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WO2003104390A2 - Acides nucleiques et proteines adamts - Google Patents

Acides nucleiques et proteines adamts Download PDF

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Publication number
WO2003104390A2
WO2003104390A2 PCT/US2002/018221 US0218221W WO03104390A2 WO 2003104390 A2 WO2003104390 A2 WO 2003104390A2 US 0218221 W US0218221 W US 0218221W WO 03104390 A2 WO03104390 A2 WO 03104390A2
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Prior art keywords
adamts
procouagen
sequence
peptidase
collagen
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PCT/US2002/018221
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English (en)
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WO2003104390A3 (fr
Inventor
Suneel Apte
Satoshi Hirohata
David Eyre
Russel Fernandes
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The Cleveland Clinic Foundation
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Priority to AU2002314999A priority Critical patent/AU2002314999A1/en
Publication of WO2003104390A2 publication Critical patent/WO2003104390A2/fr
Publication of WO2003104390A3 publication Critical patent/WO2003104390A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)

Definitions

  • the present invention relates generally to isolate nucleic acid molecules encoding proteins, and the proteins themselves, belonging to a subfamily of zinc metalloproteases referred to as "ADAMTS", an abbreviation for A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs.
  • ADAMTS zinc metalloproteases
  • proteins in the ADAMTS subfamily all possess a Zn protease catalytic site consensus sequence (HEXXH+H), which suggests an intact catalytic activity for each of these proteins.
  • the ADAMTS proteins also have putative N-terminal signal peptides and lack transmembrane domains, which suggests that the proteins in this subfamily are secreted.
  • the proteins in the ADAMTS subfamily also possess at least one thrombospondin type (TSP 1) motif, which suggests a binding of these proteins to components of the extracellular matrix (ECM) or to cell surface components.
  • ECM extracellular matrix
  • Collagens comprise the major structural proteins of the extracellular matrix (ECM) and exist in both fibril-forms (e.g., collagens I, TJ, IE, V and XI) and nonfibrillar forms.
  • Molecules belonging to both categories are homo-trimeric (e.g., collagen H) or hetero-trimeric (e.g., collagen I) assemblies of specific ⁇ -chains, each the product of a single gene.
  • the molecular forms of collagens, as well as the specific supramolecular aggregates and assemblies they form, are often tissue specific, and provide specialized functions.
  • collagen I the principal collagen of skin, is arranged in randomly oriented bundles which form a sheet in the dermis, but is arranged in parallel bundles in tendons. This reflects the different mechanical demands on these two tissues.
  • Collagen IT a specific component of cartilage ECM, is arranged in an open, intercrossing pattern which traps glycosaminoglycans and facilitates resistance to compression.
  • Fibrillar collagens I, II and m are secreted as soluble procollagens comprising a long, continuous triple helical "collagenous" region with smaller polypeptide extensions (propeptides) at their amino (N) and carboxyl (C) ends.
  • propeptides polypeptide extensions
  • the procoUagen C-propeptidase is identical to bone morphogenetic protein-1 (BMP-1) and processes all three of these fibrillar collagens.
  • N-propeptidases with specificity for procoUagen I and procoUagen II or for procoUagen HI are known.
  • One bovine and human procoUagen I/H N-propeptidases has been cloned. It is known as ADAMTS-2.
  • the ADAMTS-2 protein (EC 3.4.24.14), also known as procoUagen I/H amino-propetide processing enzyme or PCINP, catalyzes cleavage of native triple-helical procoUagen I and procoUagen U.
  • the ADAMTS-2 protein also has an affinity for collagen XIN. Lack of the ADAMTS-2 protein is known to cause dermatosparaxis in cattle, or Ehlers-Danlos syndrome type V ⁇ C (EDS-N ⁇ C) in humans. EDS-NEC is characterized clinically by severe skin fragility, and biochemically by the presence in skin of procoUagen which is incompletely processed at the amino terminus.
  • the molecular hallmark is the presence of irregular, thin, branched collagen fibrils in the dermis which appear "hieroglyphic" in cross section and contain procoUagen I with an intact ⁇ -propeptide, termed p ⁇ -collagen I. Similar findings have been described very recently in Adamts2 transgenic knockout mice. Thus, it is believed that the ADAMTS-2 protein plays a role in processing of procoUagen to mature collagen, an essential step for correct assembly of collagen into collagen fibrils. num er of diseases have been shown to be caused by: synt es s o nsu ic ent amounts of collagen; synthesis of defective collagen; or over-production of normal collagen in a form referred to as either fibrotic tissue or scars.
  • amino acids amino acids
  • EDS Ehlers-Danlos syndrome
  • BMP-1 is used herin to mean bone morphogenetic protein- 1;
  • PCR is used herein to mean polymerase chain reaction
  • RT-PCR is used herein to mean reverse transcriptase-PCR
  • kbp is used herein to mean kilobase pairs
  • base pairs are used herein to mean base pairs
  • nt is used herein to mean nucleotides
  • ECM extracellular matrix
  • TS is used herein to mean thrombospondin
  • Glyceraldehyde 3-phosphate dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase
  • UV ultraviolet
  • ADAMTS2 and ADAMTS3 are human genes.
  • Adamts2 and Adamts3 are the corresponding mouse orthologs.
