WO1992013000A1 - Nouvel antigene thyroïdien - Google Patents

Nouvel antigene thyroïdien Download PDF

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Publication number
WO1992013000A1
WO1992013000A1 PCT/AU1992/000024 AU9200024W WO9213000A1 WO 1992013000 A1 WO1992013000 A1 WO 1992013000A1 AU 9200024 W AU9200024 W AU 9200024W WO 9213000 A1 WO9213000 A1 WO 9213000A1
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Prior art keywords
tap
peptide
antibody
protein
nucleic acid
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PCT/AU1992/000024
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English (en)
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Peter Leedman
Leonard Harrison
David Cram
Ross Leon Coppel
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Amrad Corporation Limited
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Publication of WO1992013000A1 publication Critical patent/WO1992013000A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the identification, cloning and sequencing of a novel thyroid autoantigen, hereinafter designated Truncated Actin-binding Protein (TAP), and to the preparation of synthetic or recombinant TAP or fragments or derivatives thereof.
  • TAP Truncated Actin-binding Protein
  • the invention further extends to antibodies to this factor (both polyconal and monoclonal), to pharmaceutical compositions comprising TAP or antibodies thereto and to the therapeutic or diagnostic use of TAP or the antibodies thereto.
  • TSH thyrotropin
  • Platelets contain a membrane skeleton comprised predominantly of actin filaments, an actin-binding protein (PABP) and three membrane glycoproteins GB lb, GB la and another glycoprotein of M r 250,000 (Fox 1985).
  • Actin is polymerised into filaments in unstimulated platelets, which are in close association, specifically, with the inner surface of the plasma membrane, and often co-isolate with plasma membranes during purification.
  • Purified nonmuscle platelet-derived PABP has been shown to have three main functions: it nucleates the polymerisation of purified actin (Hartwig et.al.1980).
  • HABP Human endothelial cell actin-binding protein 280
  • TSH-induced actin disruption is mediated via a thyroid HABP or PABP-equivalent (TABP), which serves as a linkage molecule between actin filaments and the TSH receptor (TSH-R).
  • TSH receptor TSH receptor
  • GPIG platelet membrane glycoprotein
  • TAP thyroid autoantigen
  • TAP tissue-derived autoantibody
  • immunoscreening represents the first description of the existence of autoantibodies to an actin- binding-like protein in patients with autoimmune thyroid disease, and therefore represents an important tool in the diagnosis of autoimmune thyroid disease.
  • TAP acts in concert with a TABP, and serves as a linkage molecule between the thyroid membrane glycoproteins, such as TSH-R and actin microfilaments, thus providing an explanation for the TSH-induced actin disruption described above.
  • TAP or its recombinant or synthetic equivalents to modulate the binding of membrane glycoprotein to the cytoskeleton, particularly thyroid member proteins including the TSH receptor could thus have important therapeutic implications for the treatment of a number of disease states particularly thyroid and blood clotting and coagulation disorders.
  • a biologically pure peptide from a mammal said peptide isolatable from the thyroid and having actin- binding-like properties.
  • the mammal is a human.
  • the peptide is truncated actin-binding protein (TAP) or TAP-like protein.
  • nucleic acid molecule encoding the above mentioned peptide.
  • the nucleic acid sequence encodes TAP or TAP- like protein.
  • Yet another aspect of the present invention contemplates a method for detecting antibodies to TAP is serum of a mammal comprising contacting said serum with an antibody-binding effective amount of TAP, TAP-like protein and/or antigenic fragment thereof for a time and under conditions sufficient to form a complex comprising an antibody-TAP, antibody-TAP- like protein and/or antibody-antigenic fragment of TAP or TAP-like protein and then subjecting said complex to a detecting means.
  • kits for detecting antibodies to TAP in serum of a mammal comprising a first container adapted to contain TAP, TAP-like protein and/or antigenic fragment thereof; and a second container adapted to contain an antibody labelled with a reporter molecule.
  • Still yet a further aspect of the present invention contemplates a method of modulating thyrotropin (TSH) induced membrane glycoprotein binding in a mammal comprising the administration of a therapeutically effective amount of TAP or TAP-like protein or therapeutically active fragments thereof for a time and under conditions sufficient to mediate said binding.
  • TSH thyrotropin
  • a method of purifying the invention which comprises (a) screening a ⁇ gtll human thyroid carcinoma cDNA library with serum from a patient having Graves disease, (b) selecting positive clones, (c) hybridizing the clones of (b) against TSH-R-Pj, (d) selecting positive clones from (c), and (e) determining nucleotide sequence of clones from (d).
  • the present invention also extends to the use of the synthetic peptide or polypeptide, or fused peptide or polypeptide or fragment, of this invention as an antigen in a diagnostic test for autoimmune thyroid disease by detection or determination of the titre of circulating antibodies to TAP in patient sera using ELISA or RIA technology or an agglutination assay using antigen-coated beads or the like.
  • a diagnostic test for the detection of thyroid autoimmune disease comprising synthetic antigen alone or in combination with one or more other previously known thyroid antigens including but not limited to Thyroid Stimulating Hormone Receptor (TSH-R), Thyroid Peroxidase (TPO), Thyroglobulin, and the thyroid equivalent of HABP and PABP previously referred to as TABP.
