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WO1999058565A1 - Proteine relative a la cystatine - Google Patents

Proteine relative a la cystatine Download PDF

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Publication number
WO1999058565A1
WO1999058565A1 PCT/SE1999/000764 SE9900764W WO9958565A1 WO 1999058565 A1 WO1999058565 A1 WO 1999058565A1 SE 9900764 W SE9900764 W SE 9900764W WO 9958565 A1 WO9958565 A1 WO 9958565A1
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acid sequence
protein
testatin
nucleic acid
seq
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WO1999058565A8 (fr
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Katarina Nordqvist
Virpi TÖHÖNEN
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Karolinska Innovations AB
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Karolinska Innovations AB
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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
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 generally to the fields of organogenesis and tumor disease. More specifically, the present invention relates to the potential use of a novel gene and a corresponding protein, denominated testatin, in testis development and testis tumor diagnostics and treatment. Pharmaceutical compositions containing the protein are also claimed.
  • the gonads serve as an excellent model system for analyzing functions associated with the process of cellular differentiation and organogenesis.
  • the indifferent gonad is the only organ rudiment which can differentiate into two different tissues, the ovary and the testis. By introducing mutations in the testicular pathway, the gonads will develop as ovaries and the net result will be a living individual with a female phenotype. This is an advantage compared when working with other organ rudiments, where introduction of mutations in important genes normally lead to lethality.
  • Studies of gonad development using mouse model system will therefore help us to understand the basis for dysfunctions in diseases associated with the reproductive organs. This include different cancer forms as well as genetically inherited diseases (K. Nordqvist, Int. J. Dev. Biol. vol. 39: 727-736, (1995); A. Schafer, Adv. in Genetics, vol. 33: 275-329, (1995)).
  • Tumors of the testis can be divided into three different groups: germ cell tumors, gonadal tumors and neoplasm not unique to the gonads (i.e., mesenchymal tumors). 90% of all cancer forms associated with the testis are originating from the germ cells. Germ cell tumors are classified into two major subgroups: seminoma (most frequent among men in their forties) and non-seminoma (most frequent among younger men). Both Leydig and Sertoli cell tumors are rare. Most cases (90%) are benign. However, it is very difficult to distinguish between a malign and a benign neoplasm and it would therefore be very valuable if tumor markers were available (R. Oliver: Curr. Op. in Oncology, vol. 9: 287- 294 (1997); G. Bosi et al., Cancer: Principles & Practice of Oncology, 5th Ed., Lippincott-Raven Publ. Philadelphia, USA, p. 1397-1425 (1997)).
  • Cancer of the ovary is the third most frequent gynecological malignancy in women and have a higher mortality rate than all other genital cancer forms combined.
  • the reason for the high mortality is the difficulty in detecting the neoplasm in an early stage.
  • patients with tumors of low malignancy have an excellent prognosis even if the tumors are of an advanced stage. This underscores the need to be able to distinguish between low malignant and high malignant tumors.
  • tumor markers are therefore important tools for the diagnosis (D. Gallup: J. Med. Assoc. Georgia 86: p. 181- 185, 1997; R. Ozols et al. Cancer: Principles & Practice of Oncology, 5th Ed., Lippincott-Raven Publ., p. 1502-1539 (1997)
  • a novel cystatin-related protein has been isolated from fetal male mouse gonad.
  • the corresponding human protein has also been isolated.
  • the novel protein has a potential role in inducing testis formation in fetal gonads. It is also highly probable that the protein inhibits testis tumor growth because of structural and functional similarities with tumor inhibiting cy statins.
  • the corresponding nucleic acid sequence, as well as vectors and host cells containing the nucleic acid sequence, and pharmaceutical compositions containing the protein are also claimed. Detailed description Cystatins
  • the cystatins comprise a superfamily of related proteinase inhibitors which can be divided into three subfamilies, 1 to 3 (Rawlings and Barrett, 1990, J.Mol.Evol. 30, 60- 71).
