping of the Human Cathepsin Z Gene

      To establish the chromosomal localization of the human cathepsin Z gene, we first isolated PAC clones containing this gene by screening a genomic library with the full-length cDNA as probe. DNA from one of the isolated clones was then employed in fluorescent in situhybridization experiments using human male chromosome metaphases. After diamidine-2-phenylindole dihydrochloride banding of the metaphase cells with specific hybridization signals (more than 90% of the cells), fluorescent yellow signals corresponding to the biotinylated PAC clone were clearly mapped to the telomeric region of chromosome 20, in the q13 region (Fig. 3). As can be also seen in Fig. 3, no other chromosome site was labeled above background. A number of genes have been already mapped to this region, including those encoding adenosine deaminase, prostacyclin synthase, or phosphoenolpyruvate carboxykinase (
      • Honig J.
      • Martiniuk F.
      • D'Eustachio P.
      • Zamfirescu C.
      • Desnick R.
      • Hirschhorn L.R.
      • Hirschhorn R.
      ,
      • Yokoyama C.
      • Yabuki T.
      • Inoue H.
      • Tone Y.
      • Hara S.
      • Hatae H.S.
      • Nagata M.
      • Takahashi E.I.
      • Tanabe T.
      ,
      • Yu H.
      • Chandrasekharappa S.
      • Trent J.M.
      • Zhang J.
      • Meisler M.H.
      ). However, no cysteine proteinase genes of any of the different subfamilies have been previously found to map at this chromosome site.
      Figure thumbnail gr3
      Figure 3Chromosomal location of the human cathepsin Z gene. Fluorescent in situ hybridization with a biotinylated probe specific for human cathepsin Z. Metaphase cells were counterstained with diamidine-2-phenylindole dihydrochloride.

      Production of Recombinant Cathepsin Z in E. coli and Analysis of Its Enzymatic Activity

      To elucidate whether the isolated cathepsin Z cDNA codes for a functional cysteine proteinase, we expressed the cloned cDNA in a bacterial system following the strategy described for other cysteine proteinases (
      • Petanceska S.
      • Devi L.
      ). To do that, we first prepared by PCR amplification a 727-bp fragment containing the entire coding sequence for the predicted mature cathepsin Z. After confirming the nucleotide sequence of the amplified fragment, it was inserted in the polylinker region of the expression vector pGEX-3X. The resulting plasmid, called pGEX-3X CathZ, as well as the original vector, were transformed into E. coli BL21(DE3), and the transformed bacteria were induced with isopropyl-1-thio-β-d-galactopyranoside. SDS-PAGE analysis of protein extracts prepared from the induced bacteria revealed that the bacteria transformed with the recombinant plasmid contained a fusion protein of about 52 kDa, which was not present in the control extracts (Fig. 4 A). By contrast, a 29-kDa band corresponding to the parental glutathioneS-transferase was detected in the control extracts but not in those prepared from the recombinant bacteria (Fig. 4 A). To purify the recombinant cathepsin Z, we performed an affinity chromatography in a glutathione-Sepharose 4B column, which was eluted with a reduced glutathione-containing buffer. After elution and SDS-PAGE analysis of the protein material present in the chromatographic eluate, a single band of the expected size was detected (Fig. 4 A).
      Figure thumbnail gr4
      Figure 4Production of recombinant cathepsin Z inE. coliand analysis of its enzymatic activity. A, 5-μl aliquots of bacterial extracts (pGEX-3X and pGEX-3X cathZ) as well as 1 μl of purified fusion protein (cathZ) were analyzed by SDS-PAGE. Arrowsindicate parental glutathione S-transferase (29 kDa) and fusion protein (about 52 kDa). The size in kilodaltons of the molecular size markers (MWM) is shown at the right.B, recombinant cathepsin Z (•) and cathepsin O (▪) were incubated with 20 μm Z-Phe-Arg-AMC, and the substrate hydrolysis at 30 °C was monitored in the presence (□, ○) or in the absence (▪, •) of 20 μm E-64 at the indicated times.
      To examine the enzymatic activity of the affinity-purified cathepsin Z, we investigated its degrading activity against fluorescent substrates commonly used for analysis of cysteine proteinases, including Z-Phe-Arg-AMC, Z-Arg-Arg-AMC, and Z-Arg-AMC (Fig. 4 B). These enzymatic analyses revealed that recombinant cathepsin Z displayed a significant proteolytic activity (3 μmol/min/mol of enzyme) against the synthetic peptide Z-Phe-Arg-AMC, which is an optimal substrate for different cysteine proteinases (
      • Mason R.W.
      • Green G.D.J.
      • Barrett A.J.
      ). This activity value was slightly higher than that obtained for cathepsin O produced in E. coli and assayed under the same conditions (Fig. 4 B). In addition, the proteolytic activity of recombinant cathepsin Z against the fluorogenic substrates Z-Arg-Arg-AMC and Z-Arg-AMC was virtually undetectable. Furthermore, the proteolytic activity of cathepsin Z against Z-Phe-Arg-AMC was abolished by E-64, a widely used inhibitor of this subclass of proteolytic enzymes, but not by inhibitors of metalloproteinases (EDTA), serine proteinases (phenylmethylsulfonyl fluoride), and aspartyl proteinases (pepstatin A) (data not shown). Taken together, these preliminary enzymatic analyses indicate that the cloned cDNA codes for a bona fide cysteine proteinase with the substrate specificity and sensitivity toward inhibitors characteristic of these proteolytic enzymes.