  • a pharmaceutical composition comprising TAP alone or in combination with other actin disrupting agents including but not limited to TABP, HABP and PABP, in association with a pharmaceutically acceptable diluent.
  • actin disrupting agents including but not limited to TABP, HABP and PABP.
  • the active component of such a pharmaceutical composition may of course be derived from natural sources or be prepared by synthetic or recombinant DNA techniques.
  • expression of the cDNA insert encoding TAP may be achieved in a number of ways.
  • the detailed description herein provides examples of expression of TAP as glutathione-s-transferase (GST) fusion proteins in the vector pGEX-2T, using as host cells E coli strain JM101.
  • Successful expression of TAP as a fusion protein may also be achieved using the well-known PVC vectors or as ⁇ - Galactosidase fusion proteins in the vectors ⁇ gtll and pBTA224.
  • TAP may be expressed as a non- fused polypeptide, by using appropriate vector and host cell combinations.
  • Other vector and host cell combinations which can be used in accordance with the present invention include a number of well described yeast shuttle vectors growing in yeast cells, or enkaryotic vectors in continuous cell lines or transgenic animals.
  • TAP thyroid antigen
  • Figure 1 shows the cDNA and Predicted Amino Acid Sequence of Human TAP.
  • TAP cDNA extends 2,007 nucleotides. Two putative initiation codons are indicated by arrows. The putative open reading frame commences at the first ATG, and extends for 195 amino acid residues, coding for a protein with a predicted M r of 20,700. A consensus sequence for a Ca +2 /calmodulin phosphorylation site is underlined. A putative site for N-linked glycosylation is identified by the interrupted line.
  • Figure 2 is a photographic representation showing an Analysis of pGEX-TAP Fusion Protein
  • TAP cDNA was subcloned into pGEX 2 and the recombinant fusion protein (F3) was purified on glutathione agarose, subjected to SDS-PAGE (12.5% w/v gel) an analysed either by Coomassie blue staining (Panel A).
  • Figure 3 is a photographic representation of Polymerase Chain Reaction Analysis of TAP Expression.
  • PCR was performed on first strand cDNA from the tissue shown using oligonucleotides (TAP-1, TAP-2) designed to amplify the coding region of TAP ("600 bp). PCR products were electrophoresed on a 1.5% w/v agarose gel and stained with ethidium bromide. Size markers are ⁇ Hindlll.
  • lane A normal human brain poly(A-t-) RNA.
  • Lane B normal mouse brain poly(A+) RNA.
  • Lane C normal human skeletal muscle total RNA.
  • Lane D normal human thyroid total RNA.
  • Lane E Graves' thyroid poly (A+) RNA.
  • Lane F porcine thyroid poly(A+) RNA.
  • Lane G TAP cDNA from Bluescript II.
  • Lane H first strand cDNA after reverse transcription of TAP in the sense orientation.
  • Lane I first strand cDNA after reverse transcription of TAP in the antisense orientation.
  • Lane J cirrhotic human liver total RNA.
  • Lane K normal human liver total RNA.
  • Figure 4 shows the Amino Acid Sequence Alignment of TAP with Human Actin-Binding Protein (HABP) .
  • Numbering refers amino acid position relative to the beginning of their respective open reading frames. Asterisks denote identical residues. Residues underlined denotes a consensus sequence for a Ca +2 /calmodulin phosphorylation site. A putative site for N-linked glycosylation is identified by the interrupted line. The sequence between the two arrows represents a putative glycoprotein binding site.
  • Figure 5 is a pictorial representation showing the Structural Comparison Between TAP and HABP.
  • A Schematic diagram of TAP. Four regions are defined: a, b, c, and d. Figures within each region represent percentage amino acid homology with HABP. The amino acid position of each region boundary is shown. Regions a and c correspond to ⁇ -sheet repeat sequences in HABP. Region b is homologous with a putative glycoprotein binding site in HABP and contains a Ca *2 /calmodulin phosphorylation consensus sequence.
  • B Schematic comparison of TAP with HABP. HABP contains 3 major regions: Head, containing an actin-binding site; Backbone, comprising 23 x "96 amino acid repeats; Tail, containing self-association and glycoprotein binding sites. Two putative amino acid insertions in HABP, prior to the 16th and 24th repeats, are shown (arrows).
  • Figure 6 is a graphical representation showing the Hydropathy Profiles of TAP and HABP.
  • Figure 7 shows Amino Acid Homology Between Human TSH-R and Platelet Glycoprotein Ib « (GP Ib«). Amino acid alignment of human TSH-R with GP IB ⁇ demonstrating homology in overlapping putative transmembrane domains. Asterisks (*) denote identical residues, " denotes a conserved amino acid substitution. Numbering refers to amino acid position relative to the beginning of their respective open reading frames.
  • the present invention relates to a novel peptide having actin-binding properties.
  • the peptide is isolatable from the thyroid and is encoded for by a gene located in thyroid cells.