  • the protein products of the respective genes are designated ADAMTS-2 and ADAMTS-3.
  • Trivial names for the protein products are procoUagen ⁇ -propeptidase 1 (PC ⁇ P1) and procoUagen ⁇ -propeptidase 2 (PC ⁇ P2).
  • the preferred nucleic acids of the invention are homologs and alleles of the nucleic acids of ADAMTS3.
  • the invention further embraces functional equivalents, variants, analogs and fragments of the foregoing nucleic acids and also embraces proteins and peptides coded for by any of the foregoing.
  • variants and other defined terms, reference is made to U.S. Patent No. 6,391,610, which is hereby incorporated in its entirety by reference thereto.
  • the present invention also provides isolated polynucleotides which encode an ADAMTS-3 protein or a variant thereof, polynucleotide sequences complementary to such polynucleotides, vectors containing such polynucleotides, and host cells transformed or transfected with such vectors.
  • the present invention also relates to antibodies which are immunospecific for one or more of the ADAMTS-3 proteins.
  • an isolated nucleic acid molecule is provided.
  • the invention further embraces nucleic acid molecules that differ from the foregoing isolated nucleic acid molecules in codon sequence due to the degeneracy of the genetic code.
  • the invention also embraces complements of the foregoing nucleic acids.
  • Preferred isolated nucleic acid molecules are those comprising mammallian cDNAs or gene corresponding to ADAMTS3. Even more preferably the present invention relates to isolated nucleic acid molecules comprising human cDNA or genes corresponding to ADAMTS - 3.
  • isolated polypeptides e.g. ADAMTS-3 coded for by the isolated nucleic acid molecules described above also are provided as well as functional equivalents, variants, analogs and fragments thereof.
  • the polypeptide is a human procoUagen U N-propeptidase protein or a functionally active fragment or variants thereof.
  • Another embodiment of the present invention relates to , isolated, substantially purified, mammalian proteins belonging to the ADAMTS-3 subfamily.
  • substantially purified refers to a protein that is removed from its natural environment, isolated or separated, and at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated.
  • Another embodiment of the present invention is comprised of a method of preventing, treating or ameliorating a tissue-related disorder or condition in a patient comprising the steps of administering an effective amount of ADAMTS3 or ADAMTS-3 to a site in need of repair, regeneration, or procoUagen processing.
  • This embodiment may preferably include the steps of combining an effective amount of ADAMTS3 or ADAMTS-3 and a delivery system to form a mixture; molding said mixture to form an implant; and implanting said implant into said patient.
  • the implant is preferably suitable as a tissue substitute, and even more preferably is biodegradable.
  • the delivery system may be comprised of any suitable delivery system including natural polymers, systhetic polymers, collagen, hydroxyapatite, calcium phosphate ceramics, bioglass, hydrogels and mixtures thereof.
  • the effective amount of ADAMTS-2 may be delivered as a protein, a nucleic acid (e.g. a nucleic acid is selected from the group consisting of a gene, a cDNA, a vector, an RNA molecule, an antisense molecule, a ribozyme, and a peptide nucleic acid (PNA) molecule).
  • a nucleic acid e.g. a nucleic acid is selected from the group consisting of a gene, a cDNA, a vector, an RNA molecule, an antisense molecule, a ribozyme, and a peptide nucleic acid (PNA) molecule.
  • PNA peptide nucleic acid
  • Embodiments of the present invention also contemplate gene therapy, wherein defective cells of a donor are genetically engineered to include an isolated nucleic acid expressing a functional ADAMTS-3 protein. The cells are then returned to the donor.
  • One aspect of the present invention involves the discovery and isolation of the ADAMTS3 cDNA and the corresponding ADAMTS-3 protein
  • the expression and biological activity of the proteins are believed to be necessary for normal procoUagen processing, and alteration of the expression or biological activity of these proteins may be used to influence propeptidase activity and thereby affect collagen properties.
  • normal procoUagen biosynthesis can be established by supplying a nucleic acid expressing ADMATS-3.
  • the invention in another aspect involves a method for decreasing procoUagen propeptidase activity in a subject.
  • An agent that selectively binds to an isolated nucleic acid molecule described herein or an expression product thereof is administered to a subject in need of such treatment, in an amount effective to decrease procoUagen propeptidase activity in the subject.
  • Preferred agents are modified antisense nucleic acids and polypeptides.
  • ADAMTS3 the KIAA0366 gene
  • the novel sequence described here has been submitted to GenBank and is available with Accession No. AF247668.
  • Figure 1 A illustrates the domain organization of ADAMTS-3 and ADAMTS-2 with a key for the domains shown at the bottom of the figure;
  • Figure IB illustrates alignment of the primary structures of ADAMTS-3 and ADAMTS-2 using the single-letter amino acid code
  • Figure 2 A illustrates quantitative RT-PCR assay of mRNA levels of ADAMTS-3 and ADAMTS-2 in skin fibroblasts
  • Figure 2B illustrates quantitative RT-PCR assay of mRNA levels of ADAMTS-3 and ADAMTS-2 in human cartilage
  • Figure 3 illustrates that ADAMTS-3 excites the N-propeptide of Collagen II;
  • Figure 4 shows an amino acid sequence (SEQ ID NO: 1) of a full-length sequence for human ADAMTS-3;
  • Figure 5 shows a nucleotide sequence (SEQ ID NO: 2) of a full-length human ADAMTS3 (coding region is in upper case) which encodes ADAMTS-3;
  • Figure 6 shows an amino acid sequence (SEQ ID NO: 3) corresponding to the portion of human ADAMTS-3 corresponding to Accession No. AF247668;
  • Figure 7 shows a Nucleotide sequence (SEQ ID NO. 4) corresponding to the portion of ADAMTS3 which is necessary to encode ADAMTS-3.