  • Family 1 are intracellular proteins lacking a signal peptide, commonly termed sefins.
  • Family 2 are abundant extracellular inhibitors and family 3 consists of the circulating kininogenes (Henskens et al., 1996, Biol. Chem. 377, 71-86)
  • cystatins Cysteine proteinases and their inhibitors, the cystatins, seem to have important regulatory roles in normal body processes such as bone resorption and embryo implantation
  • cystatins are secreted proteins of approximately 120 amino acid residues containing four characteristic cysteines located near the carboxyl terminus.
  • the human members of this family include cystatin C, D, E, S, SA and SN (reviewed in Henskens et al., 1996, Biol. Chem. 377, 71-86; Ni et al., 1997, J. Biol. Chem. 272, 10853-10858).
  • cystatin C and E have a broad distribution and are expressed in most tissues while cystatin D is more tissue-specific in its localization and is primarily found in the parotid gland (Freije et al., 1991, J. Biol. Chem. 266, 20538-43; Ni et al., 1997, J. Biol. Chem. 272, 10853-10858 ).
  • family 2 cystatins are highly conserved in three domains which are important for binding to and inhibition of cysteine proteinases. These includes an N- terminal glycine (located as Gly 11 in human cystatin C), an QXVXG region (Gln55- Gly 59) located in the first hairpin loop created by the first two cysteines and two conserved amino acids (Prol05-Trpl06) in the second hairpin loop. Together these three sites form a wedge which is complementary to the active site of papain, which is the most commonly used proteinase when looking at proteinase inhibition by cystatins.
  • cystatin C The structural basis for inhibition of different target proteinases as cathepsin B, H, L and S by human cystatin C have been determined and show that the conserved regions in cystatin C are differently important for different proteinases (Hall et al., 1995, J. Biol. Chem. 270, 5115-21). For example, both the N-terminal domain and the Trp-106 residue are of crucial importance for the cystatin C inhibition of cathepsin L and B, but less significant for the interaction with cathepsin S. It is also clear that different cystatins have different inhibitory potential on different cathepsins, For example, cystatin C inhibits most cathepsins while cystatin D preferentially inhibits cathepsin L. This is probably due to structural differences in the domains of the cystatins interacting with the proteinases.
  • testatin refers to a protein isolated from fetal male gonad, preferably fetal male mouse gonad and still more preferably adult male human testis.
  • the amino acid sequence of testatin is homologous to cystatins in several regions, and especially in the C-terminal part. The overall homology in relation to mouse cystatin C is at least 20%.
  • the protein is believed to interact with cathepsins and to play an important role in the development of testis tissue from gonads.
  • the protein shows at least 60 %, more preferably at least 80 %, and still more preferably at least 90 %, homology with the amino acid sequence according to SEQ.ID.N0.8.
  • the amino acid sequence comprises a sequence according to SEQ.ID.N0.8.
  • nucleic acids or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • Optimal alignment of sequences for comparison can be cunducted, e.g., by the local homology algorithm of Smith and Waterman ( ⁇ 9$ ⁇ ) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) JMol. Biol.
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin and Altschul (1993) Proc. Nat 'l. Acad. Sci. USA 90: 5873- 5787.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to an NBCCS gene or cDNA if the smallest sum probability in a comparison of the test nucleic acid to an NBCCS nucleic acid (e.g., SEQ ID Nos: 1,58, or 59) is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the term "substantial identity” or “substantial similarity" in the context of a polypeptide indicates that a polypeptides comprises a sequence with at least 70% sequence identity to a reference sequence, or preferably 80%, or more preferably 85% sequence identity to the reference sequence, or most preferably 90% identity over a comparison window of about 10-20 amino acids residues.
  • polypeptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
  • nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • the present invention also relates to nucleic acid sequences encoding testatin.