  • the mammal is a human but non-human mammals are also within the scope of the present invention.
  • peptide is used, not only in its normal context as a molecule comprising a few amino acids, but is also shorthand for a polypeptide or protein.
  • the peptide may comprise solely amino acids but also may be associated with other molecules, such as carbohydrates (i.e. glycosylated), lipids or even other proteins, polypeptides or peptides.
  • Bioly pure means a material comprising at least 65% by weight, preferably 75% by weight, even more preferably 85% by weight and even more preferably 95% by weight of the peptide relative to other components. In some circumstances, a preparation could be used which is less than 65% by weight of peptide but due to the inert nature of the contaminants, is till quite effective. Such preparations are encompassed by the term “biologically pure”. This term, therefore, means any preparation of the peptide sufficiently pure or separated from other components to be effective, for example, in diagnostic assays or pharmaceutical preparations.
  • the peptide exhibits substantial amino acid homology with human endothelial cell actin- binding protein 280 (HABP).
  • HABP human endothelial cell actin- binding protein 280
  • the peptide is Truncated Actin-Binding Protein (TAP), having an amino acid sequence corresponding to the amino acid sequence shown in Figure 11.
  • TEP Truncated Actin-Binding Protein
  • One skilled in the art would immediately recognise that single or multiple amino acid deletions, insertions and/or additions may be made to the sequence shown in Figure 1 without substantially altering the nature of the peptide or while still retaining its activity or even retaining its antigenicity. All such derivatives are encompassed by the present invention and are included by the term "TAP-like protein" or "TAP-like molecule”.
  • TAP functional analogues, homologues, mutants and derivatives of TAP and to molecules having substantial homology to the sequence shown in Figure 1.
  • substantially homology is meant peptides with at least 40% or at least 60% or at least 70% or at least 85% with the amino acid sequence shown in Figure 1. All such peptides are encompassed by the term "TAP-like protein”. This term also extends to fragments and derivatives of TAP such as antigenic portions and/or active site portions of the molecule, the amino acid sequence of which, is shown in Figure 1.
  • the peptide of the present invention may be naturally occurring or may be prepared by recombinant means or by chemical synthesis. An example of the latter is by the well known Merrifield solid-phase procedure. If prepared by recombinant means, the peptide may be synthesized as a single molecule or as two or more molecules to be subsequently joined or as part of a fusion protein or polypeptide.
  • the additional fused protein or polypeptide may, for example, facilitate recovery and/or inhibit protease activity or protect the subject peptide from protease activity and may or may not be subsequently cleaved off to leave just the peptide or portion thereof.
  • the peptide, such as TAP or TAP-like protein may exist free or immobilized onto a solid support.
  • Solid supports contemplated by the present invention include glass or a polymer such as cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. Typical supports may be in the form of tubes, beads, discs or microplates. Immobilisation may be by covalent or passive binding, e.g. cross-linking covalent binding or physically adsorbing the molecule to the insoluble carrier. In some circumstances, it may be desirable or convenient to adsorb or associate the peptide to cells, such as red blood cells.
  • the present invention is also directed to nucleic acid molecules encoding the subject peptide. More particularly, the present invention is directed to a nucleic acid molecule encoding TAP or TAP-like protein.
  • Nucleic acid refers here to DNA or RNA, naturally occuring or prepared by chemical synthesis which includes recombinant means.
  • the nucleic acid is cDNA.
  • the preferred cDNA has a nucleotide sequence corresponding to the sequence shown in Figure 1 and, as with the amino acid sequence described above encompasses single or multiple nucleotide substitutions, deletions and/or additions.
  • the present invention also extends to nucleic acid molecules having substantial homology with the sequence shown in Figure 1.
  • “Substantial homology” in this context means at least 40% or at least 60% or at least 80% or at least 95% homology with the sequence shown in Figure 1.
  • the nucleic acid molecules contemplated herein also cover any or all functional analogues, homologues, derivatives or mutants of the subject peptide, such as TAP or TAP-like protein.
  • the nucleic acid molecules, e.g. cDNA may also be altered with respect to chemical compositions such as methylation without departing from the scope of the present invention.
  • the nucleic acid molecules may be carried by recombinant vectors, such as expression vectors. These vectors may be expressed and/or may replicate in eukaryotic and/or prokaryotic cells. Eukaryotic cells include mammalian, yeast, insect and plant cells and also extend to transgenic animals such as mammals.
  • Prokaryotic cells include, but are not limited to, Escherlchia coli and Bacillus sp cells.
  • the nucleic acid may be operably linked to a suitable expression control sequence and additional elements may be required, for example, to facilitate export from the cell or to further control expression of the nucleic acid.
  • the present invention covers the nucleic acid molecules encoding the subject peptide (e.g. TAP or TA;-like protein), to vectors containing same and to host cells carrying said molecules.
  • the present invention also extends to pharmaceutical compositions comprising the subject peptide and preferably TAP or TAP-like protein.
  • compositions may, as an alternative, comprise an antibody against the peptide to induce anti- idiotypic antibodies against naturally occurring antibodies specific to, for example, TAP.
