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Molecular Cloning and Structural and Functional Characterization of Human Cathepsin F, a New Cysteine Proteinase of the Papain Family with a Long Propeptide Domain*

  • Iñigo Santamarı́a
    Footnotes
    Affiliations
    From the Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de Medicina, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Gloria Velasco
    Affiliations
    From the Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de Medicina, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Alberto M. Pendás
    Footnotes
    Affiliations
    From the Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de Medicina, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Ana Paz
    Affiliations
    From the Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de Medicina, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Carlos López-Otı́n
    Correspondence
    To whom correspondence should be addressed: Dept. de Bioquı́mica y Biologı́a Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo-Spain. Tel.: 34-985-104201; Fax: 34-985-103564;
    Affiliations
    From the Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de Medicina, Universidad de Oviedo, 33006-Oviedo, Spain
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  • Author Footnotes
    * This work was supported in part by Grants SAF97-0258 from Comisión Interministerial de Ciencia y Tecnologı́a, Glaxo-Wellcome, Spain, and EU-BIOMED II Grant BMH4-CT96-0017.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) AJ007331 and AJ131851
    ‡ Recipient of fellowships from Ministerio de Educación y Ciencia (Spain) and Fuji-Chemical Industries (Takaoka, Japan), respectively.
Open AccessPublished:May 14, 1999DOI:https://doi.org/10.1074/jbc.274.20.13800
      A cDNA encoding a new cysteine proteinase belonging to the papain family and called cathepsin F has been cloned from a human prostate cDNA library. This cDNA encodes a polypeptide of 484 amino acids, with the same domain organization as other cysteine proteinases, including a hydrophobic signal sequence, a prodomain, and a catalytic region. However, this propeptide domain is unusually long and distinguishes cathepsin F from other proteinases of the papain family. Cathepsin F also shows all structural motifs characteristic of these proteinases, including the essential cysteine residue of the active site. Consistent with these structural features, cathepsin F produced in Escherichia coli as a fusion protein with glutathione S-transferase degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, a substrate commonly used for functional characterization of cysteine proteinases. Furthermore, this proteolytic activity is blocked bytrans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases. The gene encoding cathepsin F maps to chromosome 11q13, close to that encoding cathepsin W. Cathepsin F is widely expressed in human tissues, suggesting a role in normal protein catabolism. Northern blot analysis also revealed a significant level of expression in some cancer cell lines opening the possibility that this enzyme could be involved in degradative processes occurring during tumor progression.
      The cysteine proteinases are a widespread group of enzymes that catalyze the hydrolysis of many different proteins and play a major role in intracellular protein degradation and turnover (
      • Bond J.S.
      • Butler P.E.
      ,
      • Chapman H.A.
      • Riese R.J.
      • Shi G.P.
      ). These proteolytic enzymes can be subdivided into more than 20 different families, including the papain family, calpains, streptopains, clostripains, viral cysteine proteinases, and caspases, the largest one being that of papain (
      • Rawlings N.D.
      • Barrett A.J.
      ). In fact, the papain family of cysteine proteinases comprises a large number of enzymes from both prokaryotes and eukaryotes, with representative members expressed in bacteria, fungi, protozoa, plants, and humans (
      • Rawlings N.D.
      • Barrett A.J.
      ,
      • Berti P.J.
      • Storer A.C.
      ). In recent years, the number of human cysteine proteinases belonging to the papain family has considerably increased, and a total of 10 different family members has been characterized at the amino acid sequence level. These human cysteine proteinases include 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 (
      • Shi G.P.
      • Munger J.S.
      • Meara J.P.
      • Rich D.H.
      • Chapman H.A.
      ,
      • Paris A.
      • Strukelj B.
      • Pungercar J.
      • Renko M.
      • Dolenc I.
      • Turk V.
      ), cathepsin O (
      • Velasco G.
      • Ferrando A.A.
      • Puente X.S.
      • 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.
      ), cathepsin L2 (
      • Santamarı́a I.
      • Velasco G.
      • Cazorla M.
      • Fueyo A.
      • Campo E.
      • López-Otı́n C.
      ), and cathepsin Z (
      • Santamarı́a I.
      • Velasco G.
      • Pendás A.M.
      • Fueyo A.
      • López-Otı́n C.