  • ADAMTS3 cDNA was originally. partially cloned from human brain and named the KIAA0366 gene. The KIAA0366 cDNA was incomplete at the 5 'end, and the translation start codon had not been identified. Through molecular cloning described herein, the complete primary structure of ADAMTS3 is identified herein as shown in SEQ ID NO: 2 and Figure 5.
  • ADAMTS-3 complete sequence shown in SEQ ID NO: 1 and Figure 4
  • RCS-LTC Swarm rat chondrosarcoma derived cell line
  • RCS-LTC cells fail to process procoUagen U beyond the stage of pN-collagen ⁇ , although the amino acid sequence of the N-propeptidase cleavage site in RCS- LTC procoUagen U is normal.
  • RCS-LTC pN-collagen II is, however, processed in vitro by addition of conditioned medium from cultures of chick chondrocytes. This suggested that RCS- LTC chondrocytes either fail to express procoUagen II N-propeptidase or lack a soluble, essential co factor.
  • RCS-LTC chondrocytes thus provide a model system for identification of genes involved in procoUagen II amino propeptide processing.
  • ADAMTS3 transfection of RCS-LTC cells with ADAMTS3 or ADAMTS2 results in conversion of a portion of the pN-collagen ⁇ to a fully processed form.
  • the results establish that N-propeptidase deficiency is responsible, at least in part, for defective collagen processing in RCS-LTC cells.
  • steady-state mRNA levels of ADAMTS2 and ADAMTS3 are different in normal human skin, in skin fibroblasts, and in cartilage, with ADAMTS-3 being expressed at higher levels than ADAMTS-2 in cartilage. Together, these data suggest that ADAMTS-3 may be a major physiological procoUagen II N- propeptidase.
  • embodiments of the present invention may include ADAMTS subfamily protein members, particularly those which are procoUagen propeptidases, genes encoding those proteins, functional modifications and variants of the foregoing, useful fragments of the foregoing, as well as therapeutics and diagnostics relating thereto. More particularly, the present invention relates to ADAMTS-3 and ADAMTS3
  • procoUagen N-propeptidase genes e.g. ADAMTS3
  • an aspect of the invention is those nucleic acid sequences which code for procoUagen N-propeptidase proteins and which hybridize to a nucleic acid molecule consisting of ADAMTS 3, under stringent conditions, preferably highly stringent conditions.
  • stringent conditions refers to parameters with which the art is familiar, and reference is again made to U.S. Patent No. 6,391,610, ehich again, is incorporated herein in its entiret by refence thereto, particularly with regard to the definitions of "stringent” and "highliy stringent”.
  • the invention also provides isolated unique fragments of ADAMTS 3.
  • a unique fragment is one that is a "signature" for the larger nucleic acid.
  • Unique fragments can be used as probes in Southern blot assays to identify family members or can be used in amplification assays such as those employing PCR.
  • Unique fragments also can be used to produce fusion proteins for generating antibodies or for generating immunoassay components.
  • unique fragments can be employed to produce fragments of the procoUagen N-propeptidase protein, useful, for example, in immunoassays or as a competitive inhibitor of the substrate of the procoUagen N- propeptidase protein in therapeutic applications.
  • Unique fragments further can be used as antisense molecules to inhibit the expression of the procoUagen N-propeptidase proteins of the invention, particularly for therapeutic purposes as described in greater detail below.
  • the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding an procoUagen N-propeptidase protein, to decrease procoUagen N-propeptidase activity (particularly procoUagen I N-propeptidase activity). This is desirable in virtually any medical condition wherein a reduction in collagen production activity is desirable.
  • antisense oligonucleotide or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and or the translation of that mRNA.
  • the antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene.
  • Antisense oligonucleotides may be administered as part of a pharmaceutical composition.
  • a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • the compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • the invention also involves expression vectors coding for procoUagen N-propeptidase proteins (particularly procoUagen II N propeptidase proteins) and fragments and variants thereof and host cells containing those expression vectors.
  • procoUagen N-propeptidase proteins particularly procoUagen II N propeptidase proteins
  • host cells containing those expression vectors may be used in the practice of the invention.
  • prokaryotic or eukaryotic which can be transformed with heterologous D ⁇ A or R ⁇ A and which can be grown or maintained in culture, may be used in the practice of the invention.
  • Examples include bacterial cells such as E.coli and mammalian cells such as mouse, hamster, pig, goat, primate, etc. They may be of a wide variety of tissue types.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids and phagemids.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or fransfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g. ⁇ -galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or fransfected cells, hots, colonies or plaques.