  • the present invention also extends to pharmaceutical compositions comprising the subject peptide and preferably TAP or TAP-like protein alone or be in combination with actin disrupting agents such as thyroid stimulating hormone receptor (TSH-R), thyroid peroxidase (TPO), thyroglobulin and the thyroid equivalent of HABP and PABP.
  • TSH-R thyroid stimulating hormone receptor
  • TPO thyroid peroxidase
  • thyroglobulin the thyroid equivalent of HABP and PABP.
  • the present invention also contemplates a method of detecting antibodies to TAP in serum of a mammal, such as a human, comprising contacting said serum with an antibody- binding effective amount of TAP or TAP-like protein and/or antigenic fragments thereof for a time and under conditions sufficient to form a complex comprising antibody-TAP, antibody-TAP-like protein and/or antibody-antigenic fragment of TAP or TAP-like protein and then subjecting said complex to detecting means.
  • the detecting means comprises contacting said complex with a binding effective amount of a second antibody labelled with a reporter molecule, said second antibody specific to TAP, a TAP-like protein or antigenic portions thereof and then detecting said reporter molecule.
  • reporter molecule a molecule which, by its chemical nature, provides an analytically identifable signal which allows the detection of antigen- bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are enzymes, fluorophores, radionuclide containing molecules (i.e. radio!sotopes), and biotintylated molecules. However, many other detection systems are available such as agglutination of cells or beads.
  • an enzyme reporter molecule an enzyme is conjugated to the antibody, generally by means of glutaraldehyde or periodate.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.
  • p- nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for perioxidase conjugates, 1,2- phenylenediamine, 5-aminosalicyclic acid or tolidine are commonly used.
  • fluorogenic substrates which yield a fluoroescent product rather than the chromogenic substrates noted above.
  • the components are arranged in a kit comprising in compartmental form a first container adapted to contain TAP, TAP-like protein and/or an antigenic fragment thereof and a second container adapted to contain an antibody labelled with a reporter molecule.
  • the kit may also contain additional containers adapted for receipt of serum sample and/or to contain buffers or a substrate for a reporter molecule (e.g. substrate for an enzyme).
  • the present invention also contemplates a method of modulating thyrotropin (TSH) induced actin distruption in a mammal, such as a human, comprising the administration of a therapeutically effective amount of TAP, TAP-like protein and/or therapeutically active fragments thereof for a time and under conditions sufficient to mediate said disruption.
  • TSH thyrotropin
  • the effective amount of active molecule is from about 0.001 ⁇ g/kg body weight to about 100 mg/kg body weight.
  • Administration may be by any suitable route such as by intraveneous or oral administration.
  • administration may be by single dose or multiple dosages.
  • the treatment may also comprise TAP, TAP-like protein and/or therapeutically active fragments thereof in combination with actin disrupting agents such as TABP, HABP and/or PABP.
  • Such therapy may be by simultaneous administration or by sequential administration or a combination thereof.
  • RNA ladder was from Bethesda Research Laboratories (MD, USA) and ⁇ DNA Hind-III was from BRESA (Australia).
  • Glutathione agarose beads, glutathione, salmon sperm DNA, RNasin (ribonuclease inhibitor) and RNase type IIIA were from Sigma Chemical Co. (St Louis, MO, USA).
  • Thermus aquaticus DNA polymerase (Taqpolymerase) was from Perkin Elmer Cetus (Norwalk, CT, USA).
  • Glutathione S- transferase cloning and sequencing kit (pGEX 1-3 cloning vectors) was from AMRAD Corporation (Australia), isopropyl ⁇ -D-thiogalactopyranoside (IPTG), T3 and T7 DNA polymerases, DRIgest III DNA markers and T4 Iigase from Pharmacia Fine Chemicals (Uppsala, Sweden), Sequenase 2.0 sequencing kit from United States Biochemical Corporation (Cleveland, Ohio, USA), pBluescript II (KS+/-) from Stratagene (LaJolla, CA, USA), DNase 1 from Amersham International (UK) and RNAse TI from Boehringer Mannheim (West Germany).
  • pGEX 1-3 cloning vectors was from AMRAD Corporation (Australia), isopropyl ⁇ -D-thiogalactopyranoside (IPTG), T3 and T7 DNA polymerases, DRIgest III DNA markers and T4 Iigase from Pharmacia Fine Chemicals (Up
  • Human thyroid carcinome cDNA ⁇ gtll library was purchased from Clontech (Palo Alto, CA, USA) and a normal human thyroid cDNA ⁇ gtll library was constructed by Invitrogen (San Diego, CA, USA) from oligo-dT-purified mRNA that was extracted from normal human thyroid.
  • Formalin- fixed protein A-bearing Staphylococcus aureus of the Cowan I strain (Staph. protein A) was purchased as a 10% suspension from the Commonwealth Serum Laboratories (Melbourne, Australia). Electrophoresis reagents were from Bio-Rad Laboratories (Richmond, CA, USA).
  • Normal human brain poly(A+) RNA was kindly provided by Professor Claude Bernard (La Trobe University, Melbourne, Australia) and normal mouse (C3H/HEJ) poly9A+) RNA by Dr Ora Bernard (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). Normal and cirrhotic human liver total RNA was from Dr Geoff McCaughan (Royal Prince Alfred Hospital, Sydney, Australia).