  • nucleic acid sequences which encode testatin are deemed to be substantially similar to those described herein if: (a) the nucleic acid sequence is derived from the coding region of a native testatin gene (including, for example, variations of the sequences disclosed herein; (b) the nucleic acid sequence is capable of hybridization to nucleic acid sequences of the present invention under conditions of either moderate or high stringency (hybridization in 5 x SSPE containing 0.1%) SDS and 0.1 mg/ml ssDNA, at 50 - 65 ° dependent on the probe length, or 10 - 20°C below the T m of the probe; washing in 1 x SSPE, 0.1% SDS at 15 - 20°C below the T m of the probe for moderate stringency, and in 0.1 x SSPE, 0.1% at 10°C below the T m of the probe for high stringency conditions) (
  • nucleic acid molecules are primarily referred to herein, as should be evident to one of skill in the art given the disclosure provided herein, a wide variety of related nucleic acid molecules may also be utilized in various embodiments described herein, including for example, RNA, nucleic acid analogues, as well as chimeric nucleic acid molecules which may be composed of more than one type of nucleic acid.
  • probes and primers are provided for detecting nucleic acid sequences encoding testatin.
  • probes are provided which are capable of hybridizing to testatin nucleic acid sequences (DNA or RNA).
  • probes are "capable" of hybridizing to testatin nucleic acids if they hybridize to SEQ.ID.NO. 2 or SEQ. ID .NO. 9 preferably SEQ.ID.NO.
  • the probe may be utilized to hybridize to suitable nucleotide sequences in the presence of 5 x SSPE, 0.1% SDS, and 0.1 mg/ml ssDNA at 10-20°C below the T m of the probe. Subsequent washes may be performed in 1 x SSPE, 0.1% SDS at 15 - 20 °C for conditions of moderate stringency, and in 0.1 x SSPE, 0.1 % SDS at 10°C below the T m of the probe for conditions of high stringency.
  • Probes of the present invention may be composed of either deoxyribonucleic acids (DNA), ribonucleic acids (RNA), nucleic acid analogues, or any combination of these, and may be as few as about 12 nudeotides in length, usually about 14 to 18 nudeotides in length, and possibly as large as the entire sequence which encodes testatin. Selection of probe size is somewhat dependent upon the use of the probe. For example, a long probe used under high stringency conditions is more specific, whereas an oligonucleotide carefully selected from the sequence can detect a structure of special interest.
  • Probes may be constructed and labeled using techniques which are well known in the art. Shorter probes of, for example, 12 or 14 bases may be generated synthetically. Longer probes of about 75 bases to less than 1.5 kb are preferably generated by, for example, PCR amplification in the presence of labeled precursors such as P-dCTP, digoxigenm- dUTP, or biotin-dATP. Probes of more than 1.5 kb are generally most easily amplified by transfecting or transforming a cell with a a phage or a plasmid containing the relevant probe, growing the transfected or transformed cell into large quantities, and purifying the relevant sequence from the transfected or transformed cells (See Sambrook, supra).
  • Probes may be labeled by a variety of markers, including, for example, radioactive markers, fluorescent markers, enzymatic markers, and chromogenic markers.
  • markers including, for example, radioactive markers, fluorescent markers, enzymatic markers, and chromogenic markers.
  • the use of 32 P is particularly preferred for marking or labeling a particular probe.
  • nucleic acid probes of the present invention may be utilized to detect the presence of testatin nucleic acid molecules within a sample. However, if such nucleic acid molecules are present in only a limited number, then it may be beneficial to amplify the relevant sequence such that it may be more readily detected or obtained.