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and ifthe nature of the linkage between the two DNA sequences does not: result in the introduction of a frame-shift mutation; interfere with the ability of the promoter region to direct the transcription of the coding sequences; or interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • RNA heterologous DNA
  • RNA heterologous DNA
  • That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
  • a defective chondrocyte or fibroblast or precursor thereof is treated with DNA in a manner to promote via homologous recombination intracellularly the correction of a defective procoUagen N- propeptidase gene.
  • the invention also contemplates screening assays to detect the presence or absence of the procoUagen N-propeptidase protein and in purification protocols to isolate procoUagen N- propeptidase proteins.
  • a therapeutically effective amount means that amount necessary to delay the onset of, inhibit the progression of, or halt altogether the particular condition being treated.
  • Therapeutically effective amounts specifically will be those which desirably influence procoUagen N-propeptidase activity, be it inhibiting or enhancing procoUagen N-propeptidase I or enhancing procoUagen N propeptidase II. When it is desired to decrease procoUagen N-propeptidase I/TI activity, then any inhibition of procoUagen N-propeptidase activity is regarded as a therapeutically effective amount.
  • any enhancement of procoUagen N-propeptidase activity is regarded as a therapeutically effective amount.
  • a therapeutically effective amount will vary with the subject's age, condition, and sex, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art.
  • the dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication.
  • the therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
  • pulmonary aerosol When antibodies are used therapeutically, a preferred route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Curie, "Aerosols," in Remington's Pharmaceutical Sciences; 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resort to undue experimentation. When using antisense preparations of the invention, slow intravenous administration is preferred.
  • the invention also contemplates gene therapy.
  • the procedure for performing ex vivo gene therapy is well known in the art. In general, it involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject.
  • the functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s).
  • Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art
  • In vivo gene therapy using vectors such as adenovirus also is contemplated according to the invention.
  • In vivo gene therapy can also be used for systemic treatement, an area in which gene therapy has broad applications.
  • Systemic treatment involves transfecting target cells with the DNA of interest, expressing the coded protein in that cell, and the capability of the transformed cell to subsequently secrete the manufactured protein into blood.
  • PCR was done with nested ADAMTS3 specific antisense oligonucleotide primers 5 'TCAAGGCCTTCCAGGTCCGACTCTC3 'and 5 'GGGAGCCTGTTCTACAGCTGATCTC3 ' and with nested adaptor primers at the 5 'end of the template.
  • the RACE products were cloned and sequenced as previously described.
  • ADAMTS3 andADAMTS2 expression constructs To generate a cDNA construct for expression of full length ADAMTS-3, we first deleted the 5'end of the KIAA0366 cDNA (in pBluescript U SK+ [Stratagene, La Jolla], provided by Dr. Takahiro Nagase of the Kazusa DNA Institute). The deleted segment extended from the 5 ' Sal I cloning (i.e., vector) site up to a unique internal ⁇ 4ccl site at nt position 598 (KIAA0366 sequence enumeration). We replaced this fragment with a PCR-derived fragment of ADAMTS3 cDNA extending from the 5 ' untranslated sequence to just downstream of the Acc ⁇ site.
  • PCR was performed with Advantage PCR reagents (Clontech, Palo Alto, CA), using the RACE cDNA clone as template, the forward primer 5 'AACTCGAGGAAAGTGAACTCGACTCGTG3 ' (Xhol site underlined) and reverse primer 5 'AGCCTGTTCTACAGCTGATC3 '.
  • the resulting amplicon was digested with Xhol and Accl (at the internal ⁇ 4ccl site) and cloned into the Sall-Accl restricted KIAA0366 cDNA. This introduced the authentic ADAMTS3 ribosome binding sequence, translation start codon and complete signal peptide into the KIAA0366 cDNA.
  • ADAMTS-3 This insert encoding full-length ADAMTS-3 was excised from pBluescript wit Xho ⁇ and Notl and ligated into the corresponding sites in pcD ⁇ A 3.1 (+) myc-His A (Invitrogen, San Diego, CA). In this mammalian expression construct, ADAMTS-3 is not in frame with the C-terminal myc and poly-histidine tags.
  • ADAMTS-2 three overlapping bovine cD ⁇ A clones which have been previously reported were appropriately digested (with Notl, EcE, E ⁇ gl, and Kpnl) and assembled to generate a construct encoding full-length bovine ADAMTS-2.
  • the ADAMTS2 cD ⁇ A was inserted into the NotVXb-1 sites of the pcD ⁇ A3 expression vector (Invitrogen, San Diego, CA) using a Xbz . adapter. These were kindly provided by Dr. A.C. Colige.
  • RNA RNA extracted from skin explants. Skin samples obtained from the forearm of healthy volunteers were used to isolate dermal fibroblasts or were stored in liquid nitrogen until use (Laboratoire de Biologie des Tissus Conjonctifs, Sart-Tilman, Belgium, Ethics Committee Approval F94/14/1871). Dermal fibroblasts grown from skin explants were cultivated in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Minimum Essential Medium
  • FBS fetal bovine serum
  • CsCl 2 cesium chloride
  • EDTA ethylene diamine tetra-acetic acid
  • RNA Quantitative reverse transcriptase -PCR (RT-PCR) analysis.
  • Total RNA (2.5 mg) was reverse-transcribed with the Superscript First-Strand Synthesis System for RT-PCR (Life Technologies, RockviUe, MD) using oligo-dT as a primer.
