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Cloning and Characterization of Human MMP-23, a New Matrix Metalloproteinase Predominantly Expressed in Reproductive Tissues and Lacking Conserved Domains in Other Family Members*

  • Gloria Velasco
    Affiliations
    Departamento de Bioquı́mica y Biologı́a Molecular and
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  • Alberto M. Pendás
    Affiliations
    Departamento de Bioquı́mica y Biologı́a Molecular and
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  • Antonio Fueyo
    Affiliations
    Biologı́a Funcional, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain and the
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  • Vera Knäuper
    Affiliations
    School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Gillian Murphy
    Affiliations
    School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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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.: 34-985-104201; Fax: 34-985-103564;
    Affiliations
    Departamento de Bioquı́mica y Biologı́a Molecular and
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  • Author Footnotes
    * This work was supported by grants from Comisión Interministerial de Ciencia y Tecnologı́a-Spain (SAF97–0258), EU-BIOMED II (BMH4-CT96-0017), Glaxo-Wellcome (Spain), the Arthritis and Rheumatism Council (to G. M.), and Wellcome Trust (to V. K.), and by a fellowship (to A. M. P.) from Fuji-Chemical Industries (Japan).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) AJ005256.
Open AccessPublished:February 19, 1999DOI:https://doi.org/10.1074/jbc.274.8.4570
      A cDNA encoding a new human matrix metalloproteinase (MMP), tentatively called MMP-23, has been cloned from an ovary cDNA library. This protein exhibits sequence similarity with MMPs, but displays a different domain structure. Thus, MMP-23 lacks a recognizable signal sequence and has a short prodomain, although it contains a single cysteine residue that can be part of the cysteine-switch mechanism operating for maintaining enzyme latency. The C-terminal domain is considerably shortened and shows no sequence similarity to hemopexin, whereas all human MMPs, with the exception of matrilysin, contain four hemopexin-like repeats. Furthermore, MMP-23 is devoid of structural features distinctive of the diverse MMP subclasses, including the specific residues located close to the zinc-binding site in collagenases, the transmembrane domain of membrane-type MMPs, or the fibronectin-like domain of gelatinases. Fluorescent in situ hybridization experiments showed that the human MMP-23 gene maps to 1p36, a location which differs from all MMP genes mapped to date. Recombinant MMP-23 produced inEscherichia coli exhibits low, but significant proteolytic activity against a synthetic substrate commonly used for assaying MMPs. Northern blot analysis demonstrated that MMP-23 is predominantly expressed in ovary, testis, and prostate, suggesting that this new MMP may play a specialized role in reproductive processes.
      Matrix metalloproteinases (MMPs)
      The abbreviations MMP
      matrix metalloproteinase
      bp
      base pair(s)
      DAPI
      diamidine-2-phenylindole dihydrochloride
      EST
      expressed sequence tag
      FISH
      fluorescentin situ hybridization
      MT-MMP
      membrane-type matrix metalloproteinase
      PAGE
      polyacrylamide gel electrophoresis
      PCR
      polymerase chain reaction
      RACE
      rapid amplification of cDNA ends
      TIMP
      tissue inhibitor of metalloproteinases
      1The abbreviations MMP
      matrix metalloproteinase
      bp
      base pair(s)
      DAPI
      diamidine-2-phenylindole dihydrochloride
      EST
      expressed sequence tag
      FISH
      fluorescentin situ hybridization
      MT-MMP
      membrane-type matrix metalloproteinase
      PAGE
      polyacrylamide gel electrophoresis
      PCR
      polymerase chain reaction
      RACE
      rapid amplification of cDNA ends
      TIMP
      tissue inhibitor of metalloproteinases
      or matrixins are a family of zinc-dependent endopeptidases that degrade the different extracellular matrix proteins at a neutral pH. These enzymes are produced by many cell types, usually in response to disease processes associated with inflammation or tumor progression. In addition, they have been implicated in the connective tissue remodeling occurring in normal processes such as embryonic development, bone growth, or wound healing (
      • Woessner J.F.
      ,
      • Matrisian L.M.
      ,
      • Birkedal-Hansen H.
      • Moore W.G.I.
      • Bodden M.K.
      • Windsor L.J.