      ). Structural analysis of these enzymes has revealed that all of them contain a series of conserved features including an essential cysteine residue in their active site. In addition, it is well established that all these cysteine proteinases 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. However, these enzymes differ in tissue distribution and in some enzymatic properties, including substrate specificities and pH stability. Functional analysis of these proteinases has shown that in addition to their intracellular role in protein recycling, they are involved in other normal processes such as antigen presentation (
      • Riese R.J.
      • Wolff P.R.
      • Brömme D.
      • Natkin L.R
      • Villadangos J.A.
      • Ploegh H.L.
      • Chapman H.A.
      ), bone remodeling (
      • Tezuka K.
      • Tezuka Y.
      • Maejima A.
      • Sato T.
      • Nemoto K.
      • Kamioka H.
      • Hakeda Y.
      • Kumegawa M.
      ), and prohormone activation (
      • Krieger T.J.
      • Hook V.Y.H.
      ). In addition, it has been suggested that cysteine proteinases are involved in a variety of disease processes such as pulmonary emphysema (
      • Mason R.W.
      • Johnson D.A.
      • Barrett A.J.
      • Chapman H.A.
      ), osteoporosis (
      • Delaisse J.M.
      • Pascale L.
      • Vaes G.
      ), Alzheimer's disease (
      • Golde T.E.
      • Estus S.
      • Younkin L.H.
      • Selkoe D.J.
      • Younkin S.G.
      ), rheumatoid arthritis (
      • Mort J.S.
      • Recklies A.D.
      • Poole A.R.
      ), and cancer invasion and metastasis (
      • Sloane B.F.
      ). Therefore, these enzymes represent primary targets for the development of inhibitors that could block its uncontrolled activity in these pathological conditions.
      As part of our work directed to look for proteolytic enzymes that could be of importance in tumor progression, we have recently identified different cysteine proteinases of the papain family overexpressed in human carcinomas from diverse sources. These proteases include cathepsin O, originally cloned from a breast carcinoma (
      • Velasco G.
      • Ferrando A.A.
      • Puente X.S.
      • Sánchez L.M.
      • López-Otı́n C.
      ), cathepsin L2, overexpressed in breast and colon carcinomas (
      • Santamarı́a I.
      • Velasco G.
      • Cazorla M.
      • Fueyo A.
      • Campo E.
      • López-Otı́n C.
      ), and cathepsin Z, ubiquitously distributed in cancer cell lines and primary tumors and characterized by containing an unusual short propeptide in its amino acid sequence (
      • Santamarı́a I.
      • Velasco G.
      • Pendás A.M.
      • Fueyo A.
      • López-Otı́n C.
      ). We have also identified 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.
      ). In this work, we describe the molecular cloning and complete nucleotide sequence of a cDNA encoding a new member of the papain family of cysteine proteinases, which has been called cathepsin F, and that is mainly characterized by possessing a unique long propeptide domain in its amino acid sequence. We also report the expression of the gene inEscherichia coli and the functional characterization of the recombinant enzyme. Finally, we determine the chromosomal location of the cathepsin F gene and analyze its expression in human tissues and cancer cell lines.

      DISCUSSION

      The availability of EST data bases represents an excellent tool to look for novel genes through computer search of short expressed DNA sequences with nucleotide sequence similarity to genes of interest. In this work, we have used this strategy as a first step to clone a new member of the papain family of cysteine proteinases, which we have called cathepsin F. The identification of this human protease was based on the finding of a series of overlapping ESTs, whose sequence was similar to previously characterized human cysteine proteinases. These sequences were used to design a DNA probe that was PCR-amplified from a human prostate cDNA and subsequently employed to screen a cDNA library from the same tissue. This screening led finally to the finding of a full-length cDNA coding for cathepsin F. Pairwise comparisons for structural similarities between the identified amino acid sequence for this protein and those for the remaining papain-like cysteine proteinases confirmed that cathepsin F displays the same domain organization as other family members. Thus, a signal peptide, a propeptide domain, and a catalytic region can be identified in the amino acid sequence deduced for this protein. The identification of this signal sequence, which is also present in the mouse andDrosophila homologs of cathepsin F (Fig. 2, and data not shown), does not support the data reported by Wang et al.(
      • Wang B.
      • Shi G.P.