  • Deoxyadenosine 5'-[ ⁇ - 32 P]triphosphate ( ⁇ - 32 P-dATP), 3000 Ci/mmol, ⁇ - 35 S-dATP, 125 Iodine ( 125 I) and 14 C-labelled M r standards for use in SDS-PAGE were purchased from Amersham International (UK).
  • Nitrocellulose membrane was purchased from Schleicher and Schuell (Dassel, West Germany).
  • the polytron homogenizer was purchased from Kienematica (Lucerne, Switzerland) and the Novablot Multiphor No. 2 electrophoresis system were from LKB (Sweden).
  • the Mighty Small gel electrophoresis system was from Hoefer Scientific Instruments (San Francisco, USA).
  • E. coli cells were infected with phage from a ⁇ gtll human thyroid carcinoma cDNA library, plated on LG-agar in 150-mm petri dishes at a density of "5 x 10 4 /plate, incubated for 3 hours at 42°C, then overlayed with nitrocellulose filters previously saturated in 10 mM IPTG and incubated for 4hours at 37°C. Filters were removed and incubated in BLOTTO overnight at room temperature and then with Graves' serum (1:100, pre-absorbed against E. coli protein extract, Huynh et al. , 1985), which had immunoprecipitated and immunoblotted a human TSH affinity- purified 50 kD protein (Leedman et al.
  • a 32 P-labelled oligonucleotide (TSH-R-P1, 5'- C A A / G GGN c / ⁇ TNGGNAAA ⁇ / c GA A / G AT c / G NAA A / G -3' , N:addition of all nucleotides), constructed from amino acid sequence derived from microsequencing of a purified porcine TSH-R 50 kD protein, was used to probe the positive clones obtained by immunoscreening as described by (Hanahan and Messelson 1983). Briefly, positive clone ⁇ phase DNA was plated as above, incubated overnight at 37°C and transferred to nitrocellulose filters.
  • Plaques were lysed in 0.5 M NaOH, 1.5 M NaCl, neutralized in 1.5 M NaCl, 0.5 M Tris-HCl, pH 7.4, rinsed in 6 x SSC buffer, dried on blotting paper and baked at 80°C for 1 hour in vacuo.
  • Pre-hybridization and hybridization with the probe (TAP) labelled with ⁇ - 32 P-ATP was performed in 7 x SSPE buffer, 10 x Denhardt's solution, 100 ⁇ g/ml salmon sperm DNA and 0.1% SDS w/v at 65°C for 16 hours. The probe was added to the hybridization mixture at "5 x 10 5 cpm/ral.
  • the DNA sequence of the TAP cDNA clone was determined by the dideoxy-termination method (Sanger et al. , 1977) using the M13 vectors and M13 primer 5'-GTTTTCCCAGTCACGAC-3' (Messing 1983), [ ⁇ - 35 S]-dATP (Biggin et al., 1983) and the Sequenase Version 2.0 sequencing kit.
  • Single stranded M13 DNA for sequencing was prepared by PEG 6000 precipitation of purified ⁇ phage DNA which had been purified on sequential cesium chloride (CsCl) gradients, ad described by Maniatis et al. (1989).
  • TAP cDNA in the pGEX 2 expression vector were also sequenced directly using an alkaline- denatured double stranded template (Hattori and Sakaki 1986), and utilizing the pGEX primer 5'-GCATGGCCTTTGCAGGG- 3' to identify in frame fusions.
  • DNA sequences were analyzed using a VAX750 model and the GCG Sequence Analysis Software package (Devereux et al.. 1984).
  • Genbank IntelliGenetics Inc., CA, USA
  • European Molecular Biology Laboratory EMBL, Heidelberg, FRG
  • National Biomedical Research Foundation NBRF, Washington, USA
  • Human Retroviruses and AIDS Hardvard University, MA, USA
  • DNA databanks and the NBRF, Swiss-Prot (Geneva, Switzerland), Newat (LaJolla, CA, USA) and the Ooi/Nakashima (Kyoto, Japan) protein data banks were searched for sequence homologies.
  • TAP phage cDNA was subcloned into the bacterial expression plasmids pGEX (Smith and Johnson, 1988).
  • TAP fusion protein was isolated by a modified version of the original method of Smith and Johnson (1988). In brief, overnight cultures were diluted 1:10 into 800 ml of L-broth containing 25 ⁇ g/ml of ampicillin and incubated at 37°C for 3 hours before the addition of IPTG to 1 mM to induce expression of fusion protein.
  • Beads were washed with 100 column vol of HT-PBS, and affinity-purified protein eluted either by competition with 5 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, (then dialyzed against HT-PBS) or with SDS-PAGE sample buffer, and then stored at -70°C.