  • RNA amplification see Lizardi et al., Bio/Technology 6: 1197-1202, 1988; Kramer et al., Nature 339: 401-402, 1989; Lomeli et al., Clinical Chem. 35(91) 1826- 1831, 1989; U.S. Patent No. 4,786,600
  • PCR Polymerase Chain Reaction
  • PCR amplification is utilized to detect or obtain testatin nucleic acids. Briefly, as described in greater detail below, a nucleic acid sample is denatured at 95 °C in order to generate single stranded nucleic acid. Specific primers, as discussed below, are then annealed at 37°C to 70°C, depending on the proportion of AT/GC in the primers. The primers are extended at 72°C with Taq polymerase in order to generate the opposite strand to the template. These steps constitute one cycle, which may be repeated in order to amplify the selected sequence.
  • Primers for the amplification of a selected sequence should be selected from sequences which are highly specific and form stable duplexes with the target sequence.
  • the primers should also be non-complimentary, especially at the 3' end, should not form dimers with themselves or other primers, and should not form secondary structures or duplexes with other regions of nucleic acid. In general, primers of about 18 to 20 nudeotides are preferred, and may easily be synthesized using techniques well-known in the art.
  • the present invention relates to vectors and host cells comprising the above mentioned nucleic acid sequences.
  • the above described nucleic acid molecules which encode testatin (or portions thereof) may be readily introduced into a wide variety of host cells.
  • Representative examples of such host cells include plant cells, eucaryotic cells, and prokaryotic cells.
  • the nucleic acid molecules are introduced into cells from a vertebrate or warm-blooded animal, such as a human, chicken, macaque, dog, cow, horse, pig, sheep, rat, hamster, mouse, or a fish or any hybrid thereof.
  • the nucleic acid molecules may be introduced into host cells by a wide variety of mechanisms, including for example calcium phosphate-mediated transfection (Wigler et al, Cell 14:725, 1978), lipofection; gene gun (Corsaro and Pearson, Somatic Cell Gen. 1 :603, 1981 ; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1 :841-845, 1982), retroviral, adenoviral, protoplast-mediated transfection or DEAE dextran-mediated transfection (Ausubel et al., (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, NY, USA, 1987).
  • the nucleic acid molecules, antibodies, and proteins of the present invention may be labeled or conjugated (either through covalent or non-covalent means) to a variety of labels or other molecules, including for example, fluorescent markers, enzyme markers, toxic molecules, molecules which are non-toxic but which become toxic upon exposure to a second compound, and radionuclides.
  • fluorescent labels suitable for use within the present invention include, for example, Fluorescein Isothiocyanate (FITC), Rhodamine, Texas Red, Green Fluorescent Protein (GRP), Luciferase and Phycoerythrin (PE).
  • FITC Fluorescein Isothiocyanate to antibodies.
  • I Experiments on the Conditions of Conjugation," Immunology 18:865-873, 1970.
  • Keltkamp Conjugation of Fluorescein Isothiocyanate to Antibodies.
  • II A Reproducible Method
  • Immunology 18:875-881, 1970 and Goding, "Conjugation of Antibodies with Fluorochromes: Modification to the Standard Methods," J. Immunol. Methods 13:215-226, 1970).
  • HRP which is preferred, may be conjugated to the purified antibody according to the method of Nakane and Kawaoi ("Peroxidase-labeled Antibody: A New Method of Conjugation," J. Histochem. Cytochem. 22: 1084-1091, 1974; see also Tijssen and Kurstak, "Highly Efficient and Simple Methods for Preparation of Peroxidase and Active Peroxidase Antibody Conjugates for Enzyme Immunoassays," Anal. Biochem. 136; 451- 457, 1984).
  • Representative examples of enzyme markers or labels include alkaline phosphatase, horse radish peroxidase, and ⁇ -galactosidase.
  • Representative examples of toxic molecules include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A.
  • Representative examples of molecules which are non-toxic, but which become toxic upon exposure to second compound include thymidine kinases such as HSVTK and VZVTK.
  • radionuclides include Cu-64, Ga-67, Ta-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-I l l, 1-123, 1-125, 1-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212.
  • nucleic acid molecules, antibodies, proteins and peptides may also be labeled with other molecules such as colloidal gold, as well as either member of a high affinity binding pair (e.g., avidin-biotin).