      • Yao P.M.
      • Li Z.
      • Chapman H.A.
      • Brömme D.
      ), after submission of this manuscript, who have proposed that cathepsin F lacks signal sequence. Furthermore, the catalytic domain contains all structural motifs characteristic of cysteine proteinases, including the nucleophilic cysteine residue involved in covalent intermediate formation during peptide hydrolysis, as well as the histidine and asparagine residues that constitute the catalytic triad of these enzymes (
      • Kamphuis I.G.
      • Drenth J.
      • Baker E.N.
      ,
      • Coulombe R.
      • Grochulski P.
      • Sivaraman J.
      • Ménard R.
      • Mort J.S.
      • Cygler M.
      ). Consistent with these structural characteristics, functional analysis of recombinant cathepsin F produced in a bacterial expression system provided additional evidence that the isolated cDNA codes for a catalytically active cysteine proteinase. In fact, the purified recombinant protein exhibits a significant proteolytic activity against fluorogenic substrates used for assaying the enzymatic activity of these proteinases. In addition, this degrading activity was abolished by inhibitors of cysteine proteinases but not by inhibitors of any other class of proteolytic enzymes. Nevertheless, this novel protease also contains in its amino acid sequence some specific features. Of special interest in this regard is the finding that its N-terminal propeptide domain is extremely long when compared with those described for all the remaining papain-like cysteine proteinases. According to structural properties, the prosegments found in these enzymes can be classified into 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 one contains cathepsin L-like enzymes with prodomains of about 90 amino acids in length and bearing 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 consensus sequence. In addition, there are two cysteine proteinases that cannot be classified into any of these groups. Thus, human cathepsin C propeptide contains 206 amino acids (
      • Paris A.
      • Strukelj B.
      • Pungercar J.
      • Renko M.
      • Dolenc I.
      • Turk V.
      ), whereas the recently described human cathepsin Z contains a proregion that is only 41 residues in length and lacks the above-mentioned conserved domains (
      • Santamarı́a I.
      • Velasco G.
      • Pendás A.M.
      • Fueyo A.
      • López-Otı́n C.
      ). Human cathepsin F markedly deviates from all of them because its prosegment contains 251 amino acids. Interestingly, both mouse andDrosophila homologs of cathepsin F also exhibit a very long prodomain (Fig. 2 and data not shown) indicating that it is a characteristic feature of this enzyme. At present, the functional significance of this extremely long prosegment is unknown. In this regard, it is well established that the propeptide found in papain-like enzymes acts as an intrinsic inhibitor of their proteolytic activity (
      • Fox T.
      • de Miguel E.
      • Mort J.S.
      • Storer A.C.
      ). In addition, this region has also been found to be essential for the proper folding of these enzymes, for stabilizing their structure upon exposure to changes in pH, or for providing the structural markers required for microsomal membrane binding or lysosomal targeting (
      • Fox T.
      • de Miguel E.
      • Mort J.S.
      • Storer A.C.
      ,
      • Tao K.
      • Stearns N.A.
      • Dong J.
      • Wu Q.
      • Sahagian G.G.
      ,
      • McIntyre G.F.
      • Godbold G.D.
      • Erickson A.H.
      ,
      • Cuozzo J.W.
      • Tao K.
      • Wu Q.I.
      • Young W.
      • Sahagian G.G.
      ,
      • Carmona E.
      • Dufour E.
      • Plouffe C.
      • Takebe S.
      • Mason P.
      • Mort J.S.
      • Ménard R.
      ). It is likely that the long prosegment of cathepsin F may play some specific role in addition to those proposed for this domain of papain-like cysteine proteinases.
      In this work, we have also analyzed the chromosomal location of the cathepsin F gene as well as its expression in normal and tumor cells. According to both FISH and somatic hybrid mapping techniques, this gene localizes to the long arm of chromosome 11, at 11q13. This position is the same as that recently reported for the cathepsin W gene, indicating that these genes are clustered in the human genome. Consistent with these results, a phylogenetic tree constructed to analyze the evolutionary relationships between all known human cysteine proteinases of the papain family demonstrated that cathepsin F and cathepsin W are closely related. 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 F gene reported here may be useful for searching putative genetic diseases associated with this gene. Interestingly, different studies have reported that the 11q13 region is frequently altered in diverse human tumors (
      • Chuaqui R.F.