  • Real-time PCR was performed in an ABI Prism 7700 Sequence Detector using SYBR Green PCR Core Reagents (Applied Biosystems, Foster City, CA). In this system, continuous, automated quantitation of the PCR product is performed by measuring the fluorescence generated by the binding of SYBR Green to double-stranded DNA. All PCR amplifications were performed in triplicate along with parallel measurements of GAPDH cDNA (an internal control).
  • PCR products were also separated on a 2.0 % agarose gel, visualized with UV light through a SYBR Green filter and photographed.
  • ADAMTS2 5 GGGAAGCACAACGACATTG3 '(forward) and 5 'CTCGGTCGTCGAGGGATTAG3 '(reverse), ADAMTS3, 5 'TCAGTGGGAGGTCCAAATGCA3 '(forward) and 5 'GCAAAGAAGGAAGCAGCAGCC3 '(reverse), GAPDH, 5 'CCACTGCCAACGTGTCAGTGG3 '(forward) and
  • RT-PCR was also performed in the absence of template.
  • Northern analysis ofADAMTS2 and ADAMTS3 expression A commercially available adult human multiple tissue northern blot and a mouse embryo northern blot (Clontech Inc. Palo Alto, CA) were hybridized as per manufacturer's instructions using ExpressHybTM hybridization fluid (Clontech, Palo Alto, CA).
  • cDNA probes were used after random-primed labeling with [ ⁇ 32 P]-dCTP: a fragment containing nucleotides 946-1379 of human ADAMTS-2 cloned in pCR4-TOPO (for human multiple tissue northern blot); a 1.1 kbp H ⁇ Tfl fragment from the KIAA0366/ ADAMTS3 cDNA (for human multiple tissue northern blot); the insert of IMAGE clone 1246561, available with GenBank accession no. AA832579 (mouse ADAMTS-2 probe for mouse embryo northern blot); the insert of IMAGE clone 727026, available with Accession no. AA402760 (mouse ADAMTS-3 probe for mouse embryo northern blot). Exposure of the blots to X-ray film was for 3-7 days.
  • RNA Poly-A+ RNA (0.8 ⁇ g) from human skin or human skin fibroblasts was electrophoresed on a formaldehyde-agarose gel and blotted to ⁇ ybond N* nylon membrane (Amersham- Pharmacia Biotech).
  • cRNA probes for human ADAMTS2 and ADAMTS3 were generated by transcription from the respective clones using the Strip-EZ RNA kit (Ambion), T3 RNA polymerase and [ ⁇ 32 P]-UTP as per manufacturer's instructions.
  • Prehybridization (1 hour) and hybridization (18 hours) were performed at 65 °C in 0.2 MNa 2 ⁇ PO 4 (pH 7.2), 1 mM EDTA, 1% bovine serum albumin, 7% sodium dodecyl sulfate (SDS) and 20% formamide. Stringency washes were carried out at 65°C in 40 mM Na 2 HPO 4 , 1 mM EDTA and 1% SDS.
  • RCS-LTC chondrocytes Monolayer cultures of RCS-LTC cells were maintained in DMEM containing 4.5 g/liter glucose (Life Technologies, Grand Island, NY) and 10% FBS (Hyclone Labs, Logan, Utah) at 37 °C in 5% CO 2 . Culture medium was changed every other day and confluent cultures were sub-cultured every 2 weeks as described previously. Lipofectamine Plus (Life Technologies, Grand Island, NY) was used for RCS-LTC transfections. Cells were plated at a density of 3 X 10 5 cells/well in 6 well plates (Falcon Franklin Lakes, N.J.).
  • fransfected cells were selected in media supp emente w th 1 mg/ml G418 sulfate (Geneticin, Life Technolog es, ran s an , Y). After 2 weeks in culture, the chondrocytes from the mock transfections did not survive selection. Geneticin-resistant ADAMTS3, ADAMTS2, and lacZ fransfected chondrocytes were expanded and maintained as pools in serial monolayer culture as previously described for the RCS-LTC cell line, but in the continued presence of 1 mg/ml Geneticin.
  • ⁇ -Galactosidase expression in the lacZ stable transfectants was detected histochemically by staining the cells with 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside (X-gal, Life Technologies, Grand Island, NY). Briefly, the cultures were rinsed with phosphate-buffered saline (150 mM NaCl, 15 mM sodium phosphate, pH 7.3), fixed with 0.2% glutaraldehyde and incubated with 1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl 2 in PBS for 24 h at 37°C.
  • phosphate-buffered saline 150 mM NaCl, 15 mM sodium phosphate, pH 7.3
  • ADAMTS3, ADAMTS2 and lacZ stable transfectants as well as untransfected RCS-LTC chondrocytes were grown in 6 well plates (Falcon, Franklin Lakes, N.J.) following sub-culture as described. For the last day of culture, the culture medium was supplemented with 10 mg/ml L-ascorbate and 100 mg/ml ⁇ - aminoproprionitrile.
  • Collagen E in extracts of cartilage and RCS-LTC transfectants was analyzed by gel electrophoresis and immunoblotting. Extracts were heated to 100°C for 3 minutes in Laemmli sample buffer containing 100 mM DTT. Collagen E chains were resolved by 6% polyacrylamide gel electrophoresis and detected after transfer to polyvinyl difluoride (PVDF) membrane (BioRad, Hercules, CA ) using a monoclonal antibody to collagen E (1C10) at a dilution of 1 :2000 and the Renaissance western blot detection reagent (NEN Lifescience Products, Boston, MA). Monoclonal antibody 1C10 (D.R.