  • a high affinity binding pair e.g., avidin-biotin
  • the present invention also provides a variety of pharmaceutical compositions, compositions for treating genital tumors, comprising testatin or a peptide portion thereof along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents.
  • a pharmaceutically or physiologically acceptable carrier such as a pharmaceutically or physiologically acceptable carrier, excipients or diluents.
  • such carriers should be non-toxic to recipients at the dosages and concentrations employed.
  • the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients.
  • Neutral buffered saline or saline mixed with non-specific serum albumin are exemplary appropriate diluents.
  • compositions of the present invention may be prepared for administration by a variety of different routes, including, for example, intraarticulary, intracranially, intradermally, intramuscularly, intraocularly, intraperitoneally, intrathecally, intravenously, subcutaneously or even directly into a tumor (for example, by stereotaxic injection).
  • pharmaceutical compositions of the present invention may be placed within containers along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangiable expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g. water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition.
  • excipient ingredients or diluents e.g. water, saline or PBS
  • Fig. 1 discloses directed mRNA differential display using total RNA from male and female fetal gonads+mesonephros. Reverse transcriptions were performed using the decamer primers 5* 13, 5* 14, 5* 15 and 5* 16. Using these primers in combination with the directed multi-Leu primer, cDNAs were amplified by PCR in the presence of [ ⁇ P] dATP for 30 cycles, resolved on a denaturing polyacrylamid gel and exposed to X-ray film. One male specific cDNA fragment, M12, is indicated with arrow;
  • Fig. 2 shows the nucleotide and predicted amino acid sequence of the testatin gene.
  • the start sites for three testatin cDNA clones, 2A1 (nt 1), 10B3 (nt 6) and 4C2 (70) are marked with arrows.
  • the two predicted translation initiation methionine codons, ATG, are underlined (nt 50-52 and 89-91), and stop codons are marked with asterisks.
  • Predicted signal cleavage site is shown with a filled triangle. Exon intron boundaries are indicated by open triangles. Potential N-glycosylation sites are marked with an open box;
  • Fig.3 presents northern blot analysis of the testatin mRNA. 5 ⁇ g of total RNA from fetal gonads, 14.5 dpc, and adult mouse and rat testis, was separated on a 1.5 % agarose gel. RNA was transferred to a nitrocellulose filter and hybridized to a testatin specific probe;
  • Fig.4 discloses in vitro translation of two testatin cDNA clones, 2A1 and 4C2;
  • Fig.5 shows alignment of the derived testatin amino acid sequence (SEQ.ID.NO.1) with mouse cystatin C (SEQ.ID.NO.5), chicken cystatin C (SEQ.ID.NO.6) and CRES (Cystatin-Related Epidymal Specific)(SEQ.ID.N0.7). Putative signal peptides are indicated in yellow. The numbering is according the predicted testatin sequence. Pink boxes indicate homologous regions between testatin and the cystatins. Red boxes represent aligned cysteine residues. The marked amino acids (*) indicate putative N- glycosylation sites of the testatin protein. The lower black line is over the three sites thought to be critical to the cystatins for cysteine proteinase inhibitory activity. The last of these domains are indicated by a grey box; and
  • Fig.6 presents semi-quantitative RT-PCR for analysis of testatin expression in different tissues.
  • 0.5 ⁇ g of total RNA isolated from adult mouse tissues were reversed transcribed with a T ⁇ NN primer, amplified by PCR using testatin specific primers in the presence of [ ⁇ 33 P] dATP for 24 cycles and then separated on a native polyacrylamide gel.
  • Fig. 7 shows alignment of human testatin amino acid sequence (SEQ.ID.No. 8) (upper sequence) and mouse testatin amino acid sequence (SEQ.ID.NO. 1) (lower sequence).
  • a vertical bar indicate identity and a colon and a dot, respectively indicate sequence similarities. The homology between the two amino acid sequences is 49%.
