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Identification and Characterization of a Novel Human Matrix Metalloproteinase with Unique Structural Characteristics, Chromosomal Location, and Tissue Distribution*

  • Alberto M. Pendás
    Footnotes
    Affiliations
    From the Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006-Oviedo, Spain the
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  • Vera Knäuper
    Affiliations
    § Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN, United Kingdom the
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  • Xose S. Puente
    Footnotes
    Affiliations
    § Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN, United Kingdom the
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  • Elena Llano
    Affiliations
    § Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN, United Kingdom the
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  • Marie-Genevieve Mattei
    Affiliations
    ∥ Hopital d'Enfants, INSERM U406, Groupe Hospitalier de la Timone, 13385-Marseille, France and the
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  • Suneel Apte
    Affiliations
    ** Department of Biomedical Engineering, Cleveland Clinic Foundation, Cleveland, Ohio 44195
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  • Gillian Murphy
    Affiliations
    § Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN, United Kingdom the
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  • Carlos López-Otín
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    From the Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006-Oviedo, Spain the
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  • Author Footnotes
    * This work was supported in part by grants from Comisión Interministerial de Ciencia y Tecnología-Spain (SAF94-0892); EU-BIOMED II (BMH4-CT96-0017), Glaxo-Wellcome, Spain; Arthritis and Rheumatism Council, U.K.; an Investigator Award from the Arthritis Foundation, the Cleveland Clinic Foundation, and National Institutes of Health Grant AR43386 (to S. A.); and by grants from Association pour la Recherche Contre le Cancer and Federation Nationale des Centers de Lutte Contre le Cancer (to M.-G. M.). 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) X92521.
    Recipient of the Severo Ochoa Fellowship from Ayuntamiento de Oviedo-Asturias (Spain).
    Recipient of a fellowship from Fundación para la Investigación Científica y Técnica (FICYT-Asturias).
    1 The abbreviations used are:MMPmatrix metalloproteinasebpbase pair(s)DAPIdiamidine-2-phenylindole dihydrochlorideESTexpressed sequence tagFISHfluorescent in situ hybridizationPAGEpolyacrylamide gel electrophoresisPCRpolymerase chain reactionTIMPtissue inhibitor of metalloproteinasesPACP1 artificial chromosomeIPTGisopropyl-1-thio-β-D-galactopyranosideDTTdithiothreitol.
Open AccessPublished:February 14, 1997DOI:https://doi.org/10.1074/jbc.272.7.4281
      We have cloned a novel member of the matrix metalloproteinase (MMP) family of proteins from a human liver cDNA library. The isolated cDNA contains an open reading frame coding for a polypeptide of 508 amino acids, which has been tentatively called MMP-19. This protein exhibits the domain structure characteristic of previously described MMPs, including a signal sequence, a prodomain with the cysteine residue essential for maintaining the latency of these enzymes, an activation locus with the zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin. However, it 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 in collagenases, the fibronectin-like domain of gelatinases, the transmembrane domain of membrane-type (MT) MMPs, and the furin-activation sequence common to stromelysin-3 and MT-MMPs. In addition, the 9-residue insertion rich in hydrophobic amino acids present at the hinge region in stromelysins is replaced in MMP-19 by a longer insertion very rich in acidic residues. On the basis of these structural characteristics, we propose that MMP-19 does not belong to any of the previously defined MMP-subclasses and may represent the first member of a new MMP subfamily. Chromosomal location of the MMP-19 gene revealed that it maps to chromosome 12q14, which is also a unique location for any MMPs mapped to date. The cDNA encoding a full-length MMP-19 was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade synthetic substrates for MMPs. MMP-19 proteolytic activity was abolished by TIMP-2 and EDTA, thus providing additional evidence that the isolated cDNA codes for an authentic MMP. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues revealed that MMP-19 is mainly expressed in placenta, lung, pancreas, ovary, spleen, and intestine, suggesting that it may play a specialized role in these tissues.

      INTRODUCTION

      The human matrix metalloproteinases (MMPs)

      The abbreviations used are:

      MMP
      matrix metalloproteinase
      bp
      base pair(s)
      DAPI
      diamidine-2-phenylindole dihydrochloride
      EST
      expressed sequence tag
      FISH
      fluorescent in situ hybridization
      PAGE
      polyacrylamide gel electrophoresis
      PCR
      polymerase chain reaction
      TIMP
      tissue inhibitor of metalloproteinases
      PAC
      P1 artificial chromosome
      IPTG
      isopropyl-1-thio-β-D-galactopyranoside
      DTT
      dithiothreitol.
      are a group of structurally related endopeptidases that degrade the different macromolecular components of the extracellular matrix and basement membranes at a neutral pH. These enzymes have been implicated in the remodeling of connective tissues occurring in normal and pathological processes (
      • 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.
      • Liota L.A.
      ). At present, the family of human MMPs is composed of 14 members that can be classified into four different families: collagenases, gelatinases, stromelysins, and membrane-type MMPs (MT-MMPs), although there are some enzymes like macrophage metalloelastase (
      • Belaaouaj A.
      • Shipley J.M.
      • Kobayashi D.K.
      • Zimonjic D.B.
      • Popescu N.
      • Silverman G.A.
      • Shapiro S.D.
      ) and 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.
      ) that do not belong to these groupings. The collagenases (fibroblast collagenase, neutrophil collagenase, and collagenase-3) cleave the native helix of fibrillar collagens at a single peptide bond, generating fragments of about three-fourths and one-fourth the size of the intact molecule (
      • Welgus H.G.
      • Jeffrey J.J.
      • Eisen A.Z.
      ,
      • Hasty K.A.
      • Jeffrey J.J.
      • Hibbs M.S.
      • Welgus H.G.
      ,
      • Freije J.M.P.
      • Díez-Itza I.
      • Balbín M.
      • Sánchez L.M.
      • Blasco R.
      • Tolivia J.
      • López-Otín C.
      ,
      • Knäuper V.
      • López-Otín C.
      • Smith B.
      • Knight G.
      • Murphy G.
      ). The gelatinases (72- and 92-kDa type IV collagenases) recognize and degrade basement membranes and denatured collagens and may act synergistically with collagenases in the degradation of fibrillar collagens (
      • 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.
      ). The stromelysins (stromelysins-1 and −2 and matrilysin) exhibit a broad substrate specificity and have the ability to degrade many extracellular proteins, including proteoglycans, laminin, and fibronectin (
      • Whitham S.E.
      • Murphy G.
      • Angel P.
      • Rahmsdorf H.J.
      • Smith B.J.
      • Lyons A.
      • Harris T.J.R.
      • Reynolds J.J.
      • Herrlich P.
      • Docherty A.J.P.
      ,
      • Wilhelm S.M.
      • Collier I.E.
      • Kronberger A.
      • Eisen A.Z.
      • Marmer B.L.
      • Grant G.A.
      • Bauer E.A.
      • Goldberg G.I.
      ,
      • Muller D.
      • Quantin B.
      • Gesnel M.-C.
      • Millon-Collard R.
      • Abecassis J.
      • Breathnach R.
      ). Finally, the MT-MMP subclass is composed of at least four members mainly characterized by the occurrence of a putative transmembrane domain in their amino acid sequences and whose proposed role is the proteolytic activation of other MMPs like 72-kDa gelatinase and collagenase-3 (
      • Sato H.
      • Takino T.
      • Okada Y.
      • Cao J.
      • Shinagawa A.
      • Yamamoto E.
      • Seiki M.
      ,
      • Okada A.
      • Bellocq J.P.
      • Rouyer N.
      • Chenard M.P.
      • Rio M.C.
      • Chambon P.
      • Basset P.
      ,
      • Strongin A.Y.
      • Collier I.
      • Bannikov G.
      • Marmer B.L.
      • Grant G.A.
      • Goldberg G.I.
      ,
      • Will H.
      • Hinzmann B.
      ,
      • Takino T.
      • Sato H.
      • Shinagawa A.
      • Seiki M.
      ,
      • Puente X.S.
      • Pendás A.M.
      • Llano E.
      • Velasco G.
      • López-Otín C.
      ,
      • Knäuper V.
      • Will H.
      • López-Otín C.
      • Smith B.
      • Atkinson S.J.
      • Stanton H.
      • Hembry R.M.
      • Murphy G.
      ).
      Because of the importance of these enzymes in both normal and pathological processes, over the last years we have been interested in examining the possibility that additional yet uncharacterized members of the MMP family could be produced by human tissues (
      • Freije J.M.P.
      • Díez-Itza I.
      • Balbín M.
      • Sánchez L.M.
      • Blasco R.
      • Tolivia J.
      • López-Otín C.
      ,
      • Puente X.S.
      • Pendás A.M.
      • Llano E.
      • Velasco G.
      • López-Otín C.
      ). In this work, we describe the molecular cloning and complete nucleotide sequence of a cDNA coding for a novel member of this family of proteolytic enzymes, which has been tentatively called MMP-19. We also report the expression of the gene in Escherichia coli and the preliminary enzymatic characterization of the recombinant enzyme. Finally, we report the chromosomal location of the MMP-19 gene in the human genome and analyze its expression in human tissues showing that it is mainly expressed in placenta, lung, pancreas, ovary, spleen, and intestine, which suggests that this novel MMP may play some specialized role in these tissues. On the basis of its structural characteristics, chromosomal location, and expression pattern in human tissues, we propose that MMP-19 may represent the first member of a new MMP subfamily.

      Acknowledgments

      We thank Dr. S. Gascón for support, Dr. P. Morán for advice in FISH studies, Drs. M. Balbín and G. Velasco for helpful comments, Drs. F. Vizoso and I. Díez-Itza for providing human tissue-samples, and S. Alvarez for excellent technical assistance. Monochromosomal somatic cell hybrid DNAs were kindly provided by the Human Genome Mapping Resource Center (Cambridgeshire, UK).

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