  • ProcoUagen cleavage analysis in dermatosparactic cartilage Nasal cartilage from a dermatosparactic cow and normal fetal bovine epiphyseal cartilage were extracted in 4 M guanidine HCI, 50 mM Tris-HCl, pH 7.4 with 2 mM EDTA, 5 mM benzamidine, 2 mM PMSF and 10 mM 1,10 phenanthroline at 40°C for 24 h. Collagen in this extract was separated by reducing SDS-PAGE gel electrophoresis and visualized by Coomassie blue staining and by western blot analysis using 1 CIO as described above.
  • ADAMTS-3 protein (1205 amino acids) is comparable in length to human and bovine ADAMTS-2 (1211 and 1205 amino acids respectively).
  • the predicted mature (furin-processed) forms of these proteases are also of comparable length, 957 residues (ADAMTS-3) and 953 residues (bovine and human ADAMTS-2) long.
  • ADAMTS3 predicts a full-length protein of molecular mass 135.6 kDa and a furin processed form of 107.5 kDa.
  • a number of N-linked glycosylation sites are predicted by the sequence (see below and Fig. 1) and thus post-translational modification is likely to increase the molecular mass of ADAMTS-3.
  • Figure 1 A shows the amino acid identity (in percent) between ADAMTS-3 and ADAMTS-2 for each domain (except the C-terminal domain).
  • ADAMTS-3 and ADAMTS-2 have a similar domain structure .
  • ADAMTS-3 and ADAMTS-2 have and an overall sequence identity of 61%.
  • each of these enzymes consists of the following domains (with percent sequence identity in parentheses): signal peptide, pro-domain (37%) demarcated from the catalytic domain (85%) by a furin cleavage site, disintegrin-like domain (77%), central thrombospondin type I (TS) repeat (63%), cysteme-rich domain (67%), spacer domain (1)6%), three additional TS repeats (64%), followed by a unique, mostly nonhomologous C-terminal extension.
  • the likely furin processing site for generation of mature ADAMTS-3 and ADAMTS- 2 is indicated by the arrowhead in Figure IB.
  • Figure IB the sequence encoded by the RACE clone is overlined; TS repeats are underlined and indicated by numbers; the zinc-binding histidine triad is enclosed in a box; and the boundaries of a region of complete amino acid sequence identity in the catalytic domain are indicated by the vertical arrows.
  • the N-termini of the disintegrin-like (Dis) and spacer domains are indicated.
  • the cysteine-rich domain extends from TS repeat 1 to the start of the spacer domain.
  • Potential cell-binding RGD/E sequences are indicated by the thick overline.
  • the PLAC domains are shown by the dashed underline. Potential sites for N-linked glycosylation are indicated by asterisks.
  • ADAMTS-3 and ADAMTS-2 The presumed furin cleavage sites in ADAMTS-3 and ADAMTS-2 are at identical locations.
  • ADAMTS-3 and ADAMTS-2 catalytic domains are strikingly similar, demonstrating complete sequence identity (but for one amino acid) in a region of 69 amino acids which includes the zinc-binding active site sequence.
  • ADAMTS-3 and ADAMTS-2 each have eight consensus sites for potential N-linked glycosylation. Of these, four are conserved absolutely between ADAMTS-2 and ADAMTS-3, two conserved sites being in the pro-domain, and one site each in the catalytic domain and the second TS domain.
  • ADAMTS-3 In contrast to ADAMTS-2, which contains the potential cell binding sequence CVRGDC, ADAMTS-3 has the sequence CVRGEC at the corresponding location.
  • the C-terminal domains of these two molecules are of comparable length (184 and 191 amino acids in ADAMTS-2 and ADAMTS-3, respectively), but show little sequence similarity other than a highly conserved PLAC (Protease and LACunin) domain (83% identity).
  • the PLAC domain was first described in an insect protein, lacunin, which contains all the ancillary domains of ADAMTS, and can therefore be considered an ADAMTS-like protein.
  • the PLAC domains of human ADAMTS-3 and bovine ADAMTS-2 each contain six cysteine residues. In a previously published human ADAMTS-2 sequence (GenBank Accession No. AJ003125), one of these cysteines (at position 1090 in Fig. IB) was substituted by serine. However, it is likely that this represents a sequence variation or error since there is a cysteine at this position in three independent ADAMTS2 EST sequences (GenBank accession nos. AI417257, AI624388 and AI089232
  • ADAMTS-2 and ADAMTS-3 constitute a structurally distinctive procoUagen N- propeptidase (PCNP) subfamily of the ADAMTS family.
  • ADAMTS-2 and ADAMTS-3 as a sub- group, are st nct om t e rest o t e am y n a num er o ways.
  • n erms o domain organization these are the only two members of the ADAMTS family to have three C- terminal TS domains. Furthermore, they are the only two members of the ADAMTS family to have a substantial C-terminal extension downstream of these TS domains.