  • Total cellular RNA was purified from tissues with the guanidinium-CsCl method of Chirgwin et al. (1979). Fresh tissue was combined with 4 M guanidinium thiocyanate, homogenized with the Polytron (1-2 min, medium speed), and the homogenate layered onto a 5.7 M CsCl cushion, and centrifuged at 35,000 rpm (115,000 g) for 16 hours at 18°C. The RNA pellet was washed with ethanol, resuspended, precipitated with ethanol, resuspended in 100-500 ⁇ l TE, heated at 65°C and centrifuged at 10,000 rpm for 5 min.
  • RNA was purified from total RNA by affinity chromatography of oligo(dT)-cellulose, as described (Aviv and Leder 1972). RNA was quantitated by spectrophotometry at 260 nm (Maniatis et al., 1989). TAP cDNA was cloned into pBluescript II (KS+/-), and the plasmid DNA purified by sequential CsCl gradients (Maniatis et al., 1989). The plasmid was linearized downstream of the RNA polymerase promoter to be used, either T7 or T3 RNA polymerase with respect to the cDNA insert, by Xhol and BamHI, respectively.
  • RNA transcripts were produced essentially as described by Melton et al. (1984). Briefly, linear plasmid DNA templates were transcribed in 40 mM Tris, pH 7.5, 6 mM MgCl2, 2 mM spermidine, 10 mM DTT, RNAsin (1 U/ml), 100 ⁇ g/ml BSA, and 500 ⁇ M ATP, GTP and TUP. 3 5s-CTP and "1 ⁇ l of the apprpriate polymerase were added to the linearized plasmid DNA and incubated for 35 min at 37°C. A further 1 ⁇ l of polymerase was then added and the reaction incubated for 40 min at 37°C.
  • RNA transcript was extracted with ⁇ /CHCL3, then precipitated immediately before use and electrophoresed in an agarose-formaldehyde gel (see below) to monitor the efficiency of the transcription reaction.
  • RNA normal human thyroid total RNA (20 ⁇ g) was electrophoresis in a 1% agarose-formaldehyde gel at 60 V, transferred to nitrocellulose by capillary blotting (Thomas, 1980) with 20 x SSC for 12 hours.
  • the filter was baked at 80°C in vacuo for 2 hours, prehybridized (in hybridization buffer without riboprobe, 0.3 M NaCl, 0.07 M Na 2 , HP04, 0.01 M Tris HC1, pH 7.4, 0.005 M EDTA, 20% w/v dextran sulphate, 50% w/v forraaraide, 7 U/ml heparin, 0.01 M DDT, 0.1 mg/ml salmon sperm DNA for 6 hours at 65°C, followed by hybridization for 16 hours at 65°C in the same buffer containing either sense or antisense 3 ⁇ S-labelled TAP riboprobe.
  • Filters were washed twice in 2 x SSC, 0.1% SDS at 65°C for 15 min, prior to addition of RNase TI (10 ⁇ g-20 ⁇ g/ml) for 15 minn at 37°C. The filter was washed twice at 65°C for 15 min in 0.1% w/v SDS, 0.1 x SSC and analyzed by autoradiography. Polymerase chain reaction (PCR; Saiki et al. , 1985) was also used as a highly sensitive method to determine the tissue distribution of TAP.
  • PCR Polymerase chain reaction
  • TAPl corresponded to the 5' end of the TAP putative open reading frame (ORF): 5'-ATTGGATCCATGTACACGCCATGGCTCC-3' .
  • TAP2 at the 3' end of the ORF 5'-CCCGAATTTCAGCCCCACAAACAGGCTG-3' .
  • TAP The complete nucleotide sequence of TAP is shown in Figure 1 and comprises 2,007 nucleotides. Both strands of TAP were sequenced with Sequenase Version 2.0. Analysis of the sequence revealed an ORF which extends for 585 bp, preceded by two methionine residues and codes for a protein of 195 amino acid residues with a predicted M r of "20,700.
  • the first ATG codon, located 109 nucleotides downstream from the 5'-end of the TAP clone, is considered a potential initiation site for translation, because the surrounding sequence is in good agreement with the consensus defined for start codons (Kozak 1987) and because a stop codon is present 29 bp upstream of this codon.
  • Another potential initiator codon is present 13 nucleotides downstream of the first ATG, but the surrounding sequence is not consistent with a start site.
  • the putative non-coding sequence ("1,250 nucleotides) downstream of the open reading frame in the TAP clone does not contain a consensus sequence for the poly(A+) addition site (AATAAA) nor a poly(A+) tail.
  • TAP fusio protein the putative coding region of TAP was subclone into the pGEX 2 expression plasmid to generate in fram fusions.
  • Transformed plasmids containing TAP were identified by restriction enzyme analysis.
  • One, clone 3 was selected for further characterization and sequence using teh pGEX sequencing primers to ensure that ligatio of the "600 bp TAP PCR product into pGEX had bee successful.
  • Purified pGEX-TAP fusion protein was subjecte to SDS-PAGE and stained with Coomassie blue (Figure 2, Panel A). Several protein bands were identified.
  • Th intense band at "43-46 kD represents the pGEX-TAP fusio protein, which is consistent with the predicted molecular mass of "21 kD for TAP.
  • the less intense bands at "26 kD represent the GST moiety and indicate, because of their weak intensity, that the fusion protein was not significantly degraded.