      • Birkedal-Hansen B.
      • DeCarlo A.
      • Engler J.A.
      ,
      • Stetler-Stevenson W.G.
      • Aznavoorian S.
      • Liotta L.A.
      ). In recent years, the number of known members of the family has grown after the discovery of a series of new family members identified in both normal or pathological conditions. To date, 16 human MMPs have been cloned and characterized at the amino acid sequence level (
      • Birkedal-Hansen H.
      • Moore W.G.I.
      • Bodden M.K.
      • Windsor L.J.
      • Birkedal-Hansen B.
      • DeCarlo A.
      • Engler J.A.
      ,
      • Llano E.
      • Pendás A.M.
      • Knäuper V.
      • Sorsa T.
      • Salo T.
      • Salido E.
      • Murphy G.
      • Simmer J.P.
      • Bartlett J.D.
      • López-Otı́n C.
      ). They can be classified into at least four main subfamilies, according to their substrate specificity, primary structures, and cellular localization: the collagenases, gelatinases, stromelysins, and membrane-type MMPs (MT-MMPs). However, there are some recently described enzymes like macrophage metalloelastase (
      • Belaaouaj A.
      • Shipley J.M.
      • Kobayashi D.K.
      • Zimonjic D.B.
      • Popescu N.
      • Silverman G.A.
      • Shapiro S.D.
      ), stromelysin-3 (
      • Basset P.
      • Bellocq J.-P.
      • Wolf C.
      • Stoll I.
      • Hutin P.
      • Limacher J.M.
      • Podhajcer O.L.
      • Chenard M.P.
      • Rio M.C.
      • Chambon P.
      ), MMP-19 (
      • Pendás A.M.
      • Knäuper V.
      • Puente X.S.
      • Llano E.
      • Mattei M.G.
      • Apte S.
      • Murphy G.
      • López-Otı́n C.
      ), and enamelysin (
      • Llano E.
      • Pendás A.M.
      • Knäuper V.
      • Sorsa T.
      • Salo T.
      • Salido E.
      • Murphy G.
      • Simmer J.P.
      • Bartlett J.D.
      • López-Otı́n C.
      ), which do not appear to fall into any of these subfamilies. In addition to all these MMPs identified in human tissues, distinct MMPs have been also cloned fromXenopus laevis (
      • Stolow M.A.
      • Bauzon D.D.
      • Li J.
      • Sedgwick T.
      • Liang V.C.T.
      • Sang Q.A.
      • Shi Y.B.
      ,
      • Yang M.
      • Murray M.T.
      • Kurkinen M.
      ), embryonic sea urchin (
      • Lepage T.
      • Gache C.
      ), green alga (
      • Kinoshita T.
      • Fuzukawa H.
      • Shimada T.
      • Saito T.
      • Matsuda Y.
      ), soybean leaves (
      • Pak J.H.
      • Liu C.Y.
      • Huangpu J.
      • Graham J.S.
      ), chicken (
      • Yang M.
      • Kurkinen M.
      ), and Caenorhabditis elegans (
      • Wada K.
      • Sato H.
      • Kinoh H.
      • Kajita M.
      • Yamamoto H.
      • Seiki M.
      ). Biochemical characterization of the diverse MMPs has opened new views on the role of these enzymes in connective tissue remodeling processes, and evidence is accumulating that MMPs are not exclusively involved in the proteolytic degradation of extracellular matrix components. Thus, MMPs have been reported to play direct roles in other essential cellular processes such as differentiation, proliferation, angiogenesis, or apoptosis (). Some of these functions are mediated by the ability of MMPs to catalyze hydrolysis of a variety of substrates including membrane-bound precursors of cytokines, growth factors, or hormone receptors (
      • Massagué J.
      • Pandiella A.
      ,
      • Couet J.
      • Sar S.
      • Jolivet A.
      • Hai M.T.V.
      • Milgrom E.
      • Misrahi M.
      ), serum-amyloid A (
      • Mitchell T.I.
      • Jeffrey J.J.
      • Palmiter R.D.
      • Brinckerhoff C.E.
      ), insulin-like growth factor-binding proteins (
      • Fowlkes J.L.
      • Enghild J.J:
      • Suzuki K.