      Expression of Human Cathepsin Z in Tissues and Cancer Cell Lines

      To investigate the presence of cathepsin Z mRNA transcripts in human tissues, Northern blots containing poly(A)+ RNAs extracted from a variety of tissues, including leukocytes, colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, and heart, were hybridized with a 270-bpFokI/EcoRI probe specific for human cathepsin Z. This probe is derived from the 3′-untranslated region of the isolated cDNA for this enzyme, which is the region showing the lowest percentage of identities with other family members (less than 20% identities), thus avoiding the possibility of cross-hybridization signals. After hybridization with the radiolabeled cathepsin Z probe, a single transcript of about 1.7 kb was observed in all examined tissues (Fig. 5 A). This widespread distribution of cathepsin Z should be consistent with the possibility that it is a lysosomal enzyme involved in the intracellular protein degradation that takes place in all cell types.
      Figure thumbnail gr5
      Figure 5Northern blot analysis of cathepsin Z mRNA in human tissues and cell lines. A, 2 μg of poly(A)+ RNA prepared from the indicated tissues were analyzed by hybridization with a 270-bp probe corresponding to the 3′-end of the cDNA for human cathepsin Z. The positions of RNA size markers are shown. Filters were subsequently hybridized with a human actin probe to ascertain the differences in RNA loading among the different samples. B, 2 μg of poly(A)+ RNA prepared from the indicated tumor cell lines or 20 μg of total RNA from diverse tumor specimens were hybridized with the above described probe specific for human cathepsin Z. Filters were finally hybridized with a human actin probe.
      Finally, and because previous studies have described the overexpression of different cysteine proteinases in human malignancies (
      • Sloane B.F.
      ,
      • Sloane B.F.
      • Dunn J.R.
      • Honn K.V.
      ,
      • Recklies A.D.
      • Poole A.R.
      • Mort J.S.
      ,
      • Gal S.
      • Gottesman M.M.
      ,
      • Chauhan S.S.
      • Goldstein L.J.
      • Gottesman M.M.
      ), we examined the possibility that cathepsin Z could be expressed by human cancer cell lines and primary tumors from different sources. To do that, we first hybridized a Northern blot containing poly(A)+ RNAs extracted from different cancer cell lines (HL-60, HeLa, K-562, MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361) with the same 3′-cathepsin Z cDNA probe used for the above studies. As can be seen in Fig. 5 B, a transcript of the expected size (about 1.7 kb) was strongly detected in most cancer cell lines, thus opening the possibility that cathepsin Z may be also overexpressed by human tumors. In fact, a survey of different primary tumors for cathepsin Z expression demonstrated that this gene is widely expressed by tumors from diverse origin (Fig. 5 B), suggesting that cathepsin Z may be added to the growing list of proteolytic enzymes associated with the malignant transformation of human cells.