  • the location of the PLAC domain within this C-terminal extension is also unique, because in other ADAMTS family members where it is present, such as in ADAMTS-7B and ADAMTS- 10 it is usually at the very carboxyl end of the protein. In the PCNP sub-family, the PLAC domain is internal.
  • the pro-domain contains only two cysteines, in contrast to other ADAMTS enzymes, which usually contain three; and the catalytic domain contains 6 cysteines as opposed to eight for the other ADAMTS.
  • the usual arrangement consists of five cysteines upstream of the zinc-binding site and three downstream.
  • cysteines suggests that the catalytic domain of the PCNP subfamily may be structurally different from that of the other ADAMTS enzymes. Additionally, the sequence of the zinc-binding triad (HETGHLGMEHD) in the PCNP subfamily is unique in that it contains threonine in the third position (underlined), whereas all other ADAMTS enzymes have a hydrophobic residue with a long side-chain (leucine or isoleucine) at this position; and the spacer domains of the ADAMTS family are quite variable in length and sequence. However, those of the PCNP subfamily are significantly similar to each other (56% amino-acid identity).
  • ADAMTS2 andADjAMTS3 Differential tissue-specific expression of ADAMTS2 andADjAMTS3.
  • Adamts2 and Adamts3 were both expressed during mouse embryogenesis.
  • Adamts2 expression was noted in mouse embryos at 7, 15 and 17 days.
  • Two mRNA species (7.8 kb and 4.0 kb) were detected.
  • a single AdamtsS mRNA species (-7.2 kb in size) was also detected in mouse embryos at 7, 15 and 17 days of gestation.
  • Restricted expression of ADAMTS2 and ADAMTS3 was seen in the eight normal human adult tissues examined by northern analysis. In human placenta, lung and liver, two ADAMTS2 transcripts were present, migrating at approximately 7.8 kb and 4.0 kb as previously described.
  • ADAMTS2 or ADAMTS3 In skin samples and in skin fibroblasts, northern analysis with equivalent amounts of cRNA probes for ADAMTS2 or ADAMTS3 demonstrated a differential prevalence of steady- state mRNA levels. Autoradiograms generated by 1 h (ADAMTS2) and 18 h exposure (ADAMTS-3) illustrated this point. Based on these different durations of exposure, it is concluded that a substantially stronger signal is obtained with an AD AMTS2 probe than with ADAMTS3 in skin and skin fibroblasts, suggesting that they contain higher steady-state levels of ADAMTS2 mRNA than ADAMTS3 mRNA.
  • ADAMTS2 transcripts 7.0 kb, 4.0 kb and 2.0 kb
  • ADAMTS3 probe a 4.8 kb band was identified, plus 2.3 kb band in skin fibroblasts.
  • the discrepancy of these ADAMTS-3 bands with those noted in multiple tissue northerns is presently unexplained, but it is possible, that like ADAMTS2, ADAMTS3 also generates multiple transcripts in a tissue specific fashion. It was also noted that while ADAMTS3 signal is stronger in skin as opposed to skin fibroblasts, the reverse was true for ADAMTS2.
  • ProcoUagen II is completely processed in dermatosparactic cartilage. Dermatosparactic animals have no functioning ADAMTS-2. Despite this, Coomassie-blue staining of collagen extracted from dermatosparactic nasal cartilage demonstrated the presence of collagen chains which migrated similarly to the processed collagen E chains in control cartilage. This was confirmed by western blot analysis of these extracts using a monoclonal antibody which recognizes procoUagen E as well as processed collagen E. This analysis showe mat essentially all of the immunoreactive collagen was fully processed. Some pN-collagen I was visible in the extracts from dermatosparactic cartilage, confirming the origin of this tissue.
  • ADAMTS3 and ADAMTS2 process procoUagen II in transfected RCS-LTC cells.
  • the efficiency of RCS-LTC transfection was monitored by ⁇ -galactosidase staining. LacZ- transfected cells also served as a negative control for analysis of procoUagen processing.
  • Figure 5 A shows Z cZ-transfected chondrocytes on day 2 and day 8 after subculture, following transfection, Geneticin selection and histochemical staining for ⁇ -galactosidase activity.
  • ADAMTS-3 was capable of enzymatically removing the N-propeptide of procoUagen E
  • lysates of ADAMTS3-, ADAMTS2- and / ⁇ cZ-transfected RCS-LTC chondrocytes were blotted and procoUagen E and collagen E were identified using monoclonal antibody 1C10.
  • the results show some processing of pN-collagen E to mature collagen E in the ADAMTS2- and ADAMTS3- transfected cells, but none in / ⁇ cZ-transfected cells or in untransfected cells.
  • the al(IJ) chains migrate faster than the pN-collagen E chains and at a position similar to the naturally processed al(E) chain or pepsinized collagen E.
  • ProcoUagen I processing in dermatosparaxis is most deficient in skin, although mature skin has some processed collagen.
  • Many collagen I-containing dermatosparactic tissues such as tendon, ligament, sclera and aorta show the presence of significant amounts of fully processed collagen I. None of these tissues, nor bone, which relies on collagen I for its mechanical strength, have been noted to be fragile. Very recently, Adamts2 knockout mice have been reported to have significant amounts of processed collagen in skin.