      • Nagase H.
      ,
      • Mañes S.
      • Mira E.
      • del Mar Barbacid M.
      • Ciprés A.
      • Fernández-Resa P.
      • Buesa J.M.
      • Mérida I.
      • Aracil M.
      • Márquez G.
      • Martı́nez-A C.
      ), proteinase inhibitors (
      • Enghild J.J.
      • Salvesen G.
      • Brew K.
      • Nagase H.
      ,
      • Sottrup-Jensen L.
      • Birkedal-Hansen H.
      ,
      • Mast A.E.
      • Enghild J.J.
      • Nagase H.
      • Suzuki K.
      • Pizzo S.V.
      • Salvesen G.
      ,
      • Pei D.
      • Majmudar G.
      • Weiss S.J.
      ), or interleukin-1β (
      • Ito A.
      • Mukaiyama A.
      • Itoh Y.
      • Nagase H.
      • Thogersen I.B.
      • Enghild J.J:
      • Sasaguri Y.
      • Mori Y.
      ).
      The identification of expanding roles for MMPs in a wide variety of biological processes has stimulated the search for new family members by using improved cloning strategies. Recently, we have utilized PCR-based methods with degenerate oligonucleotides, and expressed sequence tag (EST)-based approaches for cloning different human MMPs from both normal or tumor tissues (
      • Llano E.
      • Pendás A.M.
      • Knäuper V.
      • Sorsa T.
      • Salo T.
      • Salido E.
      • Murphy G.
      • Simmer J.P.
      • Bartlett J.D.
      • López-Otı́n C.
      ,
      • Pendás A.M.
      • Knäuper V.
      • Puente X.S.
      • Llano E.
      • Mattei M.G.
      • Apte S.
      • Murphy G.
      • López-Otı́n C.
      ,
      • Freije J.P.
      • Dı́ez-Itza I.
      • Balbı́n M.
      • Sánchez L.M.
      • Blasco R.
      • Tolivia J.
      • López-Otı́n C.
      ,
      • Puente X.S.
      • Pendás A.M.
      • Llano E.
      • Velasco G.
      • López-Otı́n C.
      ). In this work, we have examined the possibility that additional yet uncharacterized MMPs could be produced by human tissues, with the finding of a novel family member tentatively called MMP-23. We describe the molecular cloning and complete nucleotide sequence of a cDNA coding for this proteolytic enzyme. We also report the expression of the gene in Escherichia coli and perform a preliminary analysis of the enzymatic activity of the recombinant enzyme. Finally, we report the chromosomal location of the MMP-23 gene in the human genome and analyze its expression in human tissues.

      DISCUSSION

      In this work, we describe the finding of a new human proteinase belonging to the MMP family, which we have tentatively called MMP-23. The strategy followed to identify MMP-23 was first based on a computer search of the EST data base, looking for sequences with similarity to previously characterized MMP family members. A single sequence presumably encoding the C-terminal region of a new MMP was identified, PCR-amplified from human ovary cDNA, and used to screen an ovarian cDNA library. After screening of this library and further 5′-RACE experiments, a full-length cDNA coding for MMP-23 was finally isolated and characterized. Structural analysis of the identified sequence for MMP-23 shows that it exhibits a series of protein domains characteristic of MMPs, including a prodomain, a catalytic domain, a hinge region, and a C-terminal domain. However, a more detailed analysis of the sequence deduced for MMP-23 reveals a number of specific structural features for this novel human proteinase. Thus, the identified sequence lacks the recognizable signal sequence present at the N-terminal end of all the remaining MMPs, suggesting that this novel enzyme could function in an intracellular compartment. However, the use of alternative secretory mechanisms as proposed for other proteins such as basic fibroblast growth factor, which are secreted despite lacking signal peptide, can not be excluded (
      • Bikfalvi A.
      • Klein S.
      • Pintucci G.
      • Rifkin D.B.
      ). In addition, the prodomain identified for MMP-23 is unusually short although it contains a single Cys residue that can be part of the activation locus characteristic of MMPs. In this regard, it is well known that MMPs are synthesized as inactive precursors with an N-terminal propeptide that maintains the latency of the enzymes through a Cys-switch mechanism (
      • Van Wart H.E.