      DISCUSSION

      In this work we report the identification of a novel human cysteine proteinase called cathepsin Z. We also provide a structural and functional analysis of this novel enzyme and describe its pattern of expression in normal and tumor tissues. Finally, we establish the chromosomal location of the cathepsin Z gene in the human genome, with the finding that it maps to chromosome 20, which is a unique location for any human cysteine proteinase mapped to date.
      The identification of cathepsin Z was initially based on a search of the human EST data base looking for sequences with similarity to previously characterized cysteine proteinases. The identified sequences were then PCR-amplified and used to screen cDNA libraries from different human tissues. This strategy led finally to the isolation of a full-length cDNA coding for a protein with sequence similarity to human cysteine proteinases of the papain family, which was tentatively called cathepsin Z. The identified sequence for this novel enzyme exhibits the domain structure characteristic of cysteine proteinases, including a signal sequence, a prodomain, and a catalytic domain responsible for the proteolytic activity of these proteins (
      • Berti P.J.
      • Storer A.C.
      ). It also contains a series of residues proposed to be important in the catalytic mechanism of these enzymes such as the active site Cys residue which is transiently acetylated during peptide hydrolysis, as well as the Asn and His residues which form the catalytic triad of cysteine proteinases (
      • Kamphuis I.G.
      • Drenth J.
      • Baker E.N.
      ,
      • Musil D.
      • Zucic D.
      • Turk D.
      • Engh R.A.
      • Mayr I.
      • Huber R.
      • Popovic T.
      • Turk V.
      • Towatari T.
      • Katunuma N.
      • Bode W.
      ,
      • Coulombe R.
      • Grochulski P.
      • Sivaraman J.
      • Ménard R.
      • Mort J.S.
      • Cygler M.
      ). However, a more detailed structural analysis of the sequence determined for cathepsin Z revealed some specific features for this novel enzyme. Thus, this novel cysteine proteinase contains three peptide insertions unique among all family members (Fig. 2). The first of these insertions consists of the introduction of the tripeptide (His-Ile-Pro) immediately adjacent to the Gln residue of the oxyanion hole. This insertion is also present in the murine homologue of cathepsin Z
      I. Santamarı́a and C. López-Otı́n, unpublished data.
      , and would considerably increase the short distance (5 residues) separating this Gln residue from the active site Cys of the remaining cysteine proteinases of the papain family. Therefore, it is tempting to speculate that it could confer distinct catalytic properties or affect the substrate specificity of cathepsin Z. The two other insertions consist of an 8-residue peptide located in the middle of the molecule and a 14-residue peptide in the C-terminal end of the molecule, but their putative structural and functional significance on cathepsin Z properties remains unknown. Nevertheless, the most striking feature of the sequence of cathepsin Z is its unusually short propeptide when compared with those present in other cysteine proteinases. It is well known that all cysteine proteinases are synthesized as inactive precursors with an N-terminal propeptide which acts as an intrinsic inhibitor of the proteolytic activity (
      • Fox T.
      • de Miguel E.
      • Mort J.S.
      • Storer A.C.
      ). The propeptide has also found to be essential for correct folding of some cysteine proteinases and for their stabilization upon exposure to changes in pH environments (
      • Tao K.
      • Stearns N.A.
      • Dong J.
      • Wu Q.I.
      • Sahagian G.G.
      ,
      • Carmona E.
      • Dufour E.
      • Plouffe C.
      • Takebe S.
      • Mason P.
      • Mort J.S.
      • Ménard R.
      ). The overall sequence similarities among the propeptides of the different cysteine proteinases are low, but according to structural features, they can be assigned to two groups (
      • Karrer K.M.
      • Peiffer S.L.
      • DiTomas M.E.
      ,
      • Vernet T.
      • Berti P.J.
      • de Montigny C.
      • Musil R.
      • Tessier D.C.
      • Ménard R.
      • Magny M.C.
      • Storer A.C.
      • Thomas D.Y.
      ). The first group contains cathepsin L-like enzymes with proregions greater than 90 amino acids in length and two highly conserved motifs called ERFNIN and GNFD. The second group comprises the cathepsins B from different sources and is characterized by a smaller proregion of about 60 amino acids lacking the ERFNIN motif. However, the proregion of cathepsin Z cannot be assigned to any of these groups because it is only 41 residues in length and lacks both conserved domains. Furthermore, this propeptide sequence does not contain any lysine residue, despite the fact that lysine-based structures present in the proregion of different cathepsins have been proposed to act as the recognition sites for the mannose phosphorylation required for intracellular targeting of these proteins (