      • Zhuang Z.
      • Emmert-Buck M.R.
      • Liotta L.A.
      • Merino M.J.
      ,
      • Newsham I.F.
      ). Consequently, it will be of great interest to examine the possibility that cathepsin F may be a target of these genetic abnormalities associated with human carcinomas.
      On the other hand, analysis of the expression of cathepsin F in human tissues has provided some information about the putative functional significance of this protein. Thus, the finding that it is expressed in most normal tissues analyzed suggests a putative general role for this enzyme in the lysosomal protein catabolism taking place in all cell types. This expression pattern of cathepsin F classifies this enzyme within the group of widely distributed cysteine proteinases such as cathepsins B, L, H, O, and Z, as opposed to a series of recently described family members including cathepsins K, S, W, and L2, which appear to play highly specific roles in those tissues in which they are overexpressed or even exclusively expressed (see Ref.
      • Chapman H.A.
      • Riese R.J.
      • Shi G.P.
      for a review). Nevertheless, it is remarkable that cathepsin F expression levels in normal tissues exhibit a large variability, and there are tissues such as skeletal muscle and testis, in which its mRNA levels are up to 20-fold higher than in others such as kidney and colon, which also produce this novel protease, albeit at low levels. The finding of very high levels of cathepsin F mRNA in skeletal muscle is of particular interest in light of previous data reporting an essential role of cysteine proteinases in muscle proteolysis in both normal and pathological conditions, including some forms of muscular dystrophy (
      • Takeda A.
      • Jimi T.
      • Wakayama Y.
      • Misugi N.
      • Miyakes S.
      • Kumagai T.
      ,
      • Richard I.
      • Broux O.
      • Allamnd V.
      • Fougerousse F.
      • Chiannikulchai N.
      • Bourg N.
      • Brenguier L.
      • Devaud C.
      • Pasturaud P.
      • Roudaut C.
      • Hillaire D.
      • Passos-Bueno M.
      • Zatz M.
      • Tischfield J.A.
      • Fardeau M.
      • Jackson C.E.
      • Cohen D.
      • Beckmann J.S.
      ,
      • Tsujinaka T.
      • Fujita J.
      • Ebisui C.
      • Yano M.
      • Kominami E.
      • Suzuki K.
      • Tanaka K.
      • Katsume A.
      • Ohsugi Y.
      • Shiozaki H.
      • Monden M.
      ). Further studies will be required to evaluate the possibility that cathepsin F could be responsible for the catabolism of specific protein substrates in the muscle. On the other hand, its high level expression in the testis is also suggestive of a role for this novel cathepsin in fertilization processes, as proposed for other family members including the recently described cathepsin L2 (
      • Santamarı́a I.
      • Velasco G.
      • Cazorla M.
      • Fueyo A.
      • Campo E.
      • López-Otı́n C.
      ,
      • Mathur P.P.
      • Grima J.
      • Mo M.
      • Zhu L.
      • Aravindan G.R.
      • Calcagno K.
      • O'Bryan M.
      • Chung S.
      • Mruk D.
      • Lee W.M.
      • Silvestrini B.
      • Cheng C.Y.
      ). Finally, the expression analysis of cathepsin F has also revealed the presence of this enzyme in several human cancer cell lines, being especially significant in high levels in HeLa cells. This finding suggests that cathepsin F may play some role in the progression of some human carcinomas, thereby providing additional interest to the further functional characterization of this proteinase.
      In conclusion, we have identified and characterized a new human cysteine proteinase of the papain family that shows similarities and differences with the remaining family members previously described. Cathepsin F exhibits signal sequence and all structural features of cysteine proteinases as well as a profile of activity against fluorogenic substrates and sensitivity to inhibitors typical of these enzymes. However, it shows an extremely long propeptide domain which distinguishes this enzyme from other family members. Furthermore, its high level expression in certain tissues such as skeletal muscle and testis is suggestive of a specific activity in some physiological processes taking place in these tissues. The availability of recombinant cathepsin F and specific reagents for this new proteinase generated in this work will be very helpful to evaluate its precise functional role in the context of the increasingly complex pathways of protein degradation and turnover in human tissues.

      Acknowledgments

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

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