      • Birkedal-Hansen H.
      ). According to this mechanism, coordination of the unpaired cysteine residue in the propeptide with the zinc ion in the active site leads to inactivation of the enzyme. Disruption of the Cys-zinc bond by limited proteolysis or conformational perturbations leads to opening of the switch and subsequent autocatalytic cleavages, finally resulting in the generation of a catalytically competent enzyme. The single Cys residue in the propeptide region of human MMP-23, which is conserved in the putative mouse and rat homologs of this protein (GenBank accession numbers AF085742 and AB010960), could participate in maintaining the latency of this enzyme. Nevertheless, the absence of conserved residues around this Cys residue in MMP-23, when compared with other MMPs (Fig.2), could suggest that these consensus residues (P-R-C-G-V-P-D) are not essential for the appropriate functioning of the Cys-switch mechanism in all MMPs. Additionally, in relation with the activation mechanism of proMMP-23, the presence of a stretch of basic residues linking the pro- and the catalytic domains is indicative of a furin-mediated activation for this enzyme, in a similar fashion to that described for stromelysin-3 and MT-MMPs (
      • Pei D.
      • Weiss S.J.
      ,
      • Santavicca M.
      • Noel A.
      • Angliker H.
      • Stoll Y.
      • Segain J.P.
      • Anglard P.
      • Chretien M.
      • Seidah N.
      • Basset P.
      ).
      An additional distinctive feature of the structure determined for MMP-23 derives from its unique C-terminal domain. All human MMPs characterized to date, with the exception of matrilysin, contain an hemopexin-like region of about 200 amino acids organized into four recognizable repeats (,
      • Gomis-Rüth F.X.
      • Gohlke U.
      • Betz M.
      • Knäuper V.
      • Murphy G.
      • López-Otı́n C.
      • Bode W.
      ). In contrast, the C-terminal domain of MMP-23 contains a domain of only 100 residues that lacks any significant similarity with hemopexin. Because this domain has been reported to be important in defining the substrate specificity of MMPs, and in mediating interactions with inhibitors (
      • Sánchez-López R.
      • Alexander C.M.
      • Behrendtsen O.
      • Breathnach R.
      • Werb Z.
      ,
      • Knäuper V.
      • Cowell S.
      • Smith B.
      • López-Otı́n C.
      • O'Shea M.
      • Morris H.
      • Zardi L.
      • Murphy G.
      ), the occurrence of a shortened domain in MMP-23 could be relevant in determining the substrate specificity and catalytic properties of this novel enzyme. In this regard, it is also worth mentioning that MMP-23 also lacks a series of structural features distinctive of the diverse MMP subclasses, including the Asp, Tyr, and Gly residues located close to the zinc-binding site of collagenases, the fibronectin-like domain of gelatinases, and the transmembrane domain of MT-MMPs (
      • Collier I.E.
      • Wilhelm S.M.
      • Eisen A.Z.
      • Marmer B.L.
      • Grant G.A.
      • Seltzer J.L.
      • Kronberger A.
      • He C.
      • Bauer E.A.
      • Goldberg G.I.
      ,
      • Wilhelm S.M.
      • Collier I.E.
      • Marmer B.L.
      • Eisen A.Z.
      • Grant G.A.
      • Goldberg G.I.
      ,
      • Sato H.
      • Takino T.
      • Okada Y.
      • Cao J.
      • Shinagawa A.
      • Yamamoto E.
      • Seiki M.
      ,
      • Will H.
      • Hinzmann B.
      ,
      • Takino T.
      • Sato H.
      • Shinagawa A.
      • Seiki M.
      ).
      Taking all these structural data collectively, it seems clear that MMP-23 cannot be classified in any of the previously defined MMP subfamilies. Consequently, it must be placed into the growing group of “other MMPs,” which includes enzymes such as macrophage metalloelastase, stromelysin-3, MMP-19, or enamelysin, all of them having distinctive structural and/or functional properties (
      • Llano E.
      • Pendás A.M.
      • Knäuper V.
      • Sorsa T.
      • Salo T.
      • Salido E.
      • Murphy G.
      • Simmer J.P.
      • Bartlett J.D.
      • López-Otı́n C.
      ,
      • Belaaouaj A.