  • the bands of higher M r (above 70 kD) were subsequently shown to represent co-purified E__ coli proteins.
  • TAP mRNA was examined by Northern analysis and PCR.
  • an ⁇ S-labelled riboprobe (corresponding to the amino-terminal "350 bp of the TAP clone) was synthesized with T3 RNA polymerase and used to probe normal human thyroid total RNA.
  • T3 RNA polymerase One major species (“4.9 kb) was expressed in normal human thyroid tissue.
  • PCR was used as a highly sensitive method for detecting TAP mRNA expression.
  • First strand cDNA was prepared using "0.5-1 ⁇ g of total cellular RNA or "0.1-0.5 ⁇ g of poly(A+) RNA from a variety of human and mouse tissues. PCR was performed on the first strand cDNA using oligonucleotides TAP-1 and TAP-2 designed to prime the amplification of the TAP coding region ("600 bp). The amplified product was detected in Graves' thyroid total RNA ( Figure 3, Lane G ), and in the positive controls including samples containing TAP cDNA from the Bluescript vector ( Figure 3, Lane Cl) and first strand cDNA produced after reverse transcription of TAP in both orientations ( Figure 3, Lanes TI and T2).
  • No 600 bp PCR product was identified from RNA samples of human or mouse brain (poly(A+)), normal human thyroid (poly(A+)), porcine thyroid (poly(A+)), skeletal muscle (total RNA), cirrhotic liver (total RNA), normal human liver (total RNA), or HeLa cell (total RNA) preparations.
  • PCR was performed using aliquots of the same first strand cDNA but with PCR primers designed to amplify a "300 op region of U1RNP.
  • a U1RNP PCR product was amplified from HeLa cells. Graves' thyroid, normal human liver and with positive control U1RNP cDNA.
  • 32 P-TAP was used to probe a normal human thyroid ⁇ gtll cDNA library by plaque hybridization.
  • Four clones were isolated of 1.5, 0.9, 0.8 and 0.6 kb and further characterization of these clones is in progress.
  • TAP Sequence Analysis of TAP
  • the sequence of TAP is novel. Interestingly, no significant homology was found between the sequences of the TSH-Rs and LH/CG-Rs and TAP. Thus, the reason that 32 p _
  • TSH-R-P1 hybridized to TAP was due to the presence of short stretches of incidental nucleotide homology between the two sequences (3 regions of significant homology were identified, eg. 16 our of 20 nucleotide match), and not becuase TAP coded for the TSH-R. Furthermore, the TSH-R-P1 oligonucleotide was moderately degenerate which increased the possibility of non-specific hybridization. However, significant homology was identified with a recentlyrcloned cytoskeletal component, human actin-binding protein (HABP, Gorlin et al 1990) ( Figure 4). This homology was present over the majority of the TAP sequence: ie.
  • HABP human actin-binding protein
  • HABP is an actin crosslinking phosphoprotein which in platelets, links actin filaments to several membrane glycoproteins, including GP la and GP lb (Fox 1985).
  • HABP was cloned by immunoscreening a human umbilical vein endothelial cell cDNA library with a panel of anti-ABP MoAbs (Gorlin et al 1990), and consists of a polypeptide subunit chain of 2,647 amino acids, coding for a protein of 280 kD, and represented by a single RNA species of 8 kilobases ( b) ( Figure 5B).
  • actin binding domain near the amino-terminus of the subunit (first 275 amino acids) where the amino acid sequence is similar to other actin filament binding proteins including ⁇ -actinin, ⁇ -spectrin, dystrophin and Dictvostelium abp-120 (Gorlin et al 1990).
  • the remaining 90% comprises 24 repeats, each "96 residues long, predicted to have stretches of ⁇ -sheet secondary structure interspersed with turns. Sequence insertions immediately before repeat 16 and 24 predict two hinges. Calpain proteolysis studies of platelet GP Ib-ABP protein complexes indicate that glycoprotein binding takes place within the last 10 kD of HABP.
  • TAP is a truncated actin-binding-like protein, lacking the amino-terminus actin-binding domain and 22 of the 24 repeats (Figure 6B).
  • TAP does contain a region similar to the second putative insertion region, prior to the 24th repeat in HABP ( Figure 4, residues bounded by the two arrows and Figure 5A, region b).
  • sequence homology between TAP and HABP is significantly greater either side of this putative insertion sequence.
  • TAP contains the consensus sequence for a Ca +2 /calmodulin kinase phosphorylation site within this same region of the sequence ( Figure 4, underlined residues (RLVS); Figure 5A, region b).
  • region b in TAP also contains a putative N-linked glycosylation site ( Figure 4, NET, residues 43-45).
  • the least homology between TAP and HABP exists at the extreme carboxyl- terminus of both sequences ( Figure 5; Figure 6A, region d, ⁇ 15%), which, for HABP, is considered to be a region critical for self-association (dimerization) of HABP monomers (Gorlin et al 1990).
  • TAP Hydropathy profiles (Kyte and Doolittle 1982) of TAP and the last "200 amino acid residues of HABP are compared in Figure 6.