      • Cuozzo J.W.
      • Tao K.
      • Wu Q.
      • Young W.
      • Sahagian G.G.
      ). Taking together these structural data, it seems clear that the proregion of cathepsin Z markedly deviates from those of all previously characterized family members, suggesting that it could be a member of a different subfamily of enzymes. Consistent with this hypothesis, chromosomal location of the cathepsin Z gene has revealed that it maps to chromosome 20. This position differs from those reported for the remaining cysteine proteinase genes of the papain family (
      • Shi G.
      • Webb A.C.
      • Foster K.E.
      • Knoll J.H.M.
      • Lemere C.A.
      • Munger J.S.
      • Chapman H.A.
      ,
      • Rood J.A.
      • Van Horn S.
      • Drake F.H.
      • Gowen M.
      • Debouck C.
      ,
      • Santamarı́a I.
      • Pendás A.M.
      • Velasco G.
      • López-Otı́n C.
      ,
      • Chauhan S.S.
      • Popescu N.C.
      • Ray D.
      • Fleischmann R.
      • Gottesman M.M.
      • Troen B.R.
      ,
      • Gong Q.
      • Chan S.J.
      • Bajkowski A.S.
      • Steiner D.F.
      • Frankfater A.
      ,
      • Rao N.V.
      • Rao G.V.
      • Hoidal J.R.
      ,
      • Wang X.
      • Chan S.J.
      • Eddy R.L.
      • Byers M.G.
      • Fukushima Y.
      • Henry W.M.
      • Haley L.L.
      • Steiner D.F.
      • Shows T.B.
      ), providing additional support to the above structural data that suggest that this novel cysteine proteinase is not closely related to the other members of the papain family. In addition to its possible value in the context of evolutionary studies of the human cysteine proteinases, knowledge of the chromosomal location of the cathepsin Z gene may be useful in searching for putative diseases related to abnormalities in this protein, as already demonstrated for cathepsin K (
      • Gelb B.D.
      • Shi G.P.
      • Chapman H.A.
      • Desnick R.J.
      ).
      In this work we have also provided evidence that cathepsin Z is expressed in all normal tissues analyzed, which suggests a putative general role of this protein as a proteolytic enzyme involved in the normal intracellular protein turnover taking place in all cell types. This housekeeping role of cathepsin Z in human tissues would be similar to that proposed for cathepsins B, L, H, and O, but distinguishes this enzyme from a series of recently described cysteine proteinases which appear to play highly specific roles in those tissues in which they are overexpressed or even exclusively expressed. This is the case of cathepsins K, S, W, and L2, predominantly expressed in osteoclasts, lymphatic tissues, T-lymphocytes, and thymus and testis, respectively, and proposed to be involved in bone remodeling (cathepsin K), antigen presentation (cathepsin S), regulation of T-cell cytolytic activity (cathepsin W), and regulation of the immune response and fertilization processes (cathepsin L2) (
      • Linnevers C.
      • Smeekens S.P.
      • Bromme D.
      ,
      • Santamarı́a I
      • Velasco G.
      • Cazorla M.
      • Fueyo A.
      • Campo E.
      • López-Otı́n C.
      ,
      • Gelb B.D.
      • Shi G.P.
      • Chapman H.A.
      • Desnick R.J.
      ,
      • Riese R.J.
      • Wolff P.R.
      • Brömme D.
      • Natkin L.R
      • Villadangos J.A.
      • Ploegh H.L.
      • Chapman H.A.
      ). The expression analysis of cathepsin Z also revealed a ubiquitous presence of this enzyme in a series of human cancer cell lines and primary tumors from different sources. This finding suggests that cathepsin Z may be somewhat linked to the malignant transformation of human cells as already shown for other cysteine proteinases (
      • Sloane B.F.
      ,
      • Chauhan S.S.
      • Goldstein L.J.
      • Gottesman M.M.
      ) and adds a new interest to the study of this novel enzyme.
      In summary, according to the results of this work, human cathepsin Z is a novel member of the papain family of cysteine proteinases that shows clear differences with the remaining family members characterized to date. The occurrence of a series of unique features in its structure, including diverse peptide insertions and an unusually short propeptide region, together with its chromosomal location at 20q13, distinguishes this enzyme from other cysteine proteinases and suggests that it may be the first representative of a new cathepsin subfamily. The availability of recombinant cathepsin Z and specific reagents for this proteinase will be very helpful in evaluating the functional significance of these structural differences as well as in studying the potential role of this novel enzyme in the protein degradative processes occurring in normal and pathological conditions, including cancer.

      ACKNOWLEDGEMENTS

      We thank Drs. M. Balbı́n and J. P. Freije for helpful comments and S. Alvarez for excellent technical assistance.

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