      • Shipley J.M.
      • Kobayashi D.K.
      • Zimonjic D.B.
      • Popescu N.
      • Silverman G.A.
      • Shapiro S.D.
      ,
      • Basset P.
      • Bellocq J.-P.
      • Wolf C.
      • Stoll I.
      • Hutin P.
      • Limacher J.M.
      • Podhajcer O.L.
      • Chenard M.P.
      • Rio M.C.
      • Chambon P.
      ,
      • Pendás A.M.
      • Knäuper V.
      • Puente X.S.
      • Llano E.
      • Mattei M.G.
      • Apte S.
      • Murphy G.
      • López-Otı́n C.
      ). In this work, we have also examined the activity of recombinant MMP-23 produced in E. coli as a fusion with the MMP-19 prodomain, and purified and refolded following the same procedure previously used for producing other active MMPs (
      • Llano E.
      • Pendás A.M.
      • Knäuper V.
      • Sorsa T.
      • Salo T.
      • Salido E.
      • Murphy G.
      • Simmer J.P.
      • Bartlett J.D.
      • López-Otı́n C.
      ,
      • Pendás A.M.
      • Knäuper V.
      • Puente X.S.
      • Llano E.
      • Mattei M.G.
      • Apte S.
      • Murphy G.
      • López-Otı́n C.
      ,
      • Balbı́n M.
      • Fueyo A.
      • Knäuper V.
      • Pendás A.M.
      • López J.M.
      • Jiménez M.G.
      • Murphy G.
      • López-Otı́n C.
      ). This recombinant protein shows low proteolytic activity against a synthetic peptide commonly used for analyzing the enzymatic properties of MMPs. Nevertheless, the fact that the detected proteolytic activity is inhibitable by TIMP-1 suggests that it corresponds to a bona fide MMP. A likely possibility to explain this low activity can be that the majority of the recombinant protein is not correctly folded. However, the possibility that this novel enzyme may have specific substrates whose nature is currently unknown, cannot be be definitively ruled out. Also consistent with the above discussed distant relationship between MMP-23 and other human MMPs, chromosomal mapping of the MMP-23 gene has shown that it is located at chromosome 1, a unique position among all MMP genes mapped to date (
      • Huhtala P.
      • Eddy R.L.
      • Fan Y.S.
      • Byers M.G.
      • Shows T.B.
      • Tryggvason K.
      ,
      • Levy A.
      • Zucman J.
      • Delattre O.
      • Mattei M.G.
      • Rio M.C.
      • Basset P.
      ,
      • St Jean P.L.
      • Zhang X.C.
      • Hart B.K.
      • Lamlun H.
      • Webster M.W.
      • Steed D.L.
      • Henney A.M.
      • Ferrel R.E.
      ,
      • Pendás A.M.
      • Matilla T.
      • Estivill X.
      • López-Otı́n C.
      ,
      • Mignon C.
      • Okada A.
      • Mattei M.G.
      • Basset P.
      ,
      • Pendás A.M.
      • Santamarı́a I.
      • Alvarez M.V.
      • Pritchard M.
      • López-Otı́n C.
      ,
      • Mattei M.G.
      • Roeckel N.
      • Olsen B.R.
      • Apte S.S.
      ). Furthermore, the pattern of MMP-23 expression in human tissues is also somewhat unusual. Thus, in this work, we have provided evidence that this gene is abundantly expressed in a number of normal tissues, which is in marked contrast with the highly restricted expression of most MMPs in adult tissues under normal quiescent conditions. It is also of interest that MMP-23 is predominantly expressed in reproductive tissues, and particularly in ovary, suggesting that this proteinase could play some specific role in any of the matrix-remodeling processes occurring in these tissues (
      • Hulboy D.L.
      • Rudolph L.A.
      • Matrisian L.M.
      ). The availability of specific reagents for MMP-23 generated in this work will be very helpful to examine the functional relevance of this enzyme in physiological processes. Finally, the isolation of genomic clones for murine MMP-23
      G. Velasco, unpublished results.
      opens the possibility of generating animals deficient in this gene, which will contribute to clarification of its precise role in the context of other proteases produced in reproductive processes.

      Acknowledgments

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

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