  • testatin a novel cystatin-related gene, named testatin, was isolated from fetal male mouse gonad (Fig. 2). Substantial homology between the testatin and members of the cystatin family of cysteine proteinase inhibitors was observed at the amino acid level (Fig. 5).
  • testatin The amino acid sequence of the testatin gene is homologous to the cystatins in several regions, specially in the C-terminal part, having the four conserved cysteines in exact alignment in addition to the two conserved amino acids in the second hairpin loop (homologous to Prol05-Trpl06 in human cystatin C).
  • Testatin lacks both the N-terminal gly cine and the QXVXG region located in the first hairpin loop. However, all three domains may not be necessary for cysteine proteinase inhibitory activity. As mentioned above, the different domains in cystatin C are differently important for different cathepsin.
  • testatin which lacks interactive sites compared with the classical cystatins, might have a different specificity in its interaction with cathepsins.
  • testatin may inhibit cell-specific cathepsins not yet isolated or other kind of unknown proteinases.
  • testatin gene had a very restricted expression pattern. So far, the only sites for testatin expression found are in fetal gonads and adult testis (Fig.6). Cysteine proteinases participates in the intracellular catabolism of proteins and peptides, in the proteolytical conversion of prohormones, in extracellular degradation of collagen and in the penetration of normal tissues with malignant cells. As cystatins inhibits cysteine proteinases which accelerate cancer growth or metastasis-formation, it is a potential cancer-inhibiting agent. Testatin has a very restricted expression pattern and is mainly expressed in fetal and adult testis. Therefore, a potential role for testatin could be to specific inhibit different cancer forms associated with the testis without disrupting other, vital functions in the body, a problem which may arise by treatment by cystatins C which is more widely distributed.
  • testatin might have a role in organogenesis and formation of fetal testis. Therefore testatin might work as a potential target for testis disruption during organogenesis, creating a model system for testis malformations.
  • RNA from male and female gonad with attached mesonephros was isolated from 13.5 dpc mouse embryo and DNase treated to remove contaminating DNA (see Nordqvist and T ⁇ honen, Int. J. Dev. Biol., vol. 41 : 627-638 (1997)).
  • reverse transcription using a decamer primer 5 CTC TAA CAG G 3 , SEQ.ID.NO. 3 was done in the following way: Reactions were mixed on ice, each reaction containing 0.45 ⁇ g total RNA and 1 ⁇ l 3 '-primer (2 ⁇ M) in a total volyme of 14 ⁇ l. The RNA was denatured at 70°C for 8 min and transferred back to ice.
  • 5 ⁇ l 5xRT buffer 200 mM Tris pH 7.5, 150 mM KC1, 15 mM MgCl 2
  • 2.5 ⁇ l DTT 0.1 M
  • 2.5 ⁇ l dNTPs 250 ⁇ M
  • 0.3 ⁇ l rRNasin 40u/ ⁇ l, Promega, cat.# N2511
  • 0.5 ⁇ l Superscript II RT 200u/ ⁇ l GIBCOBRL, cat.# 18064- 014
  • mRNA differential display was performed essentially as described by Liang and Pardee (Science, vol. 257: 967-971 (1992)) with some modifications.
  • the following components were mixed on ice in a PCR plate with 96 wells (Techne): 1.5 ⁇ l RT reaction, 2.5 ⁇ l lOxPCR buffer (500 mM KC1, 100 mM Tris pH 9.0, 1% Triton X-100), 2.5 ⁇ l MgCl 2 (20 mM), 0.5 ⁇ l [ ⁇ - 3 P] dATP (lOmCi/ml, DuPont NEN), 1 ⁇ l multi-Leu primer ( 5 CTG CTG CTG CTG CTG 3' ,(SEQ.ID.NO.
  • reactions were kept on ice until the PCR machine had reached 85°C and then applied onto the PCR block, denatured at 92°C for 1 min and then amplified for 28 cycles at 92°C for 50 sec, 40°C for 90 sec, 72°C for 90 sec. After the amplification, reactions were immediately transferred to -20°C.