  • ADAMTS-2 can process procoUagen E. Therefore, we expected that procoUagen E processing would be abnormal in dermatosparactic cartilage. However, it appears that collagen II is normally processed in dermatosparactic cartilage, despite the absence of ADAMTS-2. This leads to the conclusion that enzyme(s) other than ADAMTS-2 that could process procoUagen E exist (implication ADAMTS-3). Although EDS-NEC patients are of short stature, they do not have chondrodysplasia or premature arthritis. A defect in collagen E processing similar to the collagen I processing defect in dermatosparactic skin would be expected to cause a severe chondrodysplasia, given the critical role of collagen E in structural stability of cartilage matrix.
  • ADAMTS-2 and ADAMTS-3 comprise a structurally and functionally distinct subfamily of ADAMTS proteases.
  • ADAMTS-3 and ADAMTS-2 are closely related in the length of the polypeptide chains, and their primary sequence and domain organization, but are located on different chromosomes.
  • ancillary domains i.e., the TS, disintegrin-like, cysteine rich and spacer domains
  • ADAMTS- 1 ancillary domains are responsible for ECM-binding and the TS domains of aggrecanase-1 (ADAMTS-4) are required for binding to native aggrecan.
  • ADAMTS-4 TS domains of aggrecanase-1
  • a splice variant of ADAMTS2 which generates a short form of ADAMTS-2 lacking the ancillary domains is functionally inactive in procoUagen I processing.
  • Gene regulation can also determine which of two or more related genes are functional in any given tissue.
  • Multiple tissue northern blots demonstrated that ADAMTS2 and ADAMTS3 are differentially regulated in various tissues.
  • Data from northern blot analysis was supported by real-time, quantitative RT-PCR analysis of skin fibroblast RNA.
  • ADAMTS-3 levels were over four-fold higher than those of ADAMTS-2.
  • RCS-LTC cells suggest a failure to produce a functional processing enzyme. This could result from a structural mutation in ADAMTS-2 or ADAMTS-3 or because of transcriptional repression of these genes in RCS-LTC cells. While ot wishing to be bound by theory, it is proposed that, either on the basis of substrate preference for procoUagen E in vivo or on e basis of higher express on n cart age t an - , - is e p nc pa collagen E N-propeptidase in vivo.
  • ADAMTS-2 and ADAMTS-3 both process procoUagen E, but ADAMTS-3 is likely to be more physiologically relevant in this context, possibly due to cartilage-preferred expression.
  • Our data provide insight into the sparing of cartilage and, perhaps, into the relative sparing of some procoUagen I-containing tissues in dermatosparaxis. Since it has been shown that ADAMTS-3 will process procoUagen E, it is also likely that it will process procoUagen I. This is supported by the fact that the related gene family member AD AMTS2 can process both procoUagen I and procoUagen E.
  • the data presented herein illustrate that ADAMTS-2 and ADAMTS-3 are roughly equivalent in terms of procoUagen E processing. Finally, the amino acid cleavage sequence in procoUagen I and procoUagen E are identical.
  • Fibrosis and scarring are conditions in which excess fibrous tissue (comprising mainly collagen) is produced following injury, inflammation or attempted repair as for example after surgical incision. In such conditions, the stability and structure of the excess collagen can be rendered inferior and thus less disruptive by preventing the action of ADAMTS-3. Since removal of the N-propeptide is a requirement for formation of collagen fibers and development of an organized collagenous matrix, inhibition of ADAMTS-3 by any means constitutes a potential interference with the fibrosis process and makes ADAMTS-3 a disease or drug target. There are also applications where it may be necessary to introduce or elevate levels of ADAMTS-3.
  • ADAMTS-3 maybe introduced into the tissue engineered product by a variety of means to provide tissue engineered cartilage of optimal function. While this invention has been esc e wt an emp ass upon preerre em o imens, i will be obvious to those of ordinary skill in the art that variations of the preferred compounds and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein.

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Abstract

L'invention porte sur l'ADAMTS3 et les protéines ADAMTS-3 correspondantes, ainsi que sur leurs variantes, homologues, and équivalents, et sur leur utilisation pour le traitement au procollagène.
PCT/US2002/018221 2002-06-06 2002-06-06 Acides nucleiques et proteines adamts WO2003104390A2 (fr)

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* Cited by examiner, † Cited by third party
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WO2000053774A2 (fr) * 1999-03-08 2000-09-14 Neurocrine Biosciences, Inc. Metalloproteinases et leurs procedes d'utilisation
JP2001017183A (ja) * 1999-07-09 2001-01-23 Yamanouchi Pharmaceut Co Ltd 新規な金属プロテアーゼ

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053774A2 (fr) * 1999-03-08 2000-09-14 Neurocrine Biosciences, Inc. Metalloproteinases et leurs procedes d'utilisation
JP2001017183A (ja) * 1999-07-09 2001-01-23 Yamanouchi Pharmaceut Co Ltd 新規な金属プロテアーゼ

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FERANDES R. J. ET AL.: 'Procollagen II amino propeptide processing by ADMTS-3' J. BIOL. CHEM. vol. 276, 24 August 2001, pages 31502 - 31509 *
NAGASE T. ET AL.: 'Prediction of the encoding sequences of unidentified human genes VII. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro' DNA RES. vol. 4, no. 2, 1997, pages 141 - 150 *

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