  • TAP contains a hydrophobic region at its extreme carboxyl-terminus (underlined) consistent with a putative transmembrane domain. No such region is present in HABP.
  • actin-binding and dimerization domains in TAP implies that it is not involved in binding or crosslinking of actin.
  • TAP would appear to comprise the last two ⁇ -sheet repeats of HABP, between which lies a putative glycoprotein binding region.
  • TAP sequence of the putative glycoprotein binding region in TAP is significantly dissimilar to that of HABP, but does contain the same consensus sequence for a potential phosphorylation site.
  • TAP would appear to be membrane bound at its carboxyl-terminus end, and may be glycosylated.
  • Thyrotropin induces changes in the morphology and the organization of microfilament structures in cultured thyroid cells.

Abstract

La présente invention concerne l'identification, le clonage et le séquençage d'un nouvel autoantigène thyroïdien, désigné ci-après protéine tronquée de fixation d'actine (TAP en anglais), ainsi que la préparation synthétique ou recombinante d'une telle protéine ou de fragments ou dérivés de celle-ci. L'invention décrit également des anticorps de ce facteur (à la fois polyclonaux et monoclonaux), des compositions pharmaceutiques comprenant ladite protéine ou des anticorps de celle-ci, ainsi que l'utilisation de ladite protéine ou des anticorps de celle-ci en thérapie ou pour les diagnostics.
PCT/AU1992/000024 1991-01-25 1992-01-24 Nouvel antigene thyroïdien WO1992013000A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0753014A1 (fr) * 1993-02-19 1997-01-15 Research Corporation Technologies, Inc ANTIGENE DE LA POLYARTHRITE RHUMATOIDE RECOMBINE SPECIFIQUE DE L'IgM
WO1997025423A1 (fr) * 1996-01-05 1997-07-17 Icos Corporation Modulateurs cytoplasmiques de liaison integrine
WO2001038382A1 (fr) * 1999-11-23 2001-05-31 Bioroad Gene Development Ltd. Shanghai Nouveau polypeptide, ou proteine 54 humaine associee a l'actine, et polynucleotide codant pour ledit polypeptide

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Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 170(1), pp. 351-358, issued 1990, T. AKAMIZU, "A Microsequencing Approach to Identify Proteins which Appear to Interact with Thyrotropin in Rat FRTL-5 Thyroid Cells". *
BIOCHEMISTRY, Vol. 22, pp. 2463-2469, R. KOBAYASHI, "Identification and Purification of Calcium Ion Dependent Modulators of Actin Polymerization from Bovine Thyroid". *
CELL BIOLOGY INTERNATIONAL REPORTS, Vol. 6(12), pp. 1109-1118, 1982, AARON AVIVI, "On the Mechanism of TSH-Induced Formation of Follicle-like Structures in Primary Cultures of Thyroid Cells". *
EUROPEAN JOURNAL BIOCHEMISTRY, Vol. 147, pp. 263-272, H. PASSAREIRO, "Thyrotropin modifies the synthesis of actin and other proteins during thyroid cell culture". *
EUROPEAN JOURNAL OF BIOCHEMISTRY, Vol. 122, pp. 153-161, 1982, FRANCOISE RENOUF, "Isolation from Thyroid Cells of Purified Plasma Membranes with Associated Actin Microfilaments". *
EUROPEAN JOURNAL OF BIOCHEMISTRY, Vol. 129, pp. 149-155, issued 1982, CLAUDE ROUSTAN, "Isolation and Structural Properties of a High-Molecular-Weight Actin-Binding Protein (Filamin-Like Protein) in Hog Thyroid Gland". *
EXPERIMENTAL CELL RESEARCH, Vol. 137, issued 1982, D. TRAMONTANO, "Thyrotropin Induces Changes in the Morphology and the Organization of Microfilament Structures in Cultured Thyroid Cells", see pages 269-275. *
FEBS LETTERS, Vol. 232 (2), pp. 409-413, 1988, PIERRE P. ROGER, "Microinjection of catalytic subunit of cyclic AMP-dependent protein Kinases triggers acute morphological changes in thyroid epithelial cells". *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0753014A1 (fr) * 1993-02-19 1997-01-15 Research Corporation Technologies, Inc ANTIGENE DE LA POLYARTHRITE RHUMATOIDE RECOMBINE SPECIFIQUE DE L'IgM
EP0753014A4 (fr) * 1993-02-19 1997-08-20 Theratech Inc ANTIGENE DE LA POLYARTHRITE RHUMATOIDE RECOMBINE SPECIFIQUE DE L'IgM
WO1997025423A1 (fr) * 1996-01-05 1997-07-17 Icos Corporation Modulateurs cytoplasmiques de liaison integrine
WO2001038382A1 (fr) * 1999-11-23 2001-05-31 Bioroad Gene Development Ltd. Shanghai Nouveau polypeptide, ou proteine 54 humaine associee a l'actine, et polynucleotide codant pour ledit polypeptide
US7273724B1 (en) 1999-11-23 2007-09-25 Shanghai Bio Road Gene Development, Ltd. Polypeptide-human actin-binding protein 54 and a polynucleotide encoding the same

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