  • the conditions were the same as before, except that the final dNTP concentration was 40 ⁇ M instead of 2 ⁇ M and no isotope was added.
  • the reamplified cDNA was purified using WizardTM PCR Preps DNA Purification System (Promega, cat.# A7170), cloned into the PCRTMII vector using the TA CloningTM Kit (Invitrogen, cat.# K2000-01) and the recombinant plasmid, containing the M12 cDNA (pM12-5), was transformed into bacteria. Plasmids were extracted using Wizard Minipreps DNA Purification System (Promega, cat.# A7510)
  • DNA sequencing was carried out using the Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer, cat.# 402079) and the samples were then processed on a 373 A automated DNA sequencer (Applied Biosystems).
  • pM12-5 was used as a probe to screen a male specific fetal gonad cDNA library (constructed by the ZAP ExpressTM cDNA synthesis kit (Stratagen), by Katarina Nordqvist in Robin Lovell-Badge laboratory, National institute for Medical Research, Mill Hill, London). The screening procedure was accoring to the manual for the ZAP ExpressTM cDNA synthesis kit (Stratagen). 5x105 recombinants were screened and about 25 positives were found. From these, three different cDNA clones were isolated, 2A1, 4C2 and 10B3, containing 680, 675 and 610 base pair (bp) long sequences respectively (Fig. 2). By sequencing these clones it was disclosed that the inserts shared the same sequence except the most 5' 70 bp.
  • Example 2 Testatin has homology to members of the cystatin family
  • FIG. 5 shows the alignments of the amino acid sequences of testatin compared to the mouse and chicken cystatin C. The amino acids are numbered relative to the coding region of testatin. A number of amino acids have been conserved, indicated in pink, and the observed homology extend further when conservative amino acid exchanges are taken into consideration (data not shown). The highest homology is found in the carboxy terminal, including the presence of four conserved cysteines in exact alignment (represented by red boxes).
  • cysteines form two disulfide loops in the native cystatins which are important for the protease inhibitory activity.
  • Another amino acid important for activity is Gly9. Neither this amino acid, nor the conserved region in the first disulfide loop, consisting of Gln53-Val55-Gly57, have been conserved in testatin.
  • the only amino acids important for cystatin activity which have been conserved in testatin are positioned in the second disulfide loop, Pro93 and Trp94 (indicated as a grey box). Two potential N-glycosylation sites were also found, Asn4 and Asn80 (indicated as grey stars). lo
  • testatin mRNA was present only in adult and fetal testis (13.5 dpc). This result suggests a highly specific expression of the testatin gene in the testis.
  • a mouse testatin probe comprising nudeotides 40-478 (SEQ.ID.NO. 10) in the mouse testatin gene sequence (SEQ.ID.NO. 2) as published by Tohonen et al, PNAS, vol. 95, p. 14208-14213 (1998), was used to screen a human adult testis cDNA library (2AP Express XR (USA) Human Testis Cdt) cDNA Library cat. #939201) according to standard methods (Sambrooke et al, Supra). 5 x 10 5 recombinants were screened and two positive recombinants were found. The cDNA clones were isolated and sequenced using the Dye Terminator Cyde Sequencing Ready Reaction Kit (Perkin Elmer (USA) cat. # 402079) and the samples were then processed on a 373 Automated DNA sequencer (Applied Biosystems, USA).

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  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Un gène codant une nouvelle protéine relative à la cystatine a été isolé de la gonade foetale d'une souris mâle et d'une testicule d'un homme adulte. Cette nouvelle protéine a le potentiel d'induire la formation des testicules dans les gonades foetales. Il est aussi fort probable que cette protéine inhibe la croissance de la tumeur des testicules en raison des similitudes structurales et fonctionnelles avec les cystatines inhibant la tumeur. L'invention concerne également la séquence d'acide nucléique correspondante, les vecteurs et les cellules hôtes contenant la séquence d'acide nucléique ainsi que des compositions pharmaceutiques contenant cette protéine.
PCT/SE1999/000764 1998-05-08 1999-05-06 Proteine relative a la cystatine Ceased WO1999058565A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9801617A SE9801617D0 (sv) 1998-05-08 1998-05-08 New compound
SE9801617-3 1998-05-08

Publications (2)

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WO1999058565A1 true WO1999058565A1 (fr) 1999-11-18
WO1999058565A8 WO1999058565A8 (fr) 2000-01-06

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001046421A3 (fr) * 1999-12-23 2002-01-03 Zymogenetics Inc Zcys5:membre de la superfamille de cystatine
WO2002012479A3 (fr) * 2000-08-07 2002-07-11 Zymogenetics Inc Zcys9: un membre de la superfamille des cystatines
WO2002020567A3 (fr) * 2000-09-01 2002-08-15 Zymogenetics Inc Cystatine-8 mammalienne et utilisation de celle-ci pour inhiber la proteine cancereuse favorisant la coagulation
EP1343813A4 (fr) * 2000-12-19 2004-11-17 Smithkline Beecham Corp Nouveaux composes
US7256262B2 (en) * 1999-03-08 2007-08-14 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
CN100368018C (zh) * 2005-05-26 2008-02-13 福建医科大学 蛇毒半胱氨酸蛋白酶抑制剂抗肿瘤侵袭与转移作用及应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHANGGOO HUH ET AL.: "Structural organization, expression and chromosomal mapping of the mouse cystatin-C-encoding gene (Cst3)", GENE,, vol. 152, 1995, pages 221 - 226, XP004042680 *
EMBL, Database Genbank/DDBJ, accession no. AB017157, Kanno Y et al, 13.01.1999. *
IBTISAM AL-HASHIMI ET AL.: "Purification, Molecular Cloning, and Sequencing of Salivary Cystatin SA-I", THE JOURNAL OF BIOLOGICAL CHEMISTRY,, vol. 263, no. 19, 5 July 1988 (1988-07-05), pages 9381 - 9387, XP000857724 *
LIBUSE A. BOBEK ET AL.: "Human salivary cystatin S, Cloning sequence analysis, hybridization in situ and immunocytochemistry", BIOCHEM. J.,, vol. 278, 1991, pages 627 - 635, XP000863691 *
VIRPI TÖHÖNEN ET AL: "Testatin: A cystatin-related gene expressed during early testis development", PROC. NATL. ACAD. SCI. USA,, vol. 95, November 1998 (1998-11-01), pages 14208 - 14213, XP000857715 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7256262B2 (en) * 1999-03-08 2007-08-14 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7427664B2 (en) * 1999-03-08 2008-09-23 Genentech, Inc. Antibodies to polypeptides that stimulate TNF-α release
WO2001046421A3 (fr) * 1999-12-23 2002-01-03 Zymogenetics Inc Zcys5:membre de la superfamille de cystatine
WO2002012479A3 (fr) * 2000-08-07 2002-07-11 Zymogenetics Inc Zcys9: un membre de la superfamille des cystatines
WO2002020567A3 (fr) * 2000-09-01 2002-08-15 Zymogenetics Inc Cystatine-8 mammalienne et utilisation de celle-ci pour inhiber la proteine cancereuse favorisant la coagulation
EP1343813A4 (fr) * 2000-12-19 2004-11-17 Smithkline Beecham Corp Nouveaux composes
CN100368018C (zh) * 2005-05-26 2008-02-13 福建医科大学 蛇毒半胱氨酸蛋白酶抑制剂抗肿瘤侵袭与转移作用及应用

Also Published As

Publication number Publication date
SE9801617D0 (sv) 1998-05-08
WO1999058565A8 (fr) 2000